National Institute of Immunology New Delhi
Scientific Reports for the Annual Meeting of Research Area Panels and Scientific Advisory Committee
April 7 & 8, 2014
(For limited circulation only)
Contents Page
Programme 1
Publications and Patents 9
Research Project Reports
A. Infection and Immunity 1. Plasmodium proteins involved in virulence and Agam P. Singh 19 host modulation: Host-parasite interactions in Plasmodium liver stages
2. Genetic and functional analyses of host and Akhil C. Banerjea 25 HIV-1 genes that affect progression of HIV-1 and development of nucleic acid based antiviral approaches
3. Understanding the regulation of intracellular Amitabha 29 transport: Role of GTPases Mukhopadhyay
4. Studies on immune response from antigen loaded Amulya K. Panda 33 biodegradable polymer particles and protein refolding from inclusion bodies
5. Study of mucosal immune responses Anna George 38
6. Analysis of Salmonella typhi-host cell interaction Ayub Qadri 43
7. Molecular basis of B cell responses Devinder Sehgal 47
8. Understanding the role of IRFs in dendritic cell Prafullakumar Tailor 51 development and innate immunity
9. Disorders of proliferation: Analysis of novel Rahul Pal 55 pathways and targets
10. Study of genetic and immune factors associated Rajni Rani 60 with autoimmune disorders: Type 1 diabetes and vitiligo
11. Study of immunotherapeutic potential of MIP and Sangeeta Bhaskar 67 the underlying mechanisms in animal models of tuberculosis and tumor
12. Analysis of antigen processing and presentation Satyajit Rath 71
13. Fine tunings of NF-κΒ signaling Soumen Basak 76
14. Biology of animal viruses Sudhanshu Vrati 81
15. Biology of T lymphocytes Vineeta Bal 83
B. Reproduction and Development
16. Cellular and molecular biology of cancer Anil Suri 88
17. Study on expansion and plasticity of bone marrow Asok Mukhopadhyay 95 stem cells
18. Regulation of cell death Chandrima Shaha 101
19. Cellular and molecular aspects of reproduction and Satish K. Gupta 105 viral infections
20. Studies of sertoli cells and spermatogonial stem Subeer S. Majumdar 112 cells of the testis and other endocrinology related research
C. Molecular Design
21. Molecular mechanism of enzymatic reactions and Apurba K. Sau 117 enzyme-ligand interactions
22. Structural and functional studies of mycobacterial Bichitra K. Biswal 121 proteins
23. Molecular modelling of proteins and protein-ligand Debasisa Mohanty 125 complexes using knowledge-based approaches and all atom simulations
24. Structure, interaction and design studies involving Dinakar M. Salunke 131 regulatory peptides and proteins
25. Ribonucleases and heat shock proteins: Janendra K. Batra 135 Involvement in host defense
26. Role of carbohydrates in modulating the structure Kanwal J. Kaur 141 and function of glycopeptides
27. Structural studies on proteins, dynamics and ligand Monica Sundd 145 interactions using NMR
28. To develop strategies for making sensors and Pramod K. Upadhyay 148 actuators for biological processes
29. Protease-catalyzed splicing of peptide bond Rajendra P. Roy 154
30. Therapeutic interventions in chronic diseases: Sarika Gupta 157 Investigations on the effects of homocysteine on bone remodeling
31. Chemical glycobiology: Glycoproteomics and S. Gopalan 164 carbohydrate-based drug design Sampathkumar
32. Biophysical and biochemical characterization of Vidya Raghunathan 171 Leishmania phosphoglycerate kinase: An enzyme in the glycolytic pathway of parasitic protozoa
D. Gene Regulation
33. Elucidating the molecular mechanisms of aging and Arnab Mukhopadhyay 174 innate immunity using Caenorhabditis elegans as a model system
34. Molecular biology of infectious diseases Lalit C. Garg 179
35. Epigenetic regulation of the eukaryotic genome: Madhulika Srivastava 184 Role of transcriptional insulators in organizing chromatin
36. Role of cell signaling in eukaryotic development Pushkar Sharma 189
37. Reconstructing the chemico-cellular trestle to Rajesh S. Gokhale 195 decipher biology of tuberculosis and vitiligo
38. Determining the signaling and repair pathways that Sagar Sengupta 198 are altered in human cancer using RecQ helicases as the model system
39. Understanding the regulation of DNA replication Sandeep Saxena 203
40. The role of tumor suppressors in stress response Sanjeev Das 209
41. Molecular analyses of the human and animal Sher Ali 215 genome(s)
42. Deciphering the role of cell signalling in M. Vinay K. Nandicoori 222 tuberculosis biology and in the function and dynamics of nucleoporins
E. Ancillary Activities
43. Production of transgenic animals and development Subeer S. Majumdar 227 of new transgenic technologies
Program for RAP/SAC Meeting (April 7, 2014)
09:00 Opening remarks M. Vijayan 09:10 Director’s address Chandrima Shaha
Presentations by Principal Investigators
Session A: Immune Response & Immune Regulation Page No.
09:30 Analysis of antigen processing and presentation 71 Satyajit Rath
09:50 Molecular basis of B cell responses 47 Devinder Sehgal
10:10 Study of mucosal immune responses 38 Anna George
10:30 Understanding the role of IRFs in dendritic cell 51 development and innate immunity Prafullakumar Tailor
10:50 Coffee break
Session B: Immune Regulation & Reproduction
11:10 Disorders of proliferation: Analysis of novel 55 pathways and targets Rahul Pal
11:30 Study of genetic and immune factors associated with 60 autoimmune disorders: Type 1 diabetes and vitiligo Rajni Rani
11:50 Cellular and molecular aspects of reproduction and 105 viral infections Satish K. Gupta
12:10 Studies of sertoli cells and spermatogonial stem cells 112 of the testis and other endocrinology related research Subeer S. Majumdar
12:30 Lunch (at New Guest House)
1 Session C: Gene Regulation & Cell Signalling
13:50 Epigenetic regulation of the eukaryotic genome: 184 Role of transcriptional insulators in organizing chromatin Madhulika Srivastava
14:10 Understanding the regulation of DNA replication 203 Sandeep Saxena
14:30 Role of cell signalling in eukaryotic development 189 Pushkar Sharma
14:50 Molecular biology of infectious diseases 179 Lalit C. Garg
15:10 Cellular and molecular biology of cancer 88 Anil Suri
15:30 Coffee break
16:00 Group Interactions (Schedule on page 4) to 18:30
19:30 Dinner (Rose Garden, India International Centre)
(A bus will leave NII campus at 19-15 hours)
2 April 8, 2014
Session D: Protein/Carbohydrate Engineering & Structural Biology
09:30 Chemical glycobiology: Glycoproteomics and 165 carbohydrate-based drug design S. Gopalan Sampathkumar
09:50 Structural studies on proteins, dynamics and ligand 145 interactions using NMR Monica Sundd
10:10 Protease-catalyzed splicing of peptide bond 154 Rajendra P. Roy
10:30 Molecular mechanism of enzymatic reactions and 117 enzyme-ligand interactions Apurba K. Sau
10:50 Role of carbohydrates in modulating the structure 141 and function of glycopeptides Kanwal J. Kaur
11:10 End of Presentations and Coffee break
11:40 Meeting with RAP/SAC Members, Directors and Coordinator
13:30 Lunch and conclusion of meeting
3 Schedule for Group Interactions
Group A: Host-Pathogen Interaction
Venue: Board Room (Third floor)
Committee members: G. Padmanabhan, V.S. Chauhan, V. Nagaraja, D. Chatterjee
16:00 Chandrima Shaha
16:25 Agam P. Singh
16:50 Janendra K. Batra
17:15 Amitabha Mukhopadhyay
17:40 Akhil C. Banerjea
18:05 Ayub Qadri
Group A: Cancer Biology & Cellular Differentiation
Venue: Seminar Room I (Ground floor)
Committee members: Subrata Sinha, Radhakrishna Pillai, S.C. Lakhotia, Uttam Surana
16:00 Sagar Sengupta
16:25 Pramod K. Upadhyay
16:50 Sanjeev Das
17:15 Asok Mukkhopadhyay
17:40 Arnab Mukhopadhyay
4 Group C: Immune Regulation & Chronic disease
Venue: Seminar room # II (First floor)
Committee members: Umesh Varshney, L.S. Shashidhara, Ranjan Sen, D.N. Rao
16:00 Vineeta Bal
16:25 Soumen Basak
16:50 Amulya K. Panda
17:15 Sangeeta Bhaskar
17:40 Sarika Gupta
Group D: Gene Regulation & Structural Biology
Venue: New Guest House (First floor)
Committee members: M. Vijayan, Lalita Ramakrishnan, Shekhar Mande, B.J. Rao
16:00 Bichitra K. Biswal
16:25 Vinay K. Nandicoori
16:50 Sher Ali
17:15 Vidya Raghunathan
17:40 Debasisa Mohanty
5 Allocation of Principal Investigators for RAP/SAC review
Reviewer Principal Investigators Page
Prof. B.J. Rao Lalit Garg 179 Pushkar Sharma 189 Subeer Majumdar 112,227 Monica Sundd 145 Bichitre K. Biswal 121 Debasisa Mohanty 125 Vidya Raghunathan 171 Vinay K Nandicoori 222 Sher Ali 215 Prof. Dipankar Chatterji Apurba K. Sau 117 Rajendra P. Roy 154 Monica Sundd 145 Lalit C. Garg 179 Chandrima Shaha 101 Agam P. Singh 19 JK Batra 135 Amitabha Mukhopadhyay 29 Akhil C. Banerjea 25 Ayub Qadri 43 Prof. D. N. Rao S Gopalan Sampathkumar 165 Kanwaljeet Kaur 141 Vineeta Bal 83 Soumen Basak 76 Amulya K Panda 33 Sangeeta Bhaskar 67 Sarika Gupta 157 Prof. G. Padmanabhan Satish K. Gupta 105 Rajni Rani 60 Chandrima Shaha 101 Agam P. Singh 19 JK Batra 135 Amitabha Mukhopadhyay 29 Akhil C. Banerjea 25 Ayub Qadri 43 Prof. Lalita Ramakrishnan Rahul Pal 55 Anna George 38 Satyajit Rath 71 Bichitra K. Biswal 121 Debasisa Mohanty 125 Vidya Raghunathan 171 Vinay K. Nandicoori 222 Sher Ali 215
6 Prof. L. S. Shashidhara Rahul Pal 55 Anna George 38 Satyajit Rath 71 Rajni Rani 60 Vineeta Bal 83 Soumen Basak 76 Amulya K Panda 33 Sangeeta Bhaskar 67 Sarika Gupta 157 Prof. M. Vijayan Apurba K. Sau 117 Rajendra P. Roy 154 Monica Sundd 145 S Gopalan Sampathkumar 165 Vidya Raghunathan 171 Bichitra K. Biswal 121 Debasisa Mohanty 125 Vinay K. Nandicoori 222 Sher Ali 215 Prof. Radhakrishna Pillai Anil Suri 88 Sandeep Saxena 203 Rajendra P. Roy 154 Sagar Sengupta 198 Sanjeev Das 209 Asok Mukhopadhyay 95 Arnab Mukhopadhyay 174 Pramod K. Upadhyay 148 Dr. Ranjan Sen Prafullakumar Taylor 51 Devinder Sehgal 47 Madhulika Srivastava 184 Ranji Rani 60 Vineeta Bal 83 Soumen Basak 76 Amulya K. Panda 33 Sangeeta Bhaskar 67 Sarika Gupta 157 Prof. S. C. Lakhotia Prafullakumar Taylor 51 Madhulika Srivastava 184 Kanwaljeet Kaur 141 Sagar Sengupta 198 Sanjeev Das 209 Asok Mukhopadhyay 95 Arnab Mukhopadhyay 174 Pramod K. Upadhyay 148
7 Dr. Shekhar Mande S Gopalan Sampathkumar 165 Kanwaljeet Kaur 141 Satish K. Gupta 105 Bichitra K. Biswal 121 Debasisa Mohanty 125 Vidya Raghunathan 171 Vinay K. Nandicoori 222 Sher Ali 215 Prof. Subrata Sinha Anil Suri 88 Sandeep Saxena 203 Subeer Majumdar 112,227 Sagar Sengupta 198 Sanjeev Das 209 Asok Mukhopadhyay 95 Arnab Mukhopadhyay 174 Pramod K. Upadhyay 148 Prof. Umesh Varshney Rahul Pal 55 Anna George 38 Satyajit Rath 71 Vineeta Bal 83 Soumen Basak 76 Amulya K Panda 33 Sangeeta Bhaskar 67 Sarika Gupta 157 Prof. Uttam Surana Pushkar Sharma 189 Lalit Garg 179 Subeer Majumdar 112,227 Devinder Sehgal 47 Sagar Sengupta 198 Sanjeev Das 209 Asok Mukhopadhyay 95 Arnab Mukhopadhyay 174 Pramod K. Upadhyay 148 Prof. V. Nagaraja Devinder Sehgal 47 Madhulika Srivastava 184 Prafullakumar Taylor 51 Chandrima Shaha 101 Agam P. Singh 19 JK Batra 135 Amitabha Mukhopadhyay 29 Akhil C. Banerjea 25 Ayub Qadri 43 Dr. V. S. Chauhan Pushkar Sharma 189 Satish K. Gupta 105 Chandrima Shaha 101 Agam P. Singh 19 JK Batra 135 Amitabha Mukhopadhyay 29 Akhil C. Banerjea 25 Ayub Qadri 43
8 Original peer-reviewed articles
#1. Agarwal S, Saini S, Parashar D, Verma A, Jagadish N, Batra A, Suri S, Bhatnagar A, Gupta A, Ansari AS, Lohiya NK, Suri A (2013) Expression and humoral response of a- kinase anchor protein 4 in cervical cancer. Int J Gynecol Can 23: 650-658.
2. Agarwal S, Saini S, Parashar D, Verma A, Sinha A, Jagadish N, Batra A, Suri S, Gupta A, Ansari AS, Lohiya NK, Suri A (2013) The novel cancer testis antigen A-kinase anchor protein 4 (AKAP4) is a potential target for immunotherapy of ovarian serous carcinoma. Oncoimmunol 2: e24270.
3. Agarwal K, Kaul, R, Garg M, Shajahan A, Jha SK, Sampathkumar SG (2013) Inhibition of mucin-type O-glycosylation through metabolic processing and incorporation of N-thioglycolyl-D-galactosamine peracetate (Ac5GalNTGc). J Am Chem Soc 135: 14189-14197.
4. Ahangar MS, Vyas R, Nasir N, Biswal BK (2013) Structures of native, substrate- bound and inhibited forms of Mycobacterium tuberculosis imidazoleglycerol-phosphate dehydratase. Acta Cryst. Section D69: 2461-2467.
5. Aich A, Shaha C (2013) Novel role of calmodulin in regulating protein transport to mitochondria in a unicellular eukaryote. Mol Cell Biol 33: 4579-4593.
6. Alam A, Mirza A, Talegaonkar S, Panda AK, Iqbal Z (2013) Development of Celecoxib complex: characterization and cytotoxicity studies in MCF-7. Pharmaceut Anal Acta 4: 211.
7. Anish C, Khan N, Upadhyay AK, Sehgal D, Panda AK (2014) Delivery of polysaccharides using polymer particles: Implications on size-dependent immunogenicity, opsonophagocytosis, and protective immunity. Mol Pharm 11: 922- 937.
8. Appaiahgari MB, Glass R, Singh S, Taneja S, Rongsen-Chandola T, Bhandari N, Mishra S, Vrati S (2014) Transplacental rotavirus IgG interferes with immune response to live oral rotavirus vaccine ORV-116E in Indian infants. Vaccine 32: 651-656.
9. Arindkar S, Bhattacharjee J, Kumar JM, Das B, Upadhyay P, Asif S, Juyal RC, Majumdar SS, Nagarajan P (2013) Antigen peptide transporter 1 is involved in the development of fructose-induced hepatic steatosis in mice. J Gastroenterol Hepatol 28: 1403-1399.
10. Bhattacharjee J, Kumar JM, Arindkar S, Das B, Pramod U, Juyal RC, Majumdar SS, Nagarajan P (2014) Role of immunodeficient animal models in the development of fructose induced NAFLD. J Nutr Biochem 25: 219-226.
11. Bhattacharya S, Sen U, Vrati S (2013) The RIDD pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication. J Gen Virol 95: 71-79.
9 12. Bose A, Huhtaniemi I, Singh O, Pal R (2013) Synergistic activation of innate and adaptive immune mechanisms in the treatment of gonadotropin-sensitive tumors. PLoS ONE 8: e61288.
13. Chatterjee P, Seal S, Mukherjee S, Kundu R, Mukherjee S, Ray S, Mukhopadhyay S, Majumdar SS, Bhattacharya S (2013) Adipocyte Fetuin-A Contributes to Macrophage Migration into Adipose Tissue and Polarization of Macrophages. J Biol Chem 288: 28324–28330.
14. Chamoli M, Singh A, Malik Y, Mukhopadhyay A (2014) A novel kinase regulates Dietary Restriction-mediated longevity in C. elegans. Aging Cell (in press).
15. Chatrath S, Gupta VK, Garg LC (2014) The PGRS domain is responsible for translocation of PE_PGRS30 to cell poles while the PE and the C-terminal domains localize it to the cell wall. FEBS Lett (doi: 10.1016/j.febslet.2014.01.059).
16. Damle NP, Mohanty D (2014) Deciphering kinase-substrate relationships by analysis of domain specific phosphorylation network. Bioinformatics (DOI: 10.1093/ bioinformatics /btu112).
17. Das DS, Wadhwa N, Dubey N, Pradhan B, Majumdar SS (2013) Dickkopf homolog 3 (DKK3) plays a crucial role upstream of WNT beta CATENIN signaling for Sertoli cell mediated regulation of spermatogenesis. PLoS ONE 8: e63603.
18. De S, Srivastava V, Hussain M, Kumari J, Muniyappa K, Sengupta S (2014) RECQL4 and p53 potentiate the activity of polymerase γ and maintain the integrity of the human mitochondrial genome. Carcinogenesis 35: 34-45.
19. Ezougou CN, Ben-Rached F, Mos DK, Lin J, Black S, Knuepfer E, Green JL, Khan SM, Mukhopadhyay A, Janse CJ, Coppens I, Yera H, Holder AA, Langsley G (2014) Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite's plasma and food vacuole membranes. PLoS One 3: e87695.
20. Gupta S, Chandra S, Priyadarshini R, Madhavan V, Tikoo S, Hussain M, Mudgal R, Modi P, Srivastava V, Sengupta S (2013) Enhancement of c-Myc degradation by BLM helicase leads to delayed tumor initiation. J Cell Sci 126(Pt 16): 3882-3795.
21. Gupta S, Neogi U, Srinivasa H, Banerjea AC, Shet A (2014) HIV-1 coreceptor tropism in India: Increasing proportion of X4-tropism in subtype C strains over two. J Acquir Immune Defic Syndr 65: 397-404.
22. Guha R, Gupta D, Rastogi R, Vikram R, Krishnamurthy G, Bimal S, Roy S, Mukhopadhyay A (2013) Vaccination with Leishmania hemoglobin-receptor-encoding- DNA protects against visceral Leishmaniasis. Science Transl. Med. 5: 202ra121.
#23. Gupta N, Shrestha A, Panda AK, Gupta SK (2013) Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development. Mol Biotechnol 54: 853-862.
10 #24. Goel P, Tailor P, Chande A, Basu A, Mukhopadhyaya R (2013) An infectious HHV- 6B isolate from a healthy adult with chromosomally integrated virus and a reporter based relative viral titer assay. Virus Research 173: 280-285.
#25. Gupta N, Chakrabarti K, Prakash K, Wadhwa N, Gupta T, Gupta SK (2013) Immunogenicity and contraceptive efficacy of E. coli-expressed recombinant porcine zona pellucida proteins. Am J Reprod Immunol 70: 139-152.
26. Gill J, Jayswal P, Salunke DM* (2014) Antigen exposure leads to rigidification of germline antibody combining site. J Bioinfor Comput Biol (in press).
27. Haider S, Alam SM, Hamid H, Shafi S, Nargotra A, Mahajan P, Nazreen S, Kalle AM, Kharbanda C, Ali Y, Alam A, Panda AK (2013) Synthesis of novel 1,2,3-triazole based benzoxazolinones: Their TNF-α based molecular docking with in-vivo anti- inflammatory, antinociceptive activities and ulcerogenic risk evaluation. Eur J Med Chem 70: 579-588.
28. Jaiswal H, Kaushik M, Sougrat R, Gupta M, Dey A, Verma R, Ozato K, Tailor P (2013) Batf3 and Id2 have a synergistic effect on Irf8-Directed classical CD8α+ dendritic cell development. J Immunol 191: 5993-6001.
29. Kanojia D, Garg M, Saini S, Seth A, Bhatnagar A, Gupta A, Kumar R, Lohiya NK, Suri A (2013) Expression of cancer testis antigen, SPAG9 in bladder transitional cell carcinoma is associated with disease progression and plays role in cellular proliferation, migration and invasion of cancer cells. PLoS ONE 8: e81348.
30. Kakumani PK, Ponia S, Rajgokul KS, Sood V, Chinnappan M, Banerjea AC, Medigeshi G, Malhotra P, Mukherjee D, Bhatnagar R (2013) Role of RNAi in dengue viral replication and identification of NS4B as a RNAi suppressor. J Virol 87: 8870 - 8883.
31. Kumar SK, Kumari A, Javed S, Ali S (2014) DYZ1 arrays show sequence variation between the monozygotic males. BMC Genetics 15: 19-31.
32. Kaushik H, Deshmukh S, Mathur DD, Tiwari A, Garg LC (2013) Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein. Bioinformation 9: 617-621.
33. Lele DS, Talat S, Kaur KJ (2013) The presence of arginine in the Pro-Arg-Pro motif augments the lethality of proline rich antimicrobial peptides of insect source. Int J Pept Res Ther 19: 323-330.
34. Majumdar SS, Bhattacharya I (2013) Genomic and post-genomic leads toward regulation of spermatogenesis. Prog Biophys Mol Biol 113: 409-422.
35. Modi M, Nutan, Pancholi B, Kulshrestha S, Rawat AKS, Malhotra S, Gupta SK (2013) Anti-HIV-1 activity, protease inhibition and safety profile of extracts prepared from Rhus pariflora. BMC Complement Altern Med 13: 158.
11 36. Nutan, Modi M, Goel T, Das T, Malik S, Suri S, Rawat AKS, Srivastava SK, Tuli R, Malhotra S, Gupta SK (2013) Ellagic acid and gallic acid from Lagerstroemia speciosa L. inhibit HIV-1 infection through inhibition of HIV-1 protease and reverse transcriptase activity. Indian J Med Res 137: 540-548.
37. Nutan, Modi M, Dezutti CS, Kulshreshtha S, Rawat AKS, Srivastava SK, Verma A, Ranga U, Malhotra S, Gupta SK (2013) Extracts from Acacia catechu suppress HIV-1 replication by inhibiting the activities of the viral protease and Tat. Virology J 10: 309.
38. Natarajan VT, Ganju P, Singh A, Vijayan V, Kirty K, Yadav S, Puntambekar S, Bajaj S, Dani P, Kar HK, Gadgil CJ, Natarajan K, Rani R, Gokhale RS (2014) IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation. Proc Natl Aca Sci, USA 11: 2301-2306.
39. Nagarajan P, Arindkar S., Singh S, Majumdar SS (2013) Effect of long-term castration on serum biochemistry in rhesus monkeys. J Med Primatol (doi:10.1111/jmp.12046).
40. Pathak, CM, Singh RR, Yadav S, Kapoor N, Raina V, Gupta S, Surolia A (2014) Evaluation of Benzothiophene carboxamides as analgesics and anti-inflammatory agents. IUBMB Life (in press).
41. Pawar R, Das T, Mishra S, Nutan, Pancholi B, Gupta SK, Bhat SV (2014) Synthesis, anti-HIV activity, integrase enzyme inhibition and molecular modeling of catechol, hydroquinone and quinol labdane analogs. Bioorg Med Chem Lett 24: 302-307.
42. Ponia SK, Arora S, Kumar B, Banerjea AC (2013) Arginine rich short linear motif of HIV-1 regulatory proteins inhibits dicer dependent RNA interference. Retrovirology 10 1: 97.
43. Raina V, Gupta S, Yadav S, Surolia A (2013) Simvastatin induced neurite outgrowth unveils role of cell surface cholesterol and acetyl CoA carboxylase in SH-SY5Y cells. PLoS ONE 8: e74547.
44. Ramchandran LS, Das S, Banerjea AC (2013) Effect on HIV-1 gene expression, Tat- Vpr interaction and Cell apoptosis by natural variants of HIV-1 Tat exon 1 and Vpr from Northern India. PLoS ONE 8: e82128.
45. Ramakrishnan M, Mathur SR, Mukhopadhyay Asok (2013) Fusion-Derived Epithelial Cancer Cells Express Hematopoietic Markers and Contribute to Stem Cell and Migratory Phenotype in Ovarian Carcinoma. Cancer Res 73: 5360-5370.
46. Ritter AD, Shen Y, Fuxman BJ, Jeyaraj S, Deplancke B, Mukhopadhyay A, Xu J, Driscoll M, Tissenbaum HA, Walhout AJ (2013) Complex expression dynamics and robustness in C. elegans insulin networks. Genome Res. 23: 954-965.
47. Ronsard L, Lata S, Banerjea AC (2013) Molecular and genetic characterization of natural HIV-1 Tat exon-1 variants from North India and their functional implications. PLoS ONE 9: e85452.
12 #48. Roy A, Singh M, Upadhyay P, Bhaskar S (2013) Nanoparticle mediated co-delivery of Paclitaxel and a TLR-4 agonist leads to tumor regression and enhanced immune response in the tumor microenvironment of mice. Int J Pharm 445: 171-180.
49. Saini S, Shenoy G, Rath S, Bal V, George A (2014) Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells. Nat Immunol 15: 275-282.
50. Saini S, Agarwal S, Sinha A, Verma A, Parashar D, Gupta N, Ansari AS, Lohiya NK, Jagadish N, Suri A (2013) Gene silencing of A-kinase anchor protein 4 inhibits cervical cancer growth in vitro and in vivo. Can Gene The 20: 413-420.
#51. Saini C, Prasad HK, Rani R, Murtaza A, Misra N, Shankar Narayan NP, Nath I (2013). Lsr 2 of Mycobacterium leprae and its synthetic peptides elicit restitution of in vitro T cell responses in erythema nodosum leprosum and reversal reactions in lepromatous leprosy patients. Clin Vaccine Immunol 20: 673-682.
52. Santhanam SK, Dutta D, Parween F, Qadri A (2014) The Virulence polysaccharide Vi released by Salmonella Typhi targets membrane prohibitin to inhibit T cell activation. J Infect Dis (in press).
53. Sharma M, Sharma V, Panda AK, Majumdar DK (2013) Development of enteric submicron particles formulation of α-amylase for oral delivery. Pharm Dev Technol 18: 560-569.
#54. Sharma N, Akhade AS, Qadri A (2013) Sphingosine-1-phosphate suppresses TLR- induced CXCL8 secretion from human T cells. J Leukoc Biol 93: 521-528.
55. Sharma R, Lomash S, Salunke DM (2013) Putative bioactive motif of tritrpticin revealed by an antibody with biological receptor like properties. PLoS ONE 8: e75582.
56. Shrestha A, Wadhwa N, Gupta SK (2014) Evaluation of recombinant fusion protein comprising dog zona pellucida glycoprotein-3 and Izumo and individual fragments as immunogens for contraception. Vaccine 32: 564-571.
#57. Satija YK, Bhardwaj A, Das S (2013) A portrayal of E3 ubiquitin ligases and deubiquitylases in cancer Inter J Cancer 133: 2759-2768.
58. Sen N, Kumari R, Singh MI, Das S (2013) HDAC5, a key component in temporal regulation of p53-mediated transactivation in response to genotoxic stress (2013). Molecular Cell 52: 406-420.
59. Shailendra A, Jashdeep B, Jerald MK, Barun D, Upadhyay P, Asif S, Ramesh CJ, Subeer SM, Nagarajan P (2013) Antigen Peptide Transporter 1 (TAP1-/-) is involved in the development of fructose-induced hepatic steatosis in mice. J Gastroenterol Hepatol 28: 1403-1409.
60. Shah U, Haquea MA, Zaidia S, Hassan MI, Islam A, Batra JK, Singh TP, Ahmad F (2013) Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast iso-1-cytochrome-c. J Biomol Struct Dyn (in press).
13 61. Sinha A, Gulati A, Saini S, Blanc C, Gupta A, Gurjar BS, Saini H, Kotresh ST, Ali U, Bhatia D, Ohri A, Kumar M, Agarwal I, Gulati S, Anand K, Vijaykumar M, Sinha R, Sethi S, Saloma M, George A, Bal V, Singh G, Dinda AK, Hari P, Rath S, Dragon-Durey M-A, Bagga A (2013) Prompt plasma exchanges and immunosuppressive treatment improves the outcomes of anti-factor H autoantibody-associated hemolytic uremic syndrome in children. Kidney Int (doi: 10.1038/ki.2013.373).
62. Sinha A, Agarwal S, Parashar D, Verma A, Sinha A, Jagadish N, Ansari AS, Lohiya NK, Suri A (2013) Down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells: possible implications in targeted therapy. J Exp Clin Can Res 32: 69 1-11.
63. Singh UP, Bhat HR, Verma A, Kumawat MK, Kaur R, Gupta SK, Singh RK (2013) Phenyl hydrazone bearing pyrazole and primidine scaffolds: Design and discovery of novel class of Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) against HIV-1 and their antibacterial properties. RSC Adv 3: 17335-17348.
64. Sreekanth V, Bansal S, Motiani RK, Kundu S, Muppu SK, Majumdar TD, Panjamurthy S, Sengupta S, Bajaj A (2013) Design, Synthesis, and Mechanistic Investigations of Bile acid-Tamoxifen Conjugates for Breast Cancer Therapy. Bioconjug Chem 24: 1468-1484.
65. Suman P, Gupta SK* (2014) STAT3 and ERK1/2 cross-talk in leukemia inhibitory facter mediated trophoblastic JEG-3 cell invasion and expression of Mucin 1 and Fos. Am J Reprod Immunol (doi:10.1111/aji.12248).
66. Tiwari G, Mohanty D (2013) An In Silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-Peptide interactions. PLoS ONE 8: e71340.
67. Tiwari G, Mohanty D (2014) Structure Based Multi-Scale Approach for Identification of Interaction Partners of PDZ Domains. J Chem Inf Model (DOI: 10.1021/ci400627y).
68. Tapryal S, Gaur V, Kaur KJ, Salunke DM (2013) Structural evaluation of a mimicry recognizing paratope: plasticity in antigen–antibody interactions manifests in molecular mimicry. J Immunol 191: 456-463.
69. Tikoo S, Madhavan V, Hussain M, Miller ES, Arora P, Zlatanou A, Modi P, Townsend K, Stewart GS, Sengupta S (2013) Ubiquitin-dependent recruitment of the Bloom Syndrome helicase in response to replication stress is required to suppress homologous recombination. EMBO J 32: 1778-1792.
70. Usmani A, Ganguli N, Sarkar H, Dhup S, Batta SR, Vimal M, Ganguli N, Basu S, Nagarajan P, Majumdar SS* (2013) A non-surgical approach for male germ cell mediated gene transmission through transgenesis. Scientific Rep 3: 3430.
71. Upadhyay M, Priya GK, Ramesh P, Madhavi MB, Rath S, Bal V, George A, Vaidya T (2014) CD40 signaling drives B lymphocytes into an intermediate memory-like state, poised between naïve and plasma cells. J Cell Physiol (doi: 10.1002/jcp.24572).
14 72. Vijayan V, Khandelwal M, Manglani K, Gupta S, Surolia A (2014) Methionine down-regulates TLR4/MyD88/NF-κB signalling in osteoclast precursors to reduce bone loss during osteoporosis. Br J Pharmacol 171: 107-121.
#73. Vijayan V, Khandelwal M, Manglani K, Ranjan Singh R, Gupta S, Surolia A (2013) Homocysteine alters osteoprotegerin/RANKL System in the osteoblast to promote bone loss: Pivotal role of redox regulator forkhead O1. Free Radic Biol Med 61C: 72-84.
74. Yadav SK, Kumari A, Ali S (2013) Fate of the human Y chromosome linked genes and loci in prostate cancer cell lines DU145 and LNCaP. BMC Genomics 14: 323-330.
#In press last year, since published
15 Reviews and Proceedings
1. Bharati K, Vrati S (2013) DNA vaccines: Getting closer to becoming a reality. Indian J Med Res 137: 1027-1028.
2. Chopra A, Batra JK (2013) Antimicrobial activity of human eosinophil granule proteins. Meth Mol Biol (in press).
3. Gupta SK, Shrestha A, Minhas V (2013) Milestones in contraceptive vaccine development and hurdles in their applications. Hum Vaccin Immunother 10 (4) PMID: 24262991.
4. Gupta SK, Nutan (2013) Clinical use of vaginal or rectally applied microbicides in patients suffering from HIV/AIDS. HIV AIDS (Auckl) 5: 295-307.
5. Gupta SK (2013) Contraceptive vaccines: past, present and future. In Immunology of Pregnancy-2013 (Eds Chaouat G, Sandra O and Ledee N). Bentham e-book, Bentham Science Publishers, pp 100-134.
6. Kamle S, Ali S (2013) Mutations in H-ras gene and Costello syndrome. The Journal of Genetics- Photon 114: 124-129.
7. Kamle S, Ali S (2013) Reverberations on Biosafety Issues pertaining to Genetically Modified Crops. Biosafety 2: 112- 114.
8. Kamle S, Ali S (2013) Genetically Modified Crops: Detection Strategies and Biosafety Issues. Gene 522: 123-132.
9. Kochat V, Baligar P, Maiwall R, Mukhopadhyay A (2013) Bone marrow stem-cell therapy for genetic and chronic liver diseases. Hepatol Int (DOI 10.1007/s12072-013- 9499-z).
#10. Mukhopadhyay A (2013) Perspective on liver regeneration by bone marrow-derived stem cells: A scientific realization or a paradox. Cytotherapy 15: 881-892.
11. Majumdar SS, Bhattacharya I, Khan M (2013) Pharmacogenomics and Personalized Medicine in Infertility D. Barh et al. (eds.), Omics for Personalized Medicine, pp. 743- 760 © Springer.
12. Nutan, Gupta SK (2013). Medicinal plants with anti-HIV activity. In Emerging Frontiers and Challenges in HIV/AIDS Research (Eds Bandivdekar A and Puri CP). National Institute for Research in Reproductive Health, Mumbai, India. pp 91-110.
13. Prabhu SB, Khalsa JK, Banerjee H, Das A, Srivastava S, Mattoo HR, Thyagarajan K, Tanwar S, Das DS, Majumdar SS, George A, Bal A, Durdik JM, Rath S (2013) Role of apoptosis-inducing factor (Aif) in the T cell lineage. Indian J Med Res 138: 577-590.
14. Rani R, Singh A (2014) Functional implications of MHC associations in autoimmune diseases with special reference to Type1 diabetes, Vitiligo and Hypoparathyroidism.
16 Accepted for publication in the book "HLA & Associated Human Diseases", InTech Open Access Publishers, Janeza Trdine 9, Rijeka, Croatia (In press).
15. Rani R (2013) MHC Applications. In W. Dubitzky, O. Wolkenhauer, K. Cho & H. Yokota (eds.), Encyclopedia of Systems Biology, Springer New York. 3: 1158-1162.
16. Rawal L, Sehgal N, Ali S (2013) Genome Analysis and Human Health: A Critical Appraisal. Global Journal of Human Genetics and Gene Therapy 1: 16-37.
17. Salam N, Rane S, Das R, Faulkner M, Gund R, Kandpal U, Lewis V, Mattoo H, Prabhu S, Ranganathan V, Durdik J, George A, Rath S, Bal V (2013) T cell ageing: Effects of age on development, survival & function. Indian J Med Res 138: 595-608.
18. Shaha C (2013) Vesicle traffic wins the Nobel. Curr Sci 105: 1651-1652.
19. Suman P, Malhotra SS, Gupta SK (2013) LIF-STAT signaling and trophoblast biology. JAK-STAT 2: e25155.
20. Suman P, Gupta SK (2013) Regulators of early invasion of trophoblast cells. In Immunology of Pregnancy-2013 (Eds. Chaouat G, Sandra O and Ledee N). Bentham e- book, Bentham Science Publishers, pp 199-217.
21. Singhal A, Arora G, Sajid A, Maji A, Bhat A, Virmani R, Upadhyay S, Nandicoori VK, Sengupta S, Singh Y (2013) Regulation of homocysteine metabolism by Mycobacterium tuberculosis S-adenosylhomocysteine hydrolase. Sci Rep 3: 2264.
22. Salunke DM, Gill J, Dwevedi A (2013) Comparative structural proteomics of allergenic proteins from plant pollen. J Ind Inst Sci 94: 119-126.
23. Sharma P, Chitnis CE (2013) Key molecular events during host cell invasion by Apicomplexan pathogens. Curr Opin Microbio 16: 432-437.
24. Sreenivasamurthy SK, Dey G, Ramu M, Kumar M, Gupta MK, Mohanty AK, Harsha HC, Sharma P, Kumar N, Pandey A, Kumar A, Keshava Prasad TS (2013) A compendium of molecules involved in vector-pathogen interactions pertaining to malaria. Malaria J 12: 216.
25. Upadhyay P (2013) Tuberculosis vaccine trials. The Lancet 381: 2253-2254.
26. Upadhyay P (2013) New therapeutic approaches for airway hyperimmune response are required. Indian Pediatr 50: 1087.
#In press last year, since published
17 Patent Application / Granted
1. Baligar P, Teckman J, Mukhopadhyay A. Bone marrow-derived cells ameliorates the pathological consequences of the liver in case of alpha1-antitrypsin deficiency. US Patent Application # 14/109212 (17/12/2013).
2. Garg LC, Bhatia B. Recombinant subunit vaccine against beta toxin of Clostridium perfringens. Indian Patent application # 58/DEL/2014 (8/1/2014).
3. Gupta SK, Gupta N, Chakrabarti K, Prakash K, Wadhwa N, Gupta T. Recombinant zona pellucida (ZP) proteins, vaccine composition and method of producing said vaccines. Indian Patent application # 1210/DEL/2013 (25/04/2013).
4. Majumdar SS, Usmani A, Ganguli N. A shortcut procedure of transgene integration by hypotonic shock into male germinal cells for gene expression and transgenesis. US Patent Application # 14/096,634 (4/12/2013).
5. Suri A. siRNA useful in inhibiting cellular growth/proliferation of cancerous issues. India Patent # 257434 (Granted on 1/10/2013).
6. Surolia A, Gupta S, Singh MP, Chattopadhyay T. Composition useful for treatment of diabetes & disorder. European Patent # 2133091 (Granted on 19/04/2013).
7. Surolia A, Gupta S, Singh MP, Chattopadhyay T. Composition useful for treatment of diabetes & disorder. US Patent# 12/419,669 (Granted on 23/04/2013).
18
Research Project Reports
Plasmodium proteins involved in virulence and host modulation: Host- Parasite interactions in Plasmodium liver stages
Principal Investigator Agam P. Singh Research Associate Rajesh Anand Ph.D. Students Dabbu Kumar Surabhi Dixit
Research Fellow Himanshu Singh
Collaborator Santosh Kar, KIIT University, Bhubaneswar
Theme of research
Plasmodium species introduce effector molecules into hepatocyte cytosol to manipulate host metabolic and /or signaling pathways for its own benefit. Those could prove good targets for drug development. Parasite kinases, phosphatases etc. targeted to hepatocytes are likely candidates. The host processes affected by them could also be target for intervention. Basic theme is to identify, new parasite molecules that affect the host cellular processes, and possible intervention strategies. We use reverse genetics tool to characterize the parasite proteins with regard to its function.
Objectives Objective of this study is to identify new parasite derived proteins that are involved in host modulation, and required for parasites to grow and complete their life cycle. Currently, Primaquine is the only drug available for malaria liver stages (LS) treatment, but it can’t be administered to pregnant women and people with G6PD deficiency as it causes toxicity. Alternative drugs are the need of hour. Drugs can be targeted against parasite or host processes. Using genetic, cell biology and biochemical methods, we identified that Plasmodium circumsporozoite protein (CSP) is introduced in the hepatocyte cytoplasm and hepatocyte nucleus, and ectopic expression of CSP alter thousands of host transcripts. The overall effect is improved liver schizont growth. The current project aims to identify other parasite proteins, like CSP, that play role in liver schizont development. The newly identified proteins detail interaction with host cell will provide opportunity for developing new interventions.
Work reported in 2012-2013 A. Host parasite interactions 1. Phenotypic characterization of Pb-871 (SLTRIP) knockout clones: SLTRIP, expression across blood, mosquito and liver stages were checked by IFA. SLTRIP-KO parasites were characterized for blood and liver stage growth phenotype and were found to be defective during liver-stage growth.
19 2. Phenotypic characterization of SLTRIP pexel mutant parasite line: This line showed 50% decrease in Liver-stage growth compared to SLTRIP-KO line indicating SLTRIP playing role both directly in parasite and indirectly through host manipulation. 3. Immunization with SLTRiP recombinant protein results in significant level of protection against P. bergehi sporozoite challenge. Mouse immunization with purified SLTRIP resulted in high antibody titers and a 10000 folds reduction in parasite burden when challenged with sporozoites. 4. Pb-268 (PB 122500) knockout and phenotypic characterization of clones: This knockout like showed 100 folds loss in liver-stage parasite growth and defects at multiple life stages like oocyst development, number of sporozoite, cell traversal and LS growth. 5. Expression of 122500 during parasite life stages: IFA showed expression in oocyst, sporozoites and liver-stages. No expression was detected in blood stages or gametocytes. Novel drug targets: P. berghei has two C3 type sugar phosphate transporters in the membrane of apicoplast: triose phosphate transporter (TPT) and phosphoenolpyruvate transporter (PPT). We found that P. berghei TPT knockout parasites do not survive. However, PPT knockout parasite behaved similar to the wild type in the blood stages. The absence of PPT in other life stages, leads to defects in the development of parasite and was required at both mosquito as well as liver stages.
Progress of work during the current reporting year (2013-2014) 1. Characterization of a P. berghei FIKK Kinase (PBANKA_122500): Pb122500 protein contains conserved S/T kinase domain in its C-terminal region therefore we tested the kinase activity. We used full-length Pb122500 protein obtained through immuno-precipitation (IP) from mid gut of infected mosquitoes using anti-Pb122500 antibody. Result shows that the IP lysate containing full length Pb122500 protein had kinase activity when compared to controls (Figure 1A). We also checked the kinase activity with C-terminal purified protein and results confirmed that C-terminal of Pb122500 had comparable kinase activity (Figure 1B). The western blot analysis of lysate from Oocyst and sporozoite stage shows that Pb122500 is an ~140 kDa protein (Figure 1C). Using Protein kinase-A (PKA) inhibitor (Triciribine hydrate, Sigma T3830), in an in vitro experiment with purified PbFIKK kinase, we were able to completely inhibit the kinase activity of Pb122500.
20
Figure 1: (A) Kinase activity assay in IP lysate obtained from infected mosquitoes mid gut using the Pb122500 peptide antibody. Assay was performed with a phospho-serine/threonine antibody based ELISA assay kit. (B) Same as in A but using C-terminus Pb122500 purified protein. (C) Western blot: full-length Pb122500 protein, (left) IP-lysate from infected mosquitoes mid gut (lane1, Oocyst) and uninfected mosquitoes mid gut IP lysate (lane 2) (right) IP-lysate from uninfected salivary glands (3) and infected salivary glands (lane 4, Sporozoites).
2. Transcriptome analysis of hepatocytes infected with SLTRiP-KO parasites. In order to know the function of exported SLTRIP in host modulation we performed whole transcriptome analyses of host transcript at 22 hours post sporozoite infection of HepG2 cells. We chose this time point on the basis of maximum export of SLTRIP from 12-20 hrs post infection. When compared with WT infected cells, 2840 transcripts changed more than two folds (Figure 2A). Figure 2B shows the network analysis for host genes affected during SLTRiP-KO parasite infection and figure 2C depicts functional cluster analysis of host genes, which were showing ≥3 fold changes. Figure 2D and 2E illustrates pathway analysis of affected host genes in case of SLTRiP-KO compared to WT parasite infection by KEGG pathway. Pie chart shows the major pathways affected were RNA transport/processing, metabolism, immunity and ribosome structure /function. To validate NGS data, we selected two or more genes from various pathways (both up/ down regulated) and a total of 23 genes were tested by qPCR on independent biological repeats of the experiment. Figure 5F shows the qPCR results along with NGS fold change. Quantitative-PCR results coincide with NGS data. Transcription factors: To know the transcription factors (TF) modified in SLTRiP-KO infected host cell, we searched for all the TFs present in animal TF database which include 1544 TFs in 71 families. We found that a total of 262 transcription factors were changed more than 4-folds in SLTRiP-KO infected host cell compared to WT infection. Most of these TF were not changed during WT infection. These TFs were mostly related to cellular pathways like, cell cycle, apoptosis and cancer, immune system, oxidative stress, chemokines and MAPK signaling. Three TFs MEF2C, TCF4 and SOX10 were upregulated by 1123.7, 1346 and 2300 folds respectively.
21 Manipulation of immune- metabolic switch: RNAseq based transcriptome data reveals that central molecules of MEF2 network were upregulated in SLTRiP-KO infection. The transcript levels of RPS6KA5, CREB5, ATF, MEF2C, MAPK3, MAPK11, mTOR, RPTOR and AKT3 were upregulated as compared to WT parasite infection. MEF2 network also includes other important molecules like, EP300, FOS, HDAC9, CREBBP and CREBBP-L1, which were also upregulated. NF B, STATs, IRFs, FOXO, SMADs, TFs related to immunity were either unchanged or only marginally changed in SLTRiP-KO infection. Lipid metabolism related TF ARID was upregulated in SLTRiP-KO infection. GSK3 was also upregulated in SLTRIP-KO infection.
Figure 2: Host transcriptome analysis. (A) Venn diagram for host genes affected during SLTRiP-KO infection compared to WT parasite infection. (B) Network analysis for host genes affected by SLTRiP-KO parasite compared to WT parasite, which were showing ≥3 fold change. (C) Functional cluster analysis of host genes affected by SLTRiP-KO parasite. Nodes represented in one color are involved in same biological process. (D & E) Pie chart showing pathways and % of total gene changed in up (D) and down (E) regulated during SLTRiP- KO infection compared to WT parasite. (F) Validation of NGS data, with qPCR. Beta actin and HSP90AB1 were taken as housekeeping genes. Blue and red bars represent the fold change (log10 scale) for host genes observed in qPCR and NGS respectively.
22 3. Transcriptome analysis of hepatocytes infected with Pb-FIKK Kinase-KO parasites: We did mRNA sequencing of host cells (through NGS), infected with Pb122500-KO or WT- PBANKA parasites. From NGS, we were able to get 35,517,455 and 35,678,306 reads for wild type and Pb122500-KO infected cells respectively, which maps to ~ 16000 genes of the total 39904 human genes (GRCH37.P.13). In Pb122500- KO infected host cell, when compared with WT infected cells, 288 [219 upregulated, 69 down regulated] changed more than two folds (Figure XA). Pie chart analysis shows that major pathways affected were associated with signaling, metabolism, immunity, focal adhesion and cell cycle pathways of host cells (Figure 3B). Figure 3C illustrates pathway analysis of Pb122500-KO affected host genes compared to WT parasite infection by KEGG pathway. NGS data was validated by qRT-PCR. Figure 3D shows the qPCR results along with NGS fold change. Quantitative-PCR results coincide with NGS data.
Figure 3: (A) Venn diagram summarizing the significantly affected genes (>2 folds) in PB122500-KO infection of host cell compared to WT infection. (B) Pathways analysis of PB122500-KO affected host genes compared to WT parasite infection by KEGG pathway. The host genes showing fold change of > ±2 were considered for pathways analysis. Pie chart showing pathways in which host genes were up regulated during PB122500-KO infection compared to WT parasite. (C) Functional cluster analysis of host genes affected by Pb122500-KO parasite. Nodes represented in one color are involved in same biological process. (D) Validation of NGS data with quantitative real time PCR. Blue and red bar represent the fold change in log scale for host genes observed in NGS Data and real time PCR validation.
4. Generation of S23 Knockout Parasites: Using suppression substractive hybridization of P. yoellii salivary gland sporozoites and merozoites, Kaiser et al. [Mol Microbiol. 2004] identified liver stage /sporozoite specific genes. Knockout studies of several liver stage
23 specific genes revealed their function in host invasion and parasite development during malaria parasite life cycle. We have selected for our study PbANKA _142520 also known as S23 [upregulated in sprorozoite (UIS) gene] having orthologue in P.falciparum. It contains two putative pexel motifs..On mutating the motifs using site directed mutagenesis export of protein was significantly decreased in comparison to wild type. So we decided to characterize the pexel mutant and knockout of this gene to elucidate the function of UIS23 in parsite development and host modulation. So far we have generated the knockout population and single parasite cloning is in progress.
5. New drug screening against liver-stage parasites: Curcumin has been shown to inhibit blood stages of malaria parasite. Bioavailability of Curcumin is a matter of concern. Nano form of Curcumin has been shown to be more effective against malaria blood stage parasites due its better bioavailability. We tested both Curcumin and nano-Curcumin for their potency against liver stage malaria, in in vivo condition (mouse). We did not find any in-vivo activity against liver stage parasite at the highest dose tested [10 mg/mouse/day x 3).
Future plans 1. Phenotypic characterization of PB823-KO parasite line. 2. Phenotypic characterization of S23-KO parasite line. 3. Screening of new chemical scaffolds against liver stage parasites. 4. Biochemical and functional characterization of Pb-FIKK kinase domain.
Action taken on the RAP/SAC 2013 recommendations There were no specific recommendations.
24 Genetic and functional analyses of host and HIV-1 genes that affect progression of HIV-1 and development of nucleic acid based antiviral approaches
Principal Investigator Akhil C. Banerjea
Research Associates Sanket S Ponia Amjad Ali
Ph.D. students Sakshi Arora Richa Kapoor Rameez Raja Binod Kumar Shubhendu Trivedi
Research Fellows Jyotsna Singh Larance Ronsard
Collaborators V Ramachandran, UCMS/GTB Hospital, Delhi S Das, UCMS/GTB Hospital, Delhi V Bhattacharya, BHU Medical College, Varanasi T Dhole, SGPGI, Lucknow Annapurna Vyakarnam, Indian Institute of Science Anita Shet, St John’s medical College Hospital, Bangalore
Theme of research Host cells generate their own anti-viral responses and the viruses have evolved over the years to systematically overcome restriction factors for their advantage. Understanding the mechanistic details about the host-virus interaction is crucial for understanding biology of HIV-1 or other pathogens. Virus often modulates or redirects various host proteins including various signalling pathways for its own advantage that confers viral fitness. Micro-RNAs and various other kinds of RNAs are involved controlling gene expression and are critically involved in various disease conditions. Our broad aim is to explore their role in HIV biology and pathogenesis, with special emphasis as to how the virus manages to escape such innate cellular protective mechanisms.
Objectives
Host and the pathogen (HIV) have evolved over the years and while the host cells continue to restrict the virus growth, the virus on the other hand continues to escape this by multiple mechanisms. The various small HIV-1 ORFs like Nef, Vpu, Vif and Vpr are known to play a major role in pathogenesis. We wish to understand the role of viral accessory genes in modulating cellular functions important for causing pathogenesis. Equally important is to understand how cell is using various counter measures to reduce the virus burden. It is therefore important to study (a) how key regulatory and accessory proteins of HIV-1 are modulated or degraded inside the mammalian cells (b) how they directly or indirectly modulate the key regulators of cell cycle/apoptosis etc. Finally it is important to determine how micro-RNAs (viral and cellular) influence these functions and contribute to pathogenesis 25 and study in details what strategies are adopted by HIV to suppress the RNAi machinery and thereby subvert host defences.
Work reported in 2012 - 2013
Role of HIV-1 Vpu in causing virus release and cell death was reported using natural Vpu isolates from North India. Some natural mutants (S61A) conferred intracellular stability to the Vpu protein. We also reported a natural B-TrCP mutant (S52 & 56 being substituted with Alanins) in North Indian population and this had important functional implications. We also reported that both Tat and Rev possess RNAi-suppressor activity and that the short Arginine rich motif is sufficient to confer suppressor activity to some degree. Preliminary work on the role of p53-sentive miRNA with respect to HIV-1 replication was reported. Based on our preliminary studies we also reported that Vpr possesses a novel de-ubiquitinase property.
Progress of work during the current reporting year (2013 -2014) HIV-1 Vpr redirects host ubiquitination pathway HIV-1 is known to rely heavily on modulation of host ubiquitin pathway particularly for counteraction of antiretroviral restriction factors i.e., APOBEC3G, UNG2, BST-2 etc., viral assembly and release. Reports till date have focused on molecular hijacking of ubiquitin machinery by HIV-1 at the level of E3 ligases. Interaction of a viral protein with an E3 alters its specificity to bring about selective protein ubiquitination. However, in case of infection, multiple viral proteins can interact with this multi-enzyme pathway at various levels making it much more complicated. Here, we have addressed manipulation of ubiquitination at the whole cell level post HIV-1 infection. Our results show that HIV-1 Vpr is necessary and sufficient to bring about redirection of host ubiquitin pathway towards HIV-1 specific outcomes. We, also show that the leucines rich three helical region of Vpr is critical for this effect and this ability of Vpr is conserved across circulating recombinants. Finally, we establish that this phenomenon is involved in HIV-1 mediated diversion of host ubiquitination machinery specifically towards degradation of various restriction factors during viral pathogenesis. Our work, first of its kind, provides novel insight into regulation of ubiquitin system at whole cell level by HIV-1. Role of MicroRNA34a in HIV-1 replication HIV-1 alters the host cell microRNA expression patterns. MicroRNA-34a (miR34a) is one of the cellular microRNAs found to be elevated during HIV-1 infection. The functional consequences and mechanistic details of this phenomenon, however, remain unexplored. Our data suggests that miR34a functions to promote HIV-1 replication in T cells and there exists a positive feedback loop in HIV-1 mediated activation of microRNA-34a (miR34a) expression and vice versa. Specificity of miR34a mediated effect on viral replication was checked by employing specific locked nucleic acid miR34a inhibitor. A significant decrease in HIV-1 replication was observed when endogenous miR34a was knocked down in cells by miR34a inhibitor. The possible role of HIV-1 accessory gene products (Vif, Vpr, Vpu and Nef) in miR34a mediated increase of HIV-1 replication was ruled out using a series of HIV-1 based deletion mutant viruses. Interestingly, miR34a was found to enhance basal HIV-1 LTR promoter activity. We are greatly interested in studying its role in HIV-1 mediated apoptosis especially in human T-cells and neuronal cells.
26 Role of AKT in HIV-1 pathogenesis A number of knockdown studies have reported survival pathways to be important for HIV-1. Protein kinase B/AKT is one of the crucial factors needed by cells for their survival. The exact role of this kinase which lies downstream of the PI3 kinase pathway has not been properly elucidated in HIV-1 though it is known to be important. The aim of this study is to elucidate the role of AKT and as well as its downstream effector molecules during HIV-1 infection. The main focus of this project is MDM2 (target substrate for AKT) and its regulation in presence of HIV-1. Role of metabolism in HIV-1 pathogenesis HIV-1depends on host cell for its biosynthetic demands due to small genome size using domain and motifs for protein-protein interaction. In this study we are trying to explore how HIV-1 alters the metabolic state of the T cell and if this can be used as a therapeutic target. We have checked the levels of metabolic sensors i.e. mTOR, AMPK and Akt during time course of infection. Preliminary results indicate that these sensors shows non uniform pattern. This is quite interesting because this dynamic pattern could be attributed to stage specific demands of various metabolites by HIV-1. Because these sensors are master regulators of metabolic state of the cells hence, downstream targets of these sensors will give better insights into the exact alteration in the metabolic state of the cell by HIV-1 which is currently being pursued. Genetic & Functional analysis of Tat exon 1 variation and Tat-Vpr interaction from North Indian HIV-1 infected individuals Tat and Vpr genes from HIV-1 infected individuals from North India were genetically analyzed and extensive phylogenetic analyses were carried out and some key functional activities related to gene expression were tested. Both of these genes are critically important for viral gene expression and viral fitness. We report novel Tat- B/C recombinant with a precise breakpoint in the middle of the ORF and their HIV-1 LTR promoter activating properties were studied in detail in comparison with prototype subtype B and C Tat Exon1. Functional implications of natural Tat-Vpr variants were studied for their ability to augment the HIV-1 promoter activity and apoptosis for the first time.
Future plans
Mechanistic details of how HIV-1 affects metabolism, role of AKT in HIV-1 pathogenesis; potential role of miRNA34a in natural infection of HIV-1, will be explored in detail.
Action taken on the RAP/SAC 2013 recommendations
No specific suggestions were made.
Publications Original peer-reviewed articles
1. Ponia SK, Arora S, Kumar B, Banerjea AC* (2013) Arginine rich short linear motif of HIV- 1 regulatory proteins inhibits dicer dependent RNA interference. Retrovirology 10 1: 97.
2. Kakumani PK, Ponia S, Rajgokul KS, Sood V, Chinnappan M, Banerjea AC, Medigeshi G, 27 Malhotra P, Mukherjee D, Bhatnagar R* (2013) Role of RNAi in dengue viral replication and identification of NS4B as a RNAi suppressor. J Virol 87: 8870 -8883. 3. Ronsard L, Lata S, Banerjea AC* (2013) Molecular and genetic characterization of natural HIV-1 Tat exon-1 variants from North India and their functional implications. PLOS One 9: e85452.
4. Ramchandran LS, Das S, Banerjea AC* (2013) Effect on HIV-1 gene expression, Tat-Vpr interaction and Cell apoptosis by natural variants of HIV-1 Tat exon 1 and Vpr from Northern India. PLOS One 8: e82128.
5. Gupta S, Neogi U, Srinivasa H, Banerjea AC, Shet A* (2014) HIV-1 coreceptor tropism in India: Increasing proportion of X4-tropism in subtype C strains over two J Acquir Immune Defic Syndr 65: 397-404.
*Corresponding author
28
Understanding the regulation of intracellular transport: Role of GTPases
Principal Investigator Amitabha Mukhopadhyay
Research Associates Ruchir Rastogi Manglesh K Singh Amir kumar Singh Vijay Singh
Ph.D. Students Deepika Gupta Pawan Kishor Singh Jitendra Kumar Verma Smriti Parashar Kamal K Gupta Chandni Sood
Research Fellow Anjali Kapoor
Collaborator Syamal Roy, IICB, Kolkata
Themes of Research
Major theme of the project is to understand the regulation of intracellular trafficking and its modulation by intracellular pathogens as well as in different pathological conditions. One of the main goals of this project is to understand the mechanism of survival of intracellular pathogens in macrophages by modulating the host trafficking pathway. We are also trying to understand the regulation of intracellular trafficking pathway in a protozoan pathogen like Leishmania using hemoglobin endocytic pathway as we have shown that parasite acquires heme by the degradation of internalized hemoglobin. We have also initiated the studies on cytokine mediated modulation of intracellular trafficking.
Objectives
Phagocytosis is an important process in host defense and is mediated by complex interactions between defined intracellular compartments. The final fate of the nascent phagosomes usually culminates with the fusion of lysosomes. But some invading microorganisms modulate this central process for their survival in the phagocytic cells. The major objectives of the present investigations are:
1. Modulation of phagosome maturation by various intracellular pathogens. 2. Determination of the role of various cytokines in the modulation of phagosome trafficking. Evidences from a variety of sources, have established that transport of cargo along the endocytic pathway requires a series of highly coordinated and specific vesicle fusion events regulated by small GTP binding proteins of the Rab family. Not much is known about the regulation of endocytosis and intracellular trafficking in protozoan parasites. The major objective of the project is to understand how intracellular pathways are regulated in
29 Leishmania and role of these pathways to target the hemoglobin to the degradative compartment to generate heme, require for parasite survival. 3. Mechanism of intracellular trafficking of hemoglobin in Leishmania.
Work reported in 2012-2013
Mechanism of survival of Salmonella in macrophages
It is now well evident that several intracellular pathogens modulate host cellular machinery for their benefit through some of their own proteins, collectively known as effectors. Earlier, we have identified and determined the role two such effectors namely SopE and SipC from Salmonella which modulate host cell trafficking pathways by recruiting Rab5 and Syntaxin6, respectively. Last year, we have reported that live-SCP specifically recruits significantly higher amounts of Syntaxin8 in comparison to dead-SCP. Subsequently, we have identified another effector protein from Salmonella, SipA, which specifically binds with host Syntaxin8 through N-terminal end of SipA (SipA1-242). In addition, we have also made a SipA knock out Salmonella to understand the role of SipA and syntaxin8 interaction in the modulation of intracellular trafficking of Salmonella in macrophages.
Mechanism of hemoglobin trafficking in Leishmania
Previously, we have identified a specific receptor (HbR) for hemoglobin in Leishmania and characterized the trafficking of hemoglobin from cell surface to lysosomes via Rab5 and Rab7 regulated process. Last year, we have reported that hemoglobin endocytosis in Leishmania is a clathrin-dependent process and depolymerization of clathrin by chlorpromazine (CPZ), a pharmacological agent, blocks Hb internalization in Leishmania and thereby inhibits the growth of the parasites. We have found that blocking of hemoglobin endocytosis or its intracellular degradation is detrimental to parasites. Therefore, we also initiated studies to evaluate the hemoglobin receptor as a novel target for Leishmania.
Progress of work during the current reporting year (2013-2014)
Mechanism of survival of Salmonella in macrophages
In the reporting period, we have tried to understand the nature of binding between Salmonella effector protein SipA with host syntaxin8. Bioinformatic analysis of SipA has shown that this effector protein consists of a N-terminal domain (SipA1-435) which is subdivided into Chaperone binding domain (1-267aa), SNARE domain (180-242aa), putative transmembrane domain (250-265aa) and a caspase 3 cleavage site DEVD (432-435aa) and a C-terminal domain (436-685aa). Accordingly, we have made several truncated mutants of SipA with N- 1-169 1-242 1-277 48-277 436-685 terminal His6; SipA , SipA , SipA , SipA , SipA . Our results have shown that SipA1-277 which contains putative transmembrane domain, binds with GST-Syntaxin8 like it was observed with SipA1-242. However, SipA1-169 which lacks SNARE domain, unable to bind with GST-Syntaxin8 indicating possibly SipA acts as a cognate SNARE of Syntaxin8. Interestingly, we have found that first 48aa from the N-terminal region of SipA also plays crucial role in SipA-Syntaxin8 interaction as SipA48-277 is unable to bind with GST-Syntaxin8. It might be possible that N-terminal of SipA is required for initial zippering between SipA with Syntaxin8 as it was shown that pairing between cognate SNAREs requires N-terminal interaction.
30 More recent studies have shown that Salmonella infection activates host Caspase3 and cleaves SipA into two fragments. However, role of each fragment in the trafficking of Salmonella is not clearly understood though C-terminal of SipA was found to be colocalized with host actin. To understand the function of the two fragments generated by caspase cleavage, we have ectopically expressed SipA:WT, SipA1-435 or SipA436-685. Our preliminary results have shown that SipA:WT and SipA436-685 colocalizes with phalloidin labeled actin. Whereas, SipA1-435 is found to be localize on distinct vesicular membrane. It will be interesting to determine whether SipA1-435 containing vesicles are also positive for Syntaxin8. Simultaneously, we are also trying to map the region of Syntaxin8 important for the interaction with SipA. Previous studies have shown that residues 145-209aa is the SNARE binding domain of Syntaxin8. Therefore, we have made three truncated mutants of Syntaxin8 namely Syntaxin81-144, Syntaxin81-209 and Syntaxin8145-209. We have also initiated studies to understand the regulation of Leishmania-containing phagosome maturation in macrophages. Our preliminary results have shown that Leishmania infection in macrophages triggers the expression of Rab5a in the host cells. Currently, we are trying to elucidate how Leishmania infection overexpresses Rab5a in the host cells and its significance in Leishmania trafficking in macrophages. Mechanism of hemoglobin trafficking in Leishmania
In the reporting period, we have carried out detail studies to evaluate HbR as potential vaccine candidate against leishmaniasis in two different animal models of visceral leishmaniasis (VL) namely, hamster and mice. Our results have shown that immunization with HbR-DNA induces complete protection against virulent Leishmania donovani infection in both BALB/c mice and hamster. Moreover, we have found that HbR-DNA immunization stimulates the production of protective cytokines like IFN-γ, IL-12, and TNF-α with concomitant down- regulation of disease promoting cytokines like IL-10 and IL-4. HbR-DNA vaccination also induces protective response by generating multifunctional CD4+ and CD8+ T-cells. Most importantly, all HbR-DNA vaccinated hamsters show sterile protection and survived during an experimental period of 8 months. These findings demonstrate that HbR is a potential vaccine candidate against visceral leishmaniasis.
Interestingly, our results have also shown that hemoglobin-receptor (HbR) of Leishmania is conserved across various strains of Leishmania causing diverse forms of diseases like visceral leishmaniasis, cutaneous leishmaniasis and mucocutaneous leishmaniasis; indicating possible use of HbR as a vaccine candidate against various leishmaniasis. In addition, we have detected anti-HbR antibody in confirm Kala-azar patients’ sera indicating that HbR might be evaluate as a possible diagnostic marker for VL.
Previously, we have reported that haemoglobin endocytosis is a clathrin-dependent process in Leishmania. Therefore, we have initiated studies to find out an adaptor molecule which not only binds with cytoplasmic domain of HbR but also has the ability to bind with clathrin in Leishmania. Thus, we have tried to identify a molecule from Leishmania which can bind both clathrin as well as HbR using yeast two hybrid system. Consequently, we have identified one clone corresponding to the putative epsilon subunit of ATP-synthase which interacts with both clathrin and HbR. In order to determine the role of epsilon subunit of ATP-synthase as an adaptor molecule, we have amplified the ORF (546bp) of the ATP synthase sequence using appropriate forward (5/-GCGGATCCATGTTCCGCTTCTGTGGTCG-3/) and reverse primers (5/-GCGAATTC TTACGCGTGCTTGAGGCTCTG-3/) by RT-PCR from L. donovani cDNA. Subsequently, we have expressed epsilon subunit of ATP-synthase (Ld-ATPSy) either as N- terminal His6 or GST tag. Our results have shown that GST-LdATPSy binds with His-HbR. In addition, we have also found that His-LdATPSy binds with terminal domain of Ld-clathrin. 31 These results suggests that epsilon subunit of ATP-synthase is a novel adaptor molecule in hemoglobin endocytosis in Leishmania.
Next question we have asked how HbR containing clathrin coated pits are pinched off from the cell surface. Is there any dynamin homologue present in the Leishmania? Subsequently, we have designed appropriate forward (5/-GTCCCGGGATGGACCAGTTGATCAGCGT GATCAATG-3/ and reverse primers (5/-GTGCGGCCGCTTAGGCGCCGGCTTGCATG GACGAGG-3/ based on the putative Dynamin like sequence of L. donovani identified in gene bank and amplified a 2103 bp fragment by RT-PCR using L. donovani cDNA. Subsequently, we have expressed Ld-dynamin either as N-terminal GST or GFP tag. Comparison of Ld- Dynamin sequence by CLUSTAL W multiple sequence alignment has demonstrated that the cloned protein has about 91% similarity with L. major, 70% with T. brucei and 44% with human Dynamin sequence. However, PRD domain which is present in mammalian dynamin is completely absent in Ld-Dynamin sequence. Therefore, it will be interesting to determine the function of LdDynamin in parasite. Interestingly, we have found that GFP-Ld-dynamin colocalize with HbR in the flagellar pocket of Leishmania. Detail studies to determine the role of dynamin in hemoglobin endocytosis are in progress.
Action taken on RAP/SAC 2011 recommendations
Clarifications sought were provided to the satisfaction of the RAP/SAC members. No specific follow up actions was suggested.
Future plans
1. Studies are in progress to determine role of Syntaxin8 in intracellular trafficking of Salmonella in macrophages. 2. We are also evaluating the role of dynamin in the regulation of HbR trafficking in Leishmania. 3. We are also characterizing the epsilon subunit of ATP-synthase as a novel adaptor in hemoglobin endocytosis in Leishmania.
Publications Original peer-reviewed articles
1. Guha R, Gupta D, Rastogi R, Vikram R, Krishnamurthy G, Bimal S, Roy S, Mukhopadhyay A* (2013) Vaccination with Leishmania hemoglobin-receptor-encoding-DNA protects against visceral Leishmaniasis. Science Transl. Med. 5: 202ra121.
2. Ezougou CN, Ben-Rached F, Mos DK, Lin J, Black S, Knuepfer E, Green JL, Khan SM, Mukhopadhyay A, Janse CJ, Coppens I, Yera H, Holder AA, Langsley G* (2014) Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite's plasma and food vacuole membranes. Plos One 3: e87695.
*Corresponding author
32 Studies on immune response from antigen loaded biodegradable polymer particles and protein refolding from inclusion bodies
Principal Investigator Amulya K. Panda
Ph.D. Students Prasad S Admane Anupam Singh Vaibhav Upadhyay Jairam Meena Divya Jha Robin Kumar
Research Fellows Md. Aftab Alam Sudeepa Srichandan
Collaborators Lalit C. Garg, NII Pramod K Upadhyay, NII Ayub Qadri, NII Devinder Sehgal, NII SK Gupta, NII Alok R. Ray, IIT, Delhi SP Vyas, DHG Vishwavidyalaya, Sagar, MP DK Majumdar, DIPSAR, New Delhi Zeenat Iqbal, Jamia Hamdard, New Delhi KC Gupta, IITR, Lucknow RC Kuhad, DUSC, New Delhi GP Talwar, TRF, New Delhi I CARE Eye Hospital, Noida
Theme of research The theme of the project is to evaluate polymeric particle based delivery system for improved immunogenicity of different antigens such as Tetanus Toxoid (TT), Hepatitis B surface antigen (HBsAg), viral and carbohydrate (Vi polysaccharide and S. pneumoniae polysaccharides) based vaccines. Another major research activity of the laboratory is the analysis of inclusion body formation and development of mild solubilization processes for improved recovery of bioactive proteins.
Objectives The main objective of the project is to improve the immunogenicity of antigens entrapped in biodegradable polymer particles. High-throughput refolding of inclusion body proteins into bioactive form is another objective of the research group. Researches in the following areas are conducted in the laboratory to achieve the objectives:
1. Analysis of immune response from antigen loaded polymer particles and evaluation of adjuvant properties associated with polymeric particle formulation. Evaluation of memory antibody response from polymer particle based immunization.
33 2. Development of polymeric membrane as a scaffold for three dimensional growths of animal cells and its application as an artificial skin equivalent. 3. Solubilization and refolding of inclusion body proteins from Escherichia coli. This involves analysis of inclusion body formation during protein expression and understanding of protein aggregation with an aim to recover higher amount of bioactive protein.
Work reported in 2012-2013 We have been reporting improved immunogenicity of antigens by entrapping them in polylactic acid (PLA) particles. It was observed that single dose immunization with polymeric particles entrapping antigens leads to development of immunological memory. This concept of developing memory antibody response from single point immunization was extended to carbohydrate vaccine such as Pneumococcal capsular polysaccharides. Entrapment of capsular polysaccharides in polymer particles improved its immunogenicity and generated memory antibody titer from single point intramuscular immunization. The protective nature of antibody generated was tested in a mouse infection model while challenging the animal with virulent Pneumococci. PLA nanoparticles entrapping capsular polysaccharide alone were able to elicit protective antibody titer from a single immunization dose. Nanoparticles were internalized more efficiently than microparticle by the APCs. This may be one of the reasons for enhance immunogenicity of nanoparticle based delivery system for polysaccharide antigens. This was opposite to what was observed with protein antigens where microparticles elicited better antibody response. Polymeric particle formulation process was optimized using spray drying. The process engineering and formulation parameters were optimized to get desired sized particles in reproducible way. Spray drying was also used to produce dry powder alum for immunization. Process engineering analysis for entrapping both water soluble and water insoluble drugs in polymer particles were investigated. The idea was to develop large scale process for the manufacturing of polymeric particles entrapping drug/biomolecules. Polymeric particle were also used to form scaffold to grow cells in three dimensional modes. We have developed mild solubilization of inclusion body aggregates using organic solvents such as propanol/ β mercaptoethanol. This resulted in improved recovery of bioactive protein from inclusion bodies of E. coli. It was observed that these organic solvents protect the native like secondary structure of protein during solubilization and thus help in improved recovery of bioactive protein. Different biophysical methods were employed to understand the structure of protein at different stages of solubilization and refolding.
Progress of work during the current reporting year (2013-2014) A. Immune response from polymeric particles entrapping antigens Modulation of immune response using different sized polymer particles Polymeric particles entrapping protein/carbohydrate antigens are being routinely used in the laboratory to improve its immunogenicity. Vi polysaccharide and ZP3 recombinant protein entrapped in polymer particle are being evaluated for the generation of both primary and memory antibody response from single point immunization. Detailed studies involving in vitro release of protein antigens from polymer particles, cellular uptake by macrophages and DCs are being studied to understand the mechanism of antigen processing and presentation. Cytokine analyses were carried out to delineate the role of particle size in elicitation of 34 immune response by particles entrapping protein antigen. It was observed that both nanoparticles and microparticles by themselves were shown to induce production of inflammatory cytokines upon interaction with APCs. The levels were different with nanoparticles leading to production of significantly higher pro-inflammatory cytokines (TNF- α and IL-6). However, production of significantly higher IL-1β upon stimulation with a TLR agonist (LPS) was observed only in case of microparticles and it was uptake dependent. The uptake and processing of protein antigen was found to be more for microparticles in comparison to nanoparticles. The fact that microparticles deliver more antigens to APCs could be one of the major factors contributing to the enhanced humoral response in comparison to nanoparticles. In case of nanoparticles lesser antigen uptake and processing was observed. These results along with our previous data provide more information about the immune- stimulatory potential of different sized polymer particles. Microparticles induced better humoral response in comparison to nanoparticles. This could be due to multiple effects such as (i) delivery of high antigen concentration into APCs and (ii) better processing of the antigens through MHC II pathways and (iii) sustained release of antigens from particles for a longer period of time
Formulation and adjuvant effect of dry powder alum Extensive studies are being carried out using spray dried alum powder as an adjuvant. Alum adsorbed vaccines are known to be unstable to dehydration and lose their immunogenicity when lyophilized. Antigens (DT, TT and Lysozyme) mixed with alum were spray dried to make dry powder vaccine formulations. The size of particle were around 2-5 µm. Alum particles were characterized in detail and compared with liquid alum preparation. In spite of the fact that the antigen was released quickly from spray-dried alum particles, antibody response in mice was found to be similar to that observed with traditional alum adsorbed antigen. This was true for both primary and secondary antibody responses. The vaccine formulation powder possessed good long-term stability. This opens up new possibilities of making dry powder, room temperature stable vaccine containing alum as an adjuvant.
Formulation of dry powder Pneumococcal prot e in v a ccines and its evaluation for needle free delivery of protein antigen Spray drying process was optimized for the formulation of polymer particle entrapping recombinant Pneumococcal surface protein (PspA) as a candidate antigen. By carefully designing the process and formulation parameters, a dry powder formulation of a pneumococcal surface protein antigen (PspA) was developed. Spray dried microparticles entrapping PspA showed good aerodynamic properties. Entrapping PspA in polylactide microparticles by spray drying retained its immunogenicity and proved the potential for non‐invasive vaccine delivery. Both non‐porous hollow as well as porous particles showed acceptable aerodynamic properties suitable for aerosol immunization. In vitro phagocytic uptake studies with murine macrophages indicated the effective phagocytosis of antigen loaded particles. In vitro lung deposition studies suggested that, microparticles prepared using spray drying were suitable for pulmonary delivery of antigen. In vivo immunization by dry powder needle free vaccination by pulmonary route elicited robust anti‐PspA antibody responses. The antibody response was comparable to that achieved with intramuscular immunization of PspA loaded PLA particles. Our results demonstrate the potential of dry powder polylactide particles for needle free delivery of protein antigens.
35 B. Formulation of large porous PLA particles for tissue culture and formation of polymer membranes Our study aims at developing a novel method of scaffold fabrication using polymeric particles fusing into different types of structures. Evaluation of this polymeric membrane towards transplantation and wound healing is also being studied. Attempts have been made to formulate polymer particles entrapping antibiotics which will lead to scaffold formation. Polymer scaffold releasing gentamycin were fabricated and evaluated for wound healing. Cancer cell lines were grown on these polymer scaffolds and evaluated for drug sensitivity. It was observed that these in vitro 3D cell culture models have different drug sensitivity behaviors as compared to that observed with 2D cultures in flaks. C. Solubilization and refolding of inclusion body proteins Our focus on inclusion bodies (IBs) has been on two aspects: (1) to improve the recovery of bioactive protein and (2) to understand the nature of aggregation during inclusion body formation. Previously we have reported that different sized inclusion body aggregates are formed during expression of recombinant protein in E. coli. In past we have showed that the nature of inclusion body aggregates depends upon the polypeptide sequence of the protein as well as expression conditions used. To obtain native protein from these aggregates, it is necessary to solubilize these aggregates followed by refolding of the solubilized protein by appropriate refolding method. A novel solubilization method using n-propanol in presence of low concentration of urea was developed. N-Propanol based solubilization agent was compared with traditional solubilization agents like high concentration of urea and GdmCl for solubilization efficiency, structure and stability using recombinant human growth hormone (hGH). N-propanol in combination with 2 M urea was found to be sufficient for the efficient solubilization of hGH inclusion bodies. Aggregation during refolding was also studied and it was found that solubilization with n-propanol based buffer resulted into bioactive hGH without aggregation in comparison to those obtained with urea and GdmCl based buffers. From the results obtained, it can be concluded that solubilization of hGH IBs in n-propanol based buffer results in a partially folded folding intermediate of hGH which readily folds into native form during dilution with reduced chances of protein getting into aggregation pathway. We are also screening other organic solvents for their solubilization potential. We hypothesize that organic solvents with a high log Poctanol/water value (partition coefficient) are efficient in inclusion body solubilization. Presence of enzyme activity in inclusion bodies along with other structural studies indicates the presence of native-like secondary and tertiary structures. There are no detailed reports on the presence of enzyme activity in inclusion bodies of oligomeric proteins. One of such proteins is E. coli Asparaginase II which is active only in its tetrameric form where the active site is formed by intimate interaction of two subunits. Inclusion bodies of Asparaginase are found to be of non-classical type which displays significant enzyme activity when solubilized. This observation strongly suggests the presence of native-like quaternary structures in inclusion bodies. Currently we are exploring the structural basis of enzyme activity in these IBs employing various biophysical and biochemical techniques. We are also studying the effect of expression conditions on the nature of inclusion body aggregates.
Future plans Standardization of spray drying process for formulation of alum particles as an adjuvant and delivery system will be investigated in detail. Entrapment of conjugated polysaccharide vaccine in polymer particle will be explore and compared with that of particle with only 36 polysaccharide. More challenge experiments will be carried out to understand the potential of polysaccharide entrapped polymer particle in providing protection. Molecular mechanism of improving the immunogenicity of polymer particle entrapped antigen will be studied in detail.
Action taken on RAP/SAC 2013 recommendations
No specific recommendation was received during RAP/SAC 2013 presentation
Publications Original peer-reviewed articles
#1. Gupta N, Shrestha A, Panda AK, Gupta SK* (2013) Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development. Mol Biotechnol 54: 853- 862.
2. Sharma M, Sharma V, Panda AK, Majumdar DK* (2013) Development of enteric submicron particles formulation of α-amylase for oral delivery. Pharm Dev Technol 18: 560-569.
3. Alam A, Mirza A, Talegaonkar S, Panda AK, Iqbal Z* (2013) Development of Celecoxib complex: characterization and cytotoxicity studies in MCF-7. Pharmaceut Anal Acta 4: 211.
4 Haider S, Alam SM, Hamid H, Shafi S, Nargotra A, Mahajan P, Nazreen S, Kalle AM, Kharbanda C, Ali Y, Alam A*, Panda AK (2013) Synthesis of novel 1,2,3-triazole based benzoxazolinones: Their TNF-α based molecular docking with in-vivo anti-inflammatory, antinociceptive activities and ulcerogenic risk evaluation. Eur J Med Chem 70: 579-588.
5. Anish CK, Khan N, Upadhyay AK, Sehgal D, Panda AK* (2014) Delivery of polysaccharides using polymer particles: Implications on size dependent immunogenicity, opsonophagocytosis and protective immunity. Mol Pharmaceutics 11: 922-937.
*Corresponding author #In press last year, since published
37 Study of mucosal immune responses
Principal Investigator Anna George
Ph.D. Students Ritesh Kumar Tiwari (till July 2013) Ankur Singh Saini Lucas D’Souza Srijani Basu Snehlata Gupta (from December 2013)
Collaborators Vineeta Bal, NII Satyajit Rath, NII Uma Chandra, THSTI, Gurgaon Mouli Natchu, THSTI, Gurgaon Tushar Vaidya, CCMB, Hyderabad
Theme of research
The laboratory works in the broad area of lymphoid cell activation, differentiation and survival and on immune-mediated microbial homeostasis in the intestine. We attempt to identify and characterize signals that can modulate cellular differentiation pathways, study factors that promote plasma cell longevity and try to identify adjuvants that promote the generation of mucosal immunity following systemic priming. We are also attempting to determine how the microbial flora of the gut and the immune system interact with and influence each other.
Objectives Ongoing research efforts in the laboratory revolve around analyses of the following broad areas:
1. B cell differentiation: We are assessing the role of CD40 ligation and B cell-mediated antigen presentation on B cell responses. Approaches include stimulating B cells in vitro with LPS in the presence or absence of anti-CD40 and the use of CD40-null mice to assess the role of CD40 ligation under homeostatic conditions in setting differentiation thresholds. We have also been looking at the effect of the Lyst mutation on B cell antigen presentation and differentiation. 2. Plasma cell survival: We are attempting to characterize signaling intermediates that are involved in promoting plasma cell survival. 3. Generation of IgA responses and microbial homeostasis in the gut: We are attempting to circumvent the problems associated with oral immunization by identifying adjuvants that will generate IgA antibodies measurable in serum, saliva and fecal pellets of mice following systemic immunization. We are also interested in estimating the microbial diversity in the intestine of various inbred and immunodeficient mouse strains.
38 Work reported in 2012-2013 In our analysis of B cell differentiation we have previously reported that CD40-mediated enhancement of memory B cell generation involves JNK activation. Last year we reported that many genes downstream of JNK are modulated as early as 24h after CD40 ligation. These included Rbp4, JunB, ATF2, Elk-1, NFATC2, Foxo3a, STAT3, Pax2, Bcl-2, RxRalpha and Mcl-1 (and a number of related genes). The rapid kinetics suggests that cell fate decisions can be made even before cell division has been initiated. We also found that CD40 ligation leads to a substantial reduction in Blimp-1 transcript levels by 12h in LPS-stimulated B cells, suggesting that CD40 signaling may have a prominent early role in suppressing plasma cell generation. In our analysis of plasma cell (PC) survival we reported that iNOS-deficiency led to a reduced antibody response to NP-Ficoll immunization. We also reported that Nos2-deficient plasma cells show relatively poor survival in vitro and in vivo. iNOS inhibitors decreased the survival of wild-type PCs in vitro while addition of an NO donor increased the survival of Nos2-/- PCs. Dissection of the biochemical pathway involved indicated that nitric oxide induced by iNOS led to the activation of soluble guanylyl cyclase, leading further to activation of protein kinase G, modulation of endoplasmic reticulum stress components and inhibition of caspase activation. Neither reactive oxygen species nor mitochondrial dysfunction was noted. We also reported that IL-6, a pro-survival cytokine for PCs that is made by bone marrow stromal cells, feeds directly into this pathway by inducing IL-6. When other factors were tested, APRIL promoted the survival of wild-type PCs only, while CD44 ligation was found to enhance the survival of iNOS-sufficient as well as iNOS-deficient PCs. Thus, iNOS was identified as an intermediate in some signaling pathways for PC survival. In our attempts to induce mucosal priming following systemic immunization, we reported that vitamin D3 supplementation of mice at the time of subcutaneous immunization leads to a measurable IgA response. A reported effect of vitamin D3 is to allow dendritic cells to leave the draining lymph node and traffic to distal sites. We tested this and reported that antigen presenting cells could be detected in the spleen and mesenteric lymph nodes of mice 48-72 h after subcutaneous immunization of mice provided the mice were treated with vitamin D3. Further, bone marrow chimera experiments showed that VDR-/- cells were unable to migrate to distal organs.
Progress of work during the current reporting year (2013-2014) In our analysis of B cell differentiation, we have reported previously that B cells from beige mice, which carry the Lyst mutation, show delayed antigen presentation to T cells on the one hand and to enhanced and more sustained BCR-mediated signaling on the other. Over the past year, we tracked the internalized BCR by confocal microscopy and report that while movement of the endocytosed BCR to LAMP-1+ compartments was rapid in wild-type B cells, a substantial fraction remained outside such compartments in beige B cells even at 120 min. The data suggested that peptide generation and loading onto MHC-II molecules likely occurred in different compartments in B cells from the two strains. We tested this by pulsing activated B cells with E -GST and staining with the YAe antibody to pick up E -I-Ab complexes. We found that while YAe staining was restricted largely to LAMP-1+ compartments in wild-type cells, they were mostly outside such compartments in beige B cells. We also assessed co-localization of the BCR with p-Erk and the early endosomal marker Rab5 following treatment of B cells with anti-BCR antibody and found no differences in the co- localization till 30 min. However, the association of p-Erk with early endosomes fell thereafter in wild-type B cells, but was seen in beige B cells at 45 and 60 min. Together, the data 39 indicate that BCR-antigen complexes that are retained in early endosomal compartments can continue to signal and can also load MHC-II molecules, although the process is less efficient than in lysosomes. We have previously reported that the enhanced signaling in beige B cells led to higher frequencies of memory B cells and of long-lived plasma cells. Over the current reporting year, we have also looked at affinity maturation following immunization with a T- dependent antigen as well as affinity heterogeneity in the two strains. No significant differences were seen. Over the past year we have also confirmed and extended previous data reported from our laboratory that indicated a role for iNOS in promoting plasma cell (PC) survival. Our earlier experiments aimed at assessing PC frequencies relied on LPS stimulation of splenocytes or purified B cells from wild-type and Nos2-/- mice in vitro or intraperitoneal administration of LPS to the two strains and we found that PC frequencies were consistently two-fold lower following stimulation of NOS2-/- B cells. We have now extended these data to BCR-mediated stimulation and show a similar difference in PC frequencies in response to anti-IgM stimulation. We have previously reported that LPS-mediated proliferation of B cells, assessed by thymidine incorporation, was not compromised by lack of iNOS and we have now supplemented these data with CFSE dilution experiments. We report that the number of cells in all division peaks is greater in stimulated NOS2-/- cultures than in wild-type cultures. In addition to tracking the survival of purified PCs in vitro by viability assays, we have also now scored their function as antibody secreting cells (ASCs) by ELISPOT assays and we report that iNOS deficiency leads to greater loss of ASC numbers over time. To confirm that iNOS deficiency did not affect the initiation of terminal differentiation, we estimated transcript levels of the molecules Pax5, Bach2, IRF4 and Blimp1 that are known to be modulated during PC generation by qRT-PCR over time. We report that wild-type B cells and NOS2-/- B cells showed similar down-modulation of Pax5 and Bach 2, and similar induction of IRF4 and Blimp-1 over a 72h period. Further, when PCs were tracked over time by flow cytometry, similar frequencies were seen in cultures from wild-type and NOS2-/- B cells until at 24 h, 48 h and 72 h post-stimulation. By 96 h however, frequencies dropped in the NOS2-/- cultures. Together with our activation and proliferation data, these results confirm that it is plasma cell survival rather than their generation that is affected by iNOS deficiency. We have also been analyzing the effect of NOS2-deficiency in vivo. Last year, we reported that when PCs purified from the two strains were adoptively transferred into Igh congenic mice, donor IgM titers declined more sharply in recipients given NOS2-/- PCs. This is an indirect readout of PC survival and hence, over the current reporting year we have done two kinds of experiments to more directly score PC death in vivo. First, we tracked antigen- specific ASC numbers following immunization of the two strains with the T-independent antigen NP-Ficoll over time. We report that the temporal drop in ASC numbers is sharper in the spleens and bone marrow of NOS2-/- mice. To look at T-dependent responses without possible confounding effects of iNOS deficiency on T cells, we generated mixed marrow chimeras with bone marrow from wild-type (CD45.1) and NOS2-/- (CD45.2) mice and immunized the chimeras with NP-OVA. At various times, CD45.1 and CD45.2 cells were sorted from the spleens and bone marrow of the chimeras and NP-specific ASC numbers were estimated. The NOS2-/- compartment showed a greater decline in splenic ASCs and relatively poor accumulation in bone marrow. Thus, PCs generated from NOS2-/- B cells show compromised survival in vitro and in vivo. Finally, we tested whether iNOS inhibition could lead to inhibition of an established antibody response. To do this, we treated wild-type and NOS2-/- mice daily with the iNOS inhibitor aminoguanidine from day 28 post immunization. This allowed a good primary response that included germinal center formation, generation of IgG antibodies and localization of PCs to the bone marrow before iNOS inhibition. We report that while aminoguanidine treatment had not effect on the antibody response of NOS2-/- mice, 40 it led to a dramatic fall in titers in wild-type mice. We confirmed these experiments in mixed marrow chimeras that were immunized with NP-OVA and treated with aminoguanidine as above. At various times wild-type and NOS2-/- cells were sorted from the spleens and bone marrow of untreated and treated mice and scored for NP-specific ASC numbers. Aminoguanidine treatment led to a sharp fall in wild-type ASCs in the spleen and to their significantly lower accumulation in the bone marrow, while having no effect on the NOS2-/- compartment. Collectively, our data show that iNOS promotes the survival of PCs in vitro and in vivo via a biochemical pathway that involves soluble guanylyl cyclase mediated activation of PKG, leading to modulation of endoplasmic stress and caspase activation and that pro- survival mediators such as IL-6 feed into this pathway by inducing iNOS. They also show that iNOS inhibition can lead to down-modulation of an established antibody response by adversely affecting PC survival.
Future plans We have reported previously that vitamin D supplementation augments IgA responses to systemic immunization and we have preliminary data to indicate that some mouse strains (ICAM-/-, beige and TCR -/- mice) are resistant to the effects of vitamin D. We aim to characterize these data further. We would also like to extend our finding that iNOS inhibition in vivo leads to a fall in plasma cell numbers and in circulating antibody levels to disease models and will therefore initiate experiments to see whether autoimmune conditions can be ameliorated by iNOS inhibition. We have standardized genomic PCRs for estimating microbial diversity in fecal pellets of mice and would like to carry out such estimations in multiple mouse strains, and also determine whether specific members of the intestinal flora are coated with IgA.
Action taken on the RAP/SAC 2013 recommendations Progress of work in the laboratory was presented to RAPSAC members and was met with their approval. The members looked carefully at the reviews we received for a manuscript on plasma cell survival that we had submitted, and we discussed specific experiments that would address the concerns of the reviewers. These experiments were done during the current reporting year and the manuscript was accepted for publication. No other recommendations were made.
Publications Original peer-reviewed articles
1. Sinha A, Gulati A, Saini S, Blanc C, Gupta A, Gurjar BS, Saini H, Kotresh ST, Ali U, Bhatia D, Ohri A, Kumar M, Agarwal I, Gulati S, Anand K, Vijaykumar M, Sinha R, Sethi S, Saloma M, George A, Bal V, Singh G, Dinda AK, Hari P, Rath S, Dragon-Durey M-A, Bagga A* (2013) Prompt plasma exchanges and immunosuppressive treatment improves the outcomes of anti-factor H autoantibody-associated hemolytic uremic syndrome in children. Kidney Int (doi: 10.1038/ki.2013.373).
2. Saini S, Shenoy G, Rath S*, Bal V*, George A* (2014) Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells. Nat Immunol 15: 275-282.
41 3. Upadhyay M, Priya GK, Ramesh P, Madhavi MB, Rath S, Bal V, George A, Vaidya T* (2014) CD40 signaling drives B lymphocytes into an intermediate memory-like state, poised between naïve and plasma cells. J Cell Physiol (doi: 10.1002/jcp.24572). Reviews / proceedings 1. Salam N*, Rane S, Das R, Faulkner M, Gund R, Kandpal U, Lewis V, Mattoo H, Prabhu S, Ranganathan V, Durdik J, George A, Rath S, Bal V* (2013) T cell ageing: Effects of age on development, survival & function. Indian J Med Res 138: 595-608. 2. Prabhu SB*, Khalsa JK, Banerjee H, Das A, Srivastava S, Mattoo HR, Thyagarajan K, Tanwar S, Das DS, Majumdar SS, George A, Bal A, Durdik JM, Rath S* (2013) Role of apoptosis-inducing factor (Aif) in the T cell lineage. Indian J Med Res 138: 577-590.
*Corresponding author(s)
42 Analysis of Salmonella Typhi-host cell interaction
Principal investigator Ayub Qadri
PhD students Ajay Suresh Akhade Farhat Parveen Sonia Jitender Yadav Mohd. Anees Ahmed
Theme of research
Pathogenic Salmonella species produce different clinical manifestations depending upon Salmonella serovar and the type of host. In humans, these manifestations range from self limiting localized gastroenteritis caused by Salmonella Typhimurium to systemic disease produced by Salmonella Typhi. During systemic dissemination, bacteria can be found in many organs including spleen, liver, bone marrow and gall bladder. Salmonella Typhi does not however establish infection in normal mice. On the other hand, Salmonella Typhimurium infection in mice results in a systemic outcome that is analogous to human typhoid. The reasons for different clinical outcomes produced by these two closely related Salmonella serovars are not understood. Further, the mechanisms by which pathogenic Salmonella modulates host immune defenses in order to establish systemic infection are also not very clear. Work in our laboratory addresses these two aspects of Salmonella-host cell interaction.
Objectives Broadly, our studies are aimed at 1. Identifying differences in host-pathogen interactions which take place during infection with S.Typhi versus S.Typhimurium We have previously shown that one of the molecules, Vi (capsular virulence polysaccharide), which is present in S. Typhi but not in non-typhoidal Salmonella serovars, can engage membrane prohibitin complex and downregulate inflammatory responses. Molecular details of this downregulation are currently being worked out in our laboratory. 2. Understanding modulation of immune responses during infection with pathogenic Salmonella This includes analysis of changes that Salmonella might undergo upon sensing host cues and studying their relevance to inflammation and immunity. 3. Understanding the role and regulation of TLRs in T cells We are also investigating involvement of host factors in regulating inflammatory responses produced by TLRs.
43 Work reported in 2012-2013 We have been for the last several years studying the role of capsular polysaccharide, Vi (for virulence), which distinguishes Salmonella Typhi from Salmonella Typhimurium, in host- pathogen interaction. Vi interacts specifically with membrane associated prohibitin complex and engagement of this complex results in downregulation of inflammatory responses during infection with S. Typhi, a ploy that could facilitate establishment of infection with this pathogen. We also showed that circulating hemoglobin has the capability to abrogate anti- inflammatory ability of Vi and transform it into a proinflammatory TLR2 agonist. In our last report, we showed that this inflammatory activity might be important for generating IgG kind of antibodies against this T-independent antigen in mice. IgG anti-Vi antibodies have been previously shown to correlate with efficacy of Vi vaccine in humans. These results suggested a possible mechanism by which IgG antibodies might be generated during vaccination with Vi. We also reported that knock-down of prohibitin expression in epithelial cells considerably reduces induction of inflammatory responses upon infection with Salmonella. We are investigating the mechanism by which prohibitin might regulate inflammatory responses. In our studies on understanding modulation of immune responses with pathogenic Salmonella, we showed that infection with S. Typhimurium almost completely abrogates the ability of T cells to produce IL-2 upon in vitro stimulation with anti-TCR antibodies. This was associated with increased secretion of IFN-γ and IL-17. The latter two cytokines were also produced following stimulation of cells with S. Typhimurium – derived bacterial extract. This response did not require presentation of Salmonella antigens on MHC class II as secretion of IFN-γ was also observed when T cells from infected mice were incubated with Salmonella antigens in presence of accessory cells from MHC class II deficient mice. These findings suggested that IFN-γ and IL-17 might be produced by T cells as a result of innate activation. The significance of these findings is currently under investigation in our laboratory.
Progress of work during the current reporting year (2013-2014) A. Caspase-1 regulates expression of TLR/NLR ligand from pathogenic Salmonella Previous work from our laboratory showed that sensing of lysophospholipids derived from intestinal epithelial cell (IEC) activates release of proinflammatory flagellin from Salmonella, thereby revealing a previously unappreciated mechansim by which inflammatory responses could be regulated during infection with this pathogen. Post intestinal invasion, pathogenic Salmonella species infect and reside within mononuclear phagocytes which can recognize bacterial flagellin not only through membrane TLR-5 but also through the intracellular inflammasome, Nlrc4; the responses through these sensors play a vital role in innate immunity against Salmonella infection. We therefore thought it pertinent to investigate regulation of flagellin secretion during infection of macrophages with Salmonella. The results showed significant release of flagellin during interaction of Salmonella with macrophages. This release was not seen when cells were infected with this bacterium in presence of caspase-1 inhibitor or infected with Salmonella strains which lack effectors (e.g. SipB) required for activating caspase-1. Importantly, the release of flagellin was also observed during infection of macrophages with a Salmonella strain which expressed flagellin intracellularly but did not make surface flagella clearly demonstrating that the release of flagellin was not an outcome of depolymerisation of flagella but a result of de novo production of flagellin following host sensing. This was in line with our previous findings with IECs. We now show that this host- mediated increase in flagellin expression increases the ability of Salmonella to bring about pyroptotic cell death that is accompanied by increased IL-1β secretion. Mass spectrometric analysis revealed that macrophage - derived supernatant capable of triggering flagellin release 44 from Salmonella contained lysophopsholipids. These results are consistent with a recent study which showed that Salmonella infection in macrophages is associated with a lipid storm that is dependent on caspase-1 activation. To investigate if this host stimulus-dependent regulation of flagellin was also operative during intracellular residence of Salmonella, we looked at expression of this TLR/NLR ligand (flagellin) in Salmonella obtained from WT or caspase-1 deficient bone marrow macrophage derived cell lines (kindly provided by Eicke Latz, Institute of Innate Immunity, Bonn) infected in vitro with S. Typhimurium. The data showed that while Salmonella obtained from WT cells continued to express flagellin, Salmonella obtained from caspase-1 deficient cells started reducing expression of flagellin at 24 post infection. Similarly, flagellin expression was reduced when mouse macrophages were infected with SipB deficient Salmonella which is known not to activate caspase-1. Taken together, these results suggest that first host response following infection with pathogenic Salmonella might be directed at promoting pyroptotic ability of the pathogen that would lead to its clearance. That is precisely the reason why Salmonella begins to reduce expression of flagellin as the infection progresses. Our preliminary data suggest that caspase-1 activation might also bring about other modulations that have a bearing on the fitness/pathogenicity of bacteria. Our findings reveal a novel mode of regulation of inflammatory responses during infection with pathogenic Salmonella.
2. Cross-talk between Toll-like receptors and lipid - specific G-protein coupled receptor(s) in the induction of inflammatory responses
We had previously reported that TLR5 induced CXCL8 secretion from model human intestinal epithelial cell lines (IECs) and colonic explants obtained from DSS-treated mice is considerably increased in the presence of non-proteinaceous component(s) of serum. This enhancement was not due to promotion of flagellin binding to cells but due to modulation of intracellular signaling by host-derived factor(s). Further investigations revealed that this phenomenon was not restricted to IECs as upregulation in CXCL8 secretion in presence of serum was also observed during stimulation of mononuclear phagocytes, both mouse and human, and human T lymphocytes with flagellin and other TLR agonists. Analysis of identity of the serum component revealed that the serum-mediated enhancement on TLR activation could be recapitulated to a large degree with serum-borne bioactive lipid, lysophosphatidylcholine (LPC). LPC enhanced TLR-activated inflammatory responses from human as well mouse macrophages. Investigations into the mechanism revealed that this enhancement of inflammatory responses was produced as a result of priming of immune cells by TLR-activation to respond to serum/LPC. Serum lipids are known to activate G-protein coupled receptors (GPCRs) and hence modulate a plethora of biological functions such as cell migration, proliferation and survival. We therefore carried out TLR stimulations in presence of GPCR inhibitors suramin and pertussis toxin. Both these inhibitors blocked serum/LPC- mediated enhancement of cytokine production from TLR-primed cells. Pertussis toxin blockade was also seen during induction of inflammatory responses in vivo with TLR2 agonist Pam3csk. Moreover, upregulation brought about by LPC was also inhibited with anti-G2A antibody; G2A is known to be playing a crucial role in the induction of GPCR-dependent cellular responses with LPC. Finally, we were able to show that the cross-talk between TLRs and LPC-specific GPCR was mediated through activation of JNK module of intracellular signaling. Our work presents evidence for a previously unappreciated mechanism in the induction of inflammatory responses through TLRs.
45 3. Innate immune responses from T cells are regulated by antigen receptor activation
T cells are responsible for providing adaptive immunity, yet these cells express Toll-like receptors (TLRs) which are vital to innate immunity. The functional significance of these receptors and regulation of their responses are not completely understood. We and other have shown that these TLRs on human T cells are signal competent and upon engagement with microbial ligands can produce neutrophil chemoattractant CXCL8. To understand how activation of T cell through the antigen receptor might influence the innate capability of T cells, we stimulated these cells with plate coated anti-CD3 antibody and anti-CD28 antibody and tested their responses to TLR2 (Pam3csk) and TLR5 (flagellin) ligands. While naïve CD4+ T cells isolated from peripheral blood and model human T cell line Jurkat readily secreted CXCL8 upon stimulation with flagellin or Pam3csk, TCR-activated cells secreted negligible amounts of CXCL8. TCR activation did not however affect the ability of TLRs to generate signaling intermediates of MAP-kinase and NF- B pathways indicating that signals transduced through the TCR did not alter TLR levels. Preliminary results show that while naïve T cells increased CXCL8 mRNA levels upon stimulation through TLR2, activated T cells did not increase mRNA levels further upon treatment with TLR2 agonist raising a possibility that CXCL8 locus might be unresponsive to TLR triggered intracellular signals. These findings highlight the role of TCR in shaping innate immune activation of CD4+ T cells.
Future plans Studies on modulation of innate and adaptive immune responses during infection with pathogenic Salmonella, and the role of the two way host-pathogen cross-talk in the establishment of infection with Salmonella would continue to be our focus areas. We would like to get molecular insights into regulation of TLR responses in T cells and understand functioning of membrane prohibitin complex in cell signaling.
Action taken on the RAP/SAC 2013 recommendations
The suggestions given by the members during the last RAP-SAC were valuable in our studies.
Publications Original peer-reviewed articles
#1. Sharma N, Akhade AS, Qadri A* (2013) Sphingosine-1-phosphate suppresses TLR- induced CXCL8 secretion from human T cells. J Leukoc Biol 93: 521-528.
2. Santhanam SK, Dutta D, Parween F, Qadri A* (2014) The Virulence polysaccharide Vi released by Salmonella Typhi targets membrane prohibitin to inhibit T cell activation. J Infect Dis (in press).
*Corresponding author # In press last year, since published
46
Molecular basis of B cell responses
Principal Investigator Devinder Sehgal
Research Associates Arif Tasleem Jan Preetika Arya
Ph.D. students Jaya Bhushan Ruchika Hina Jhelum Sujata Kumari Ajay Kumar
Collaborators Amulya Kumar Panda, NII Rajendra P. Roy, NII K. Natarajan, University of Delhi
Theme of research The theme of research is to decipher the molecular and cellular basis of immune responses against protein and polysaccharide antigens present on the surface of the human bacterial pathogen Streptococcus pneumoniae (also called pneumococcus). The other research interest is to find out how pneumococci cause disease and what interventions can be made to stop this from happening. The research is focused on the pneumococcal products and strategies that allow the pathogen to avoid being destroyed by the mammalian immune system, and the types of immune responses that can circumvent these strategies and products.
Objectives
The main objectives are (a) molecular analysis of immune response to pneumococcal cell surface protein and polysaccharide antigens, (b) identification and characterization of virulence factors such as toxins and adhesins from S. pneumoniae that are or may be related to pathogenesis, (c) how these virulence factors interact with the immune system and host cell to alter its cellular and molecular processes, and (d) evaluating the vaccine potential of pneumococcal cell surface proteins.
Work reported in 2012-2013
Functional characterization of monoclonal antibodies generated against Pneumococcal surface protein A
Pneumococcal surface protein A (PspA) is a polymorphic, cell surface, choline-binding protein. PspA interferes with fixation of C3 on the pneumococcal surface. PspA, a virulence factor, is present in all strains of pneumococci and is serologically variable, cross-reactive, and cross protective. PspAs have been classified into family 1 (clades 1 and 2), family 2 (clades 3, 4 and 5) and family 3 (clade 6). PspA families 1 and 2 account for the majority (94 -
47 99%) of the pneumococcal isolates. Anti-PspA monoclonal antibodies (mAbs) have been shown to be protective. The protective epitopes have been mapped to the extracellular α- helical domain of PspA. Previously, we reported some data regarding the molecular characterization of 36 mouse monoclonal antibodies generated against the extracellular domain of PspA (PspA3-286) from strain R36A (PspA family 1 / clade 2). We tested whether the mAbs raised against PspA from R36A cross-react with PspA from the other 5 clades. The reactivity patterns of the culture supernatant from the 36 hybridomas with the 6 clades of PspA were analyzed by ELISA. Out of the 36 hybridomas, only three mAbs (all 3 are IgM) exhibited some degree of cross-reactivity with recombinant PspAs from the 6 clades. Thirty anti-PspA mAbs cross-reacted with clade 1 and 2 PspA but not with the other clades. The results indicated that majority of the mAbs are family 1 specific. For subsequent analysis we restricted ourselves to the mAbs that were raised against PspA using heat-killed R36A as an immunogen and were of the IgG isotype. The cross-reactivity of the culture supernatants from these 21 IgG secreting hybridomas were tested with 3 strains that express PspA of clade 1 (BG8838, AC-094 and BG6692) and 4 strains that express PspA of clade 2 (WU2, D39, R36A and A66.1) using flow cytometry. The analysis indicated that only 8 of the 21 mAbs showed surface binding with the 7 strains that express PspA of family 1. These mAbs are B3D12, B3H8, L5C8, L5F10, M4F4, P2A4, P2B5 and P1E11. These mAbs were analyzed for bactericidal activity and their ability to confer protection in vivo. All 8 mAbs exhibited bactericidal activity against pneumococcal strains BG8838 and D39. Passive mouse protection experiments were performed with 8 short-listed mAbs. CBA/N mice were administered purified mAbs (5 mg per kg body weight) intraperitoneally. One hour later mice were challenged with ~103 cfu of the pneumococcal strain WU2. Survival of mice was monitored for 21 days. Mouse survival data suggested that M4F4, P1E11 and P2A4 provided 100% protection while P2B5 provided 87% protection. B3H8 and L5F10 conferred 50% or less protection. B3D12 and L5C8 failed to protect. Isotype controls conferred any protection.
Progress of work during the current reporting year (2013-2014) Identification and functional characterization of secreted nuclease(s) from S. pneumoniae During pneumococcal infection, neutrophils are among the first immune cell types that infiltrate the site of infection. A novel defense mechanism has been reported (referred to as NETosis) wherein neutrophils die to generate neutrophil extracellular traps (NETs). These traps are a framework of chromatin, histones and antimicrobial proteins. Bacteria get trapped in this mesh and are subsequently killed. In this way bacterial dissemination is restricted. Secreted nucleases can help bacteria in evading NETs and establishing infection. In this regard, we aim to identify potential nucleases in the pneumococcal secretome. Typically, pneumococci start undergoing lysis on reaching stationary phase. In order to prevent intracellular proteins released as a result of lysis from contaminating the secretome an autolysin (lytA) deficient mutant was constructed by overlap extension PCR. It was characterized by immunoblotting and growth kinetics studies in nutrient rich and chemically defined media. The culture supernatant from logarithmic phase lytA deficient pneumococci showed nuclease activity. The nuclease activity was completely abrogated when the secretome was treated with proteinase K, heat or EDTA thereby suggesting that the observed nuclease activity is heat labile and required divalent cations as cofactors. Next, we set out to identify the protein(s) responsible for this activity. For this purpose, in-gel nuclease activity assay was performed. Three bands corresponding to degradation of DNA were observed. Currently efforts are underway to identify these nucleases by mass spectrometry. As a second approach we are using bioinformatic tools to predict possible candidates that are secreted and have nuclease activity. The gene corresponding to the probable candidates would be cloned, 48 expressed, purified and validated for its enzymatic activity. In addition, their role in host- pneumococcal interaction would be assessed. Functional characterization of lipoproteins from S. pneumoniae The virulence factors of S. pneumoniae include capsular polysaccharide, cell wall components and surface exposed proteins. Surface proteins are known to play an important role in pneumococcal pathogenesis by functioning as adhesins, invasins and interacting with the host cells and molecular components. Lipoproteins comprise one of the most abundant classes of molecules present on the surface of S. pneumoniae. Many lipoproteins form the substrate binding component of ABC transporters. ABC transporters have been demonstrated to have an important bearing on the fitness and virulence of pneumococci. Given their surface localisation, and importance in pneumococcal fitness and virulence we focussed our study on functional characterization of pneumococcal lipoproteins. Lipoproteins of Gram positive bacteria are initially translated as pre-prolipoproteins which possess an N-terminal signal peptide with a lipobox motif. Lipobox is modified through the covalent attachment of the diacylglycerol moiety to the thiol group on the side chain of the indispensible cysteine residue. This modification is catalyzed by the enzyme lipoprotein diacylglyceryl transferase (lgt), resulting in a prolipoprotein. Post lipidation, lipoprotein signal peptidase (lsp) is responsible for cleaving the signal sequence of the lipidated prolipoprotein and leaves the cysteine of the lipobox as the new amino-terminal residue. We constructed a pneumococcal strain deficient in lipoprotein biosynthesis by deleting the gene encoding lgt by inframe gene replacement mutagenesis. A previously published study has shown that absence of lgt results in loss of retention of lipoproteins on the surface of S. pneumoniae. To study the role of pneumococcal lipoproteins in modulation of alveolar macrophage function we prepared Triton X-114 extract from wild-type and lgt deficient S. pneumoniae. We confirmed the presence of lipoproteins in the extract by immunoblotting for known lipoproteins. PspA, a choline binding protein, was found to be absent in the Triton X-114 extract from lgt deficient pneumococci by Western blotting. We have used a model murine alveolar macrophage cell line MH-S for this study. We observed that deletion of lgt abrogated adhesion of S. pneumoniae to MH-S cells by >50%. We studied the role of pneumococcal lipoproteins in modulating alveolar macrophage functions such as production of nitric oxide (NO), proinflammatory cytokines and reactive oxygen species (ROS). We observed that incubation of MH-S cells with Triton X-114 extract from lgt deficient S. pneumoniae resulted in significant decrease in nitrite production compared to the extract from the wildtype strain. We also quantified nitrite production from MH-S cells in presence of live wild-type and lgt deficient S. pneumoniae, and heat-inactivated wild-type and lgt deficient S. pneumoniae. Nitrite production in the presence of live S. pneumoniae was comparable to the untreated controls. Nitrite production from γ interferon treated MH-S cells was lower when incubated with heat-killed lgt deficient S. pneumoniae compared to heat-killed wild-type S. pneumoniae. The nitrite production from MH-S cells treated with Triton X-114 extract from the wild-type S. pneumoniae was abrogated in the presence of iNOS inhibitor L-NAME suggesting the involvement of iNOS in pneumococcal lipoprotein mediated NO production. We further studied the contribution of pneumococcal lipoproteins in the production of proinflammatory cytokines IL-6 and IL-12. We observed a significant decrease in IL-6 and IL-12 production from MH-S cells upon incubation with Triton X-114 extract from the lgt deficient S. pneumoniae compared to wildtype extract. The role of pneumococcal lipoproteins in production of intracellular ROS was studied by flow cytometry using dihydrorhodamine-123 staining. The treatment of MH-S cells with the Triton X-114 extract from wild-type S. pneumoniae resulted in 1.5 fold higher ROS production compared to untreated sample. ROS production occurred in a dose dependent manner when live wild-type and lgt deficient pneumococci were used. We observed that ROS production was enhanced in presence of lgt deficient S. pneumoniae in comparison to wild-type S. pneumoniae. Our data suggests that pneumococcal lipoproteins have important implication in 49 adhesion and modulation of innate effector and inflammatory functions of alveolar macrophages. Further we would like to investigate the role of pneumococcal lipoproteins in invasion and intracellular survival. The role of pneumocoocal lipoproteins in mouse model of pneumococcal pneumonia will be studied by infecting mice with wild-type and lgt deficient S. pneumoniae and comparing bacterial load and cytokine profile in lung and BALF, and mice survival.
Future plans
We will continue to functionally characterize the lipoproteins from S. pneumoniae. We plan to identify and characterize the protein(s) responsible for the nuclease activity observed in the S. pneumoniae secretome especially in the context of virulence and host-pathogen interaction.
Action taken on the RAP/SAC 2013 recommendations
The work was presented before the RAP-SAC members. The scientific and technical clarifications sought were provided. There was no specific recommendation from the committee members.
Publication Original peer-reviewed article
1.Anish C, Khan N, Upadhyay AK, Sehgal D, Panda AK* (2014) Delivery of polysaccharides using polymer particles: Implications on size-dependent immunogenicity, opsonophagocytosis, and protective immunity. Mol Pharm 11: 922-937.
*Corresponding author
50 Understanding the role of interferon regulatory factors (IRFs) in dendritic cell development and innate immunity
Principal Investigator Prafullakumar Tailor
Ph.D. Students Hemant Jaiswal Rohit Verma Irene Saha (Since Jan 2013) Sneha Aggarwal (Since Jan 2013) Kuldeep Singh Chauhan (Since Dec 2013)
Research Fellow Monika Kaushik
Collaborators Subeer Majumdar, NII Debasis Mohanty, NII Keiko Ozato, NIH, USA
Theme of research
Dendritic cells (DCs) are composed of multiple subsets that collectively provide early innate immunity, leading to subsequent adaptive immunity. Plasmacytoid dendritic cells (pDC), CD4+ DC, CD8α+ DC and CD4-CD8- DC are four major subtypes in the murine spleen. These subtypes of DCs express different sets of genes and assume distinct functions. We are interested in understanding the mechanisms of development of DC subsets and their functions. Members of Interferon regulatory factors (IRFs) play important role in DC subset development and their respective functions. Main area of research of the laboratory is to understand the significance of signaling pathways and contribution of IRFs and other critical transcription factors in DC subset development and functions.
Objectives
The principal aim of the project is to understand the role of IRF family members in the DC development and functions. Interferon regulatory factor 4 (Irf4) and Interferon regulatory factor 8 (Irf8) plays pivotal role in generation of diverse DC subtypes. The development of CD8α+ DC and pDC requires Irf8, whereas CD4+ DC subset is dependent on Irf4. Recent reports suggest an important role of Inhibitor of DNA binding 2 (Id2) and Basic leucine zipper transcription factor 3 (Batf3) in the CD8α+ DC development. Major areas of focus this year are
1. Understanding the significance of Irf8, Id2 and Batf3 in DC subset development. 2. Understanding contribution of TGF-β signaling in Irf8 directed DC development.
51 Work reported in 2012-2013
Understanding the significance of Irf8, Id2 and Batf3 in DC development
We had shown that expression of Irf8 in DC9 (Irf8-/- DC line) cells led to growth inhibition and guided cells towards pDC and CD8α+ DC differentiation. Our experiments suggested that expression of Irf8 led to the increase in the Id2 and Batf3 transcript levels. Further, Id2 and Batf3 expressed alone or together, can not drive differentiation of CD8α+ DCs, whereas Id2 and Batf3 expression had a synergistic effect on CD8α+ DC development when expressed along with Irf8. Though, Id2 and Batf3 when co-expressed with Irf8 led to synergistic increase in the CD8α+ DC specific transcripts; such synergistic effect was not extended to the pDC specific gene expression. A recent study of Nfil3 (nuclear factor, IL-3 regulated; also called E4BP4) gene knock out mice suggested that it indirectly controls the CD8α+ DC development by regulating Batf3 gene expression. Expression of Nfil3 in DC9 line led to expression of some of the CD8α+ DC specific gene expression and only Irf8 expression could induce complete profile of CD8α+ DC phenotype. Thus, taken together our data suggests that Irf8 is a master regulator of CD8α+ DC development.
Understanding the role of TGF-β signaling in Irf8 regulated DC development In the first step, we initiated the study towards understanding the role of TGF-β signaling in the DC development. Experimental treatment of the murine BMDCs with the TGF-β led to the increase in the Smad7 and Tgfbi genes and this increase in the transcript level was inhibited by TGF-β signaling chemical inhibitors SB431542 and LY364945. We observed that addition of TGF-β to the DCs led to SMAD2 phosphorylation as early as 30 min and addition of inhibitors completely blocked this activation, though IRF8 protein levels did not change over time period till 24 hrs. Results of BMDC experiments suggest that Irf4 and Irf8 transcript levels are not sensitive to chemical inhibition of the TGF-β signaling. Further, expression of Smad7 and dnTGFbRII led to decrease in the transcript levels of Id2 and Sis but the Irf8 and Irf4 transcript levels remained comparable. Our preliminary finding suggests that TGF-β signaling may be redundant in pDC or CD8α+ DC development and further study is needed to confirm our observations.
Progress of work during current reporting year (2013-14)
Understanding the significance of Irf8, Id2 and Batf3 in DC development
Our earlier observation suggested that Irf8 plays a central role in CD8α+ DC development and that Batf3 and Id2 have a synergistic effect on Irf8 directed CD8α+ DC development. Recent reports have identified a Zinc finger transcription factor zDC (Zbtb46), which is expressed specifically in cDC population and expression of zDC differentiates classical DCs from pDCs and monocyte-macrophage lineage population. We observed that cDC specific gene zDC (Zbtb46) is efficiently induced by Irf8 and co-expression with Id2 or Batf3 led to a synergistic increase in zDC levels. Our observation suggests that Irf8 is upstream of zDC in the cDC developmental scheme which would help to further fine tune the existing model for the DC subtype development. As our results were mainly interpretations of in vitro experiments with cell line; it was necessary to demonstrate the CD8α+ DC development using in vivo mouse model. For in vivo analysis; DC9 cells were transduced with control and Irf8 expressing retroviruses and 48 hr post transduction, equal numbers of cells were introduced into mice by retro-orbital injection. Analysis of control and Irf8 expressing DC9 populations in spleen showed that only Irf8 expressing cells developed into CD8α+ DCs and displayed very high levels of MHC II expression as compared with the control vector transduced cells. One of the 52 most important characteristics of CD8α+ DC is their ability to cross present the antigen and hence to demonstrate cross presentation; DC9 cells were transduced with control and Irf8 expressing retroviruses and cells were selected for 48 hr with puromycin. Puromycin selected control and Irf8 expressing populations were treated overnight with CpG in presence/absence of Ovalbumin. Cells were washed thoroughly and cultured with B3Z cells for 24 hrs. The B3Z line is a murine CD8+ T cell hybridoma that specifically gets activated upon recognition of Ovalbumin peptide SIINFEKL in the context of MHC I by the T cell receptor (TCR). TCR signaling of B3Z cell line leads to expression of beta-galactosidase under NF-AT elements from the interleukin-2 (IL-2) promoter (Int. Immunol. 1994, 6:369). Cells were washed and activation of B3Z cells was quantitated by LacZ assay. Intracellular production of β- galactosidase was detected using substrate Chlorophenol red-b-D-galactopyranoside (CPRG) by measuring absorbance at 570 nm. Antigen cross presentation studies using B3Z line suggested that only DC9 cells expressing Irf8 could cross-present antigen thus demonstrating an efficient development of classical CD8α+ DC subset. As Batf3, Id2 and Irf8 are essential for CD8α+ DC development; we have initiated the experiments to conduct the detailed analysis of DC subtype development by these transcription factors. We have generated the constructs for the mammalian two hybrid systems and Bimolecular fluorescence complementation assay so as to understand interactions (if any) between Batf3, Id2 and Irf8. Experiments in near future will help us to understand the interactions between these transcription factors and their role in defining DC subtype development.
Understanding the role of TGF-β signaling in Irf8 regulated DC development Transforming growth factor-β (TGF-β) signaling is shown to have important roles in DC development. In DCs, TGF-β signaling induces Id2 expression (Nat. Immunol. 2003, 4:380- 386). ID (ID1-4) proteins are members of helix-loop-helix (HLH) group of transcription factors and are termed as antagonists of another activator class of HLH members. Id2 expression is essential for CD8α+ DCs and Langerhans cells (Nat. Immunology 2003, 4:380- 386). A recent study identified Irf8 as a direct target of TGF-β signaling in DCs (Eur. J. Immunol. 2007, 37: 1174–1183); and as per our studies, Irf8 expression in DC9 cell line (Irf8-/- DC line) led to induction of Id2 gene, suggesting that IRF8 may regulate CD8α+ DC development by controlling Id2 gene expression.
Our preliminary data suggested that blocking TGF-β signaling using chemical inhibitors (SB431542 or LY364945) or by over-expressing signaling blocking proteins (Smad7 or dnTGFbRII) did not lead to change in Irf8 or Irf4 transcript levels. Thus, our observations imply that TGF-β signaling may be redundant in pDC or CD8α+ DC development and further study is needed to confirm our observations.
In our experiment, chemical inhibitor treatment or Smad7 and dnTGFbRII were expressed in the ongoing BMDC cultures and there is a possibility that commitment towards CD8α+ DC may be an early event. Hence, we procured the transgenic mice expressing dnTGFbRII. First, the BMDC developed from dnTGFbRII transgenic mice and control mice showed a comparable Irf8 or Irf4 transcripts levels. Further, detailed study of the BMDC and splenic DC population by flow cytometry studies using various pDC and CD8α+ DC markers didn’t show abnormality in different major DC subtypes. Analysis of the DC populations from TGFbRII transgenic mice confirmed our preliminary studies from previous year. Thus, we conclusively show that TGF-β signaling is redundant in pDC or CD8α+ DC development. Further, we are conducting in depth experiments to demonstrate the role of Irf8 in regulating Id2 gene expression and understanding the role of TGF-β signaling in directing the DC subtype specific gene transcription.
53 Future plans 1. To study the functional significance of co-expression of Irf8, Id2 and Batf3 to understand molecular mechanism of DC differentiation. 2. To understand the role of TGF-β signaling in CD8α+ DC development. We have preliminary observations about other independent initiatives in the lab and more emphasis would be put on understanding the cross-talk between transcription factors and signaling pathways to better define the mechanisms of DC development.
Action taken on the RAP/SAC 2012 recommendations
The scientific and technical clarifications sought by the SAC were provided during the discussions. No specific recommendations were made.
Publications Original peer-reviewed articles
1. Jaiswal H, Kaushik M, Sougrat R, Gupta M, Dey A, Verma R, Ozato K, Tailor P* (2013) Batf3 and Id2 have a synergistic effect on Irf8-Directed classical CD8α+ dendritic cell development. J Immunol 191: 5993-6001.
#2. Goel P, Tailor P, Chande A, Basu A, Mukhopadhyaya R* (2013) An infectious HHV-6B isolate from a healthy adult with chromosomally integrated virus and a reporter based relative viral titer assay. Virus Research 173: 280-285.
*Corresponding author #In press last year, since published
54 Disorders of proliferation: Analysis of novel pathways and targets
Principal Investigator Rahul Pal
Research Associates Anjali Bose Ruchi Sachdeva
Ph.D. Students Sonia Jain Poonam Singh Beneeta Kalha
Collaborator Ilpo Huhtaniemi, Imperial College, London
Theme of research
The lab focuses on two disorders characterized by proliferative aberrance, systemic autoimmune disease and tumorigenesis. In particular, the work seeks to investigate the consequences of aberrant cell death in systemic autoimmune disease as well as to delineate mechanisms and pathways by which human chorionic gonadotropin (hCG) can impact upon systemic autoimmunity and cancer.
Objectives
The pathological consequence of autoreactive immune responses are the subject of intense investigation. Systemic Lupus Erythematosus (SLE) is a prototypical non-organ specific autoimmune disease. Animals genetically modified to impair the uptake of apoptotic cells exhibit lupus-like pathology. Autoimmune cascades initiated by autoantibody responses specifically directed towards apoptotic cells are being investigated, since apoptotic debris appears to constitute the original antigenic insult. The immunological and physiological sequel arising as a result of the release of sequestered hemoglobin in animals prone to autoimmunity also forms a focus of current investigations.
Human chorionic gonadotropin (hCG) is critical for the sustenance of pregnancy, but has also been shown to be secreted by a variety of cancers; its presence has been associated with poor patient prognosis. Understanding the molecular pathways by which hCG impacts on tumor progression as well as the development of novel immunotherapeutic anti-hCG vaccination strategies form a focus of the laboratory. Given reports of pregnancy-associated lupus flares in humans and the ameliorating influence of hCG in murine models of organ-specific (Th1- mediated) autoimmune disease, the effects of the hormone in animals genetically prone to systemic autoimmunity (a state which can notionally be considered to constitute a Th2 skew) are also being investigated.
55 Work reported in 2012-2013
A. hCG and tumorigenesis
Studies in βhCG transgenic (TG) mice TG mice exhibit accelerated weight gain, and ovarian and pituitary tumors. In previous work, anti-hCG immunization prevented increases in body-weight; prolactin levels and ovarian and pituitary morphology remained normal. Serum derived from βhCG transgenic mice induced the expression of VEGF, IL-8 and MMP-9 from tumor cells. Immunization led to restoration of fertility and prevented down-modulation of the cyclin-dependent kinase inhibitors CDK1b (p27), CDK 2a (p16) and CDK 2c (p18). Further, pituitary transcript levels of ccnd1, hmga2, e2f1, galanin and prolactin, growth hormone, pttg1, fgf2 and bmp4 were comparable to those in non-transgenic litter-mates. These studies re-emphasize a potential role for hCG in tumorigenesis. hCG and chemo-resistance Pre-incubation of tumor cells with hCG reduced chemotherapeutic drug-induced apoptosis. In consonance with these results, hCG enhanced levels of several molecules known to be associated with chemo-resistance. hCG and some TLR ligands acted in synergy to mediate enhanced chemo-resistance. Macrophages incubated with supernatants of tumor cells previously incubated with hCG were found to produce significant amounts of IL6, which in turn mediated chemo-resistance. These studies indicate that the chemo-protective effects of hCG are heightened and extended in conjunction with other molecules and cells.
B. Systemic autoimmunity
The immunobiology of hemoglobin (Hb) Upon immunization of autoimmune-prone mice with Hb, antibody reactivity towards Hb and anti-double stranded DNA was heightened, accompanied by autoantibody sequestration in the kidneys. Immunized mice demonstrated increases in the mesangial matrix and glomerulosclerosis. Immunization of non-autoimmune-prone animals resulted in lower antibody responses, with kidneys remaining unaffected. These results indicate that the propensity towards generation of anti-Hb responses in autoimmune-prone mice may have pathological consequences.
The influence of hCG on systemic autoimmune responses Previous work had established that hCG enhanced the generation of distinct anti-self antibody responses when administered to autoimmune-prone mice and, to a lesser extent, in non- autoimmune-prone mice. Current studies indicated that autoantibodies directed towards β2- glycoprotein 1, prothrombin, Protein C and Protein S were heightened, as were responses to various phospholipids. hCG may therefore contribute to the generation of a pro-thrombotic milieu.
Progress of work during the current reporting year (2013-2014) A. hCG and tumorigenesis hCG and chemo-resistance Tumor-associated hCG has been linked with poor patient prognosis. Previous work had established that hCG increases viability and proliferation of tumor cells. Additionally, hCG reduced the loss of viability induced by 5-flourouracil, curcumin, cisplatin, tamoxifen and
56 etoposide. Enhanced viability in the presence of hCG was attributable to a significant reduction in drug-induced apoptosis. While incubation of tumor cells with a chemotherapeutic drug reduced levels of pro-caspase 3 as expected, incubation with hCG reduced basal levels of active caspase 3. Pre-incubation of cells with hCG reduced the pro-apoptotic effects the drug; compared with cells treated with drug alone, levels of active caspase 3 were significantly diminished whereas levels of pro-caspase 3 were not diminished to the same extent. Tumor cells incubated with hCG demonstrated increases in the levels of cIAP-1, XIAP and survivin (inhibitors of apoptotic proteins), HIF-1α, HO-1 (molecules associated with chemo- resistance and tumorigenesis), PON2 and Hsp27 (molecules associated with protection from cell stress). siRNAs against HIF-1α, HO-1, Nrf-2 and survivin decreased transcription and expression of respective targets in tumor cells. Silencing of survivin, HO-1 and Nrf-2 decreased hCG-induced resistance to both tamoxifen and curcumin respectively. Addition of siRNA to HIF-1α had no effect on hCG-induced protection against curcumin; however, there was significant reduction in protection against tamoxifen. The results revealed that, while all four molecules influenced hCG-mediated chemo-resistance, the nature of the toxic insult was also an important determinant of the extent of afforded protection. Increasing evidence links toll-like receptors (TLRs) with the development of chemo- resistance. In data reported previously, several TLRs were up-modulated upon the incubation of tumor cells with hCG, and some TLR ligands acted in synergy with hCG to provide enhanced resistance against chemo-therapeutic agents. Intra-cellular signaling pathways activated upon the combined addition of hCG and TLR ligands in the presence of drug were investigated, and showed some unique signaling patterns. Cells incubated with hCG along with ligands for either TLR2 or TLR8 showed enhanced ERK phosphorylation. Interestingly, all three components (hCG, TLR ligand and drug) were required for maximal phosphorylation of JNK and p38 via TLR3, of ERK, AKT and JNK via TLR6, of ERK via TLR8, and of JNK via TLR9. In the view of known and suspected roles of hCG in tumorigenesis, anti-hCG vaccination is increasingly considered an attractive option. In the current reporting year, studies were designed to investigate the effects of combined chemotherapy and immunotherapy. Mice implanted with syngeneic Lewis Lung cancer cells (LLC) were immunized with a βhCG- based vaccine formulation and also received either curcumin or tamoxifen. Animals receiving either drug alone, or immunotherapy alone, demonstrated a reduction tumor volumes and incidences. Co-administration of drugs and immunotherapy resulted in a further decrease in both tumor volume and incidence, and in animal survival. The data suggests that combining anti-hCG immunotherapy with standard chemotherapy may result in substantial benefit in individuals carrying gonadotropin-sensitive tumors. Studies in βhCG transgenic (TG) mice Previous reports described the role of CG in the generation of endogenous tumors in TG mice. Implantation of syngeneic tumor cells in TG animals permitted study of the effects of exogenous CG in an in vivo environment. Higher tumor incidences and larger tumor volumes were recorded in male and female TG mice upon the implantation of LLC cells. Markedly higher transcript levels of IL-6, IL-8, TNFα, VEGF, MMP-2, MMP-9, Bcl-2, Bcl-xl and survivin were observed in LLC tumors isolated from TG mice. Serum derived from TG animals induced secretion of the tumor-associated moieties VEGF, KC, TGF-β and IL-6 from LLC cells. As with endogenous tumors, anti-hCG immunization reduced the incidence of exogenous tumors.
57 B. Systemic autoimmunity The immunobiology of hemoglobin (Hb) Previous work had demonstrated that immunization of autoimmune-prone mice with Hb resulted in autoantibody sequestration in the kidneys and associated glomerulosclerosis, while similarly immunized non-autoimmune-prone mice remaining relatively unaffected. These results indicate that the propensity towards generation of anti-Hb responses in autoimmune- prone mice may result in pathological consequences. Current studies were focussed on the etiology of anti-Hb autoimmune responses. Splenocytes derived from both autoimmune-prone and non-autoimmune-prone mice responded to ferric (but not ferrous) murine Hb by the increased secretion of IL-6, IL-8 and TNF-α. Of interest was the fact that ferric (but not ferrous) murine Hb induced a significant increase in levels of maturation markers (CD80, CD86, CD83, CD40) on BMDCs from autoimmune-prone mice. The functional consequences of these phenotypic changes are under investigation. Using lipopolysaccharide-based stimulation assays, previous work had indicated an increased level of anti-Hb B cell precursor frequencies in autoimmune-prone mice. Interestingly, the addition of apoptotic blebs to splenocytes derived from autoimmune-prone (but not from non- autoimmune-prone) mice led to the generation of antibodies to Hb. The fact that elicited antibodies did not exhibit exclusive cross-reactivity between Hb and apoptotic blebs argues for a role of T cells in this phenomenon. Since previous studies had demonstrated that ferric Hb preferentially interacts with several autoantigens, whether Hb-apoptotic bleb complexes can induce additive or synergistic effects in terms of phenotypic and functional modifications on BMDC, cytokine secretion from splenic cells or antibody expression from B cells, form the focus of on-going work. The influence of hCG on systemic autoimmune responses Immunopathological outcomes in SLE are believed to be antibody-mediated. Pregnancy, characterized by a Th1 to Th2 skew, has been associated with an exacerbation of clinical symptoms in lupus patients. In previous work, in vivo administration of hCG in autoimmune- prone mice induced humoral autoreactivity towards cell surface, cytoplasmic and nuclear moieties; increases in antibodies to Protein S, Protein C, β2-Glycoprotein 1 and several phospholipids were observed. Increased levels of such auto-antibodies are strongly associated with thrombotic events. In current work, hCG enhanced the maturation of bone marrow- derived dendritic cells driven by ssRNA (a TLR ligand implicated in lupus), as assessed by cell-surface phenotype and the ability to induce allogeneic proliferative responses. hCG preferentially enhanced the pro-proliferative effects of LPS in splenic B cells derived from autoimmune-prone mice and significantly increased mixed lymphocyte-induced proliferative responses. While synergistic increases in IL-6 and IL-10 upon anti-CD3 and hCG stimulation occurred in both autoimmune and non-autoimmune murine strains, enhancements in T cell receptor-induced proliferative effects and associated increases in the phosphorylation of ERK, p38 and AKT were preferentially observed in autoimmune mice, as was the heightened generation of autoantibodies to apoptotic blebs. The data supports a role for hCG in pregnancy-associated disease flares in lupus by mediating proliferative, cytokine and differentiative responses, and possibly also in non-autoimmune conditions such as preeclampsia and ovarian hyper-stimulation syndrome, via the induction of autoantibodies against components of the clotting cascade.
58 The influence of apoptotic blebs and apoptotic cell-specific antibodies on BMDC generation and maturation Aberrant apoptosis is considered a hallmark of SLE. Whether apoptotic blebs differentially affect innate immune components of autoimmune-prone and non-autoimmune-prone animals was investigated. Purified blebs potently inhibited the generation of BMDCs, an effect more severe in autoimmune-prone animals. Blebs were also more effective than healthy cell lysate in the induction of BMDC maturation. BMDCs matured in the presence of blebs produced heightened levels of inflammatory cytokines and mediated higher allogeneic proliferative cytokine responses. Previous work had described some characteristics of monoclonal apoptotic cell-specific antibodies, including the ability to induce hyper-gammaglobulinemia and expansion of the autoantibody repertoire upon immunization into syngeneic, autoimmune- prone animals. Much like apoptotic blebs, such autoantibodies also induced both the maturation BMDCs as well as the secretion of inflammatory cytokines. Whether immune complexes comprised of the two inflammatory components express synergistic activity is under investigation.
Future plans
In exogenous tumors arising upon the implantation of LLC in βhCG TG mice, whether increases in tumor-associated transcripts are paralleled by increases in protein levels will be investigated. Immuno-histochemical analysis will enable the identification of source cells, thereby confirming or negating the premise that tumor cells and non-transformed resident cells collaborate in the elaboration of tumor-promoting factors in an βhCG/CG-driven manner. Further, tumor implantation studies employing ovarectomized, female βhCG TG mice, and/or drugs which interfere with the secretion of prolactin will permit the elucidation of individual contributions of gonadotropin, steroids and prolactin to the tumorigenesis. Whether ferric Hb-apoptotic bleb complexes can induce additive or synergistic effects in terms of phenotypic and functional modifications on BMDC, cytokine secretion from splenic cells or antibody expression from B cells, will form the focus of future studies. Whether immune complexes constituting two “self” components shown to be inflammatory in an autoimmune milieu (apoptotic blebs and anti-apoptotic cell specific antibodies) are endowed with synergistic activity will be evaluated.
Action taken on the RAP/SAC 2013 recommendation
No specific recommendations were received.
Publication Original peer-reviewed article
1. Bose A, Huhtaniemi I, Singh O*, Pal R* (2013) Synergistic activation of innate and adaptive immune mechanisms in the treatment of gonadotropin-sensitive tumors. PLoS ONE 8: e61288.
*Corresponding authors
59 Study of genetic and immune factors associated with autoimmune disorders: Type 1 diabetes and vitiligo
Principal Investigator Rajni Rani
Research Associates Jaya Singh (Till Dec. 2013) Sukanya Prathipati (Till Nov. 2013) Samrina Mehtab (from Jan, 2014) Avinash Kumar (Till Dec. 2013)
Ph.D. students Bhukya Naik Varkhande S Risha Anshu Sharma Utpraksha Vaish Ankita dabla
Collaborators Ravinder Goswami, AIIMS, New Delhi Rajesh S. Gokhale, IGIB, delhi K. Natarajan, JNU, New Delhi Anita Kamra Verma. DU, New Delhi S.K Sarin, ILBS, New Delhi H. K. Kar, Ram Manohar Lohia Hospital, New Delhi V.K. Sharma, AIIMS, New Delhi S. Vijaya, I.I.Sc., Bangalore Indira Nath, IOP, New Delhi Rasheedunnisa Begum, MSU of Baroda, Vadodara Peter Stastny, Southwestern Medical Center, Dallas, USA
Theme of Research
The project aims to decipher the Immunogenetic and autoimmune factors involved in the destruction of pancreatic beta cells and melanocytes in Type 1 diabetes (T1D) and vitiligo respectively. We aim to device ways to inhibit autoimmune responses in T1D. Vitiligo, is a multifactorial disease etiology of which is not precisely understood. While several hypotheses have been proposed including autoimmunity, it is not clear how the pigment producing melanocytes are destroyed by the autoimmune responses. So, the theme of this project is to understand the aetiopathogenesis of vitiligo with an aim to develop therapeutic approaches for the disease.
Objectives
1. To study the role of Human leukocyte antigens (HLA) in aetiopathogenesis of both T1D and vitiligo. 2. To study other Immune function related genes which may have a role in manifestation of T1D and Vitiligo. 3. To study the autoimmune factors associated with T1D and vitiligo. 4. To design and use peptides in-vitro to inhibit autoimmune T-cell responses. 5. To encapsulate the peptides which inhibit Th1 immune responses in-vitro, in nano-sized carriers for slow and targeted release.
60 6. Study delivery of peptide/vector complexes in Balb-C and C57Bl6 mice followed by NOD mice. 7. To differentiate mouse Mesenchymal stem cells into insulin producing cells. 8. To study the role of MHC restricted auto-antigen specific CD4+/CD8+T cells in autoimmune destruction of melanocytes in vitiligo. 9. To study the role of Cytokines increased in vitiligo patients in aetiopathogenesis of vitiligo.
Work reported in 2012-13
Type 1 diabetes
We had reported last year that we had studied antigen processing genes in T1D. For this purpose about 3 SNPs in LMP2 and LPM7 genes were studied in 226 T1D patients and about 700 healthy controls and the data was not analyzed. Since we wanted to use MSCs to inhibit autoimmune responses, we treated MSCs with different cytokines as their immunosuppressive properties have been reported to be enhanced by these treatments. A dose response was done to check for the expression of different immunosuppressive markers like Foxp3, indoleamine 2,3 dioxygenase (IDO) and inducible nitric oxide synthase (iNOS). We observed that TNF alpha and IFN-gamma could induce the expression of IDO and iNOS.
Vitiligo We had conducted global transcriptional profiling of epidermal cells after treatment with pro- inflammatory cytokines that were increased in the lesional skin of vitiligo patients on primary keratinocyte cultures. It was observed that pathways such as cell proliferation, nucleotide synthesis etc. were downregulated in IFN-γ treated keratinocytes. However, with a combination treatment of IL-17A and IFN-γ, epidermal differentiation genes were also strongly affected. The data needed to be verified and analysed using available softwares.
Progress of work during the current reporting year (2012-2013) A. Type 1 diabetes Genetic basis of Type 1 diabetes We have added a few more samples to study the role of antigen processing genes in Type 1 diabetes and analysed the data on 241 T1D patients and 752 healthy controls for 3 SNPs in LMP2 and LMP7 genes. LMP7 exon 2 SNP that results in an amino acid change from Glutamine to Lycine (Q49K) at position 49 showed a significant increase of CC genotype i.e. KK homozygosity and this difference was significant even after Bonferroni’s correction. Since LMP2 and 7 are localized in the MHC class-II region, we checked whether there was any linkage disequilibrium between the predisposing MHC allele and the LMP7 exon 2 predisposing alleles, however, there was no linkage disequilibrium between the two suggesting that the association of LMP7 exon 2 C allele was independent of the predisposing MHC alleles.
61 Mesenchymal stem cell treatment of non-obese diabetic mice Since we observed increased expression of immunosuppressive markers like indoleamine 2,3 dioxygenase (IDO) and inducible nitric oxide synthase (iNOS) in mesenchymal stem cells (MSCs) after treatment with TNF alpha and IFN-gamma, we wanted to study whether these treated cells would have any functional significance if given to non-obese diabetic mouse (NOD). To study the effect of cytokine treated MSCs on inhibition of autoimmune responses in NOD mice, sets of five mice were injected in the tail vein with 5X105 MSCs treated with IFN-γ, TNF-α or IL-1β or PBS for controls. Four injections at 6 weeks, 8 weeks, 10 weeks and 12 weeks were given and the blood sugar levels were monitored for 32 weeks (Figure 1).
Figure 1: Blood glucose levels of control NOD mice (a), NOD mice treated with MSCs (b), NOD mice injected with IFN-γ treated MSCs (c) NOD mice injected with TNF-α treated MSCs (d), NOD mice injected with IL-1 β treated MSCs (e) and Percent non-diabetic mice in controls and treated mice at 32 weeks of age. * in the X-axis show 4 injections given at 6, 8 10 and 12 weeks. PBS was given to control mice.
As is clear from the figures while 40% of the control mice remained non-diabetic at the end of 32 weeks, 100% of the mice injected with IFN-γ and TNF-α treated MSCs remained non- diabetic. 60% of the NOD mice injected with IL-1 β treated MSCs remained non-diabetic. While we were trying to differentiate MSCs into insulin producing cells, we realized that there was not more than 1.5 fold change in the expression of genes of pancreatic lineage in the induced versus control MSCs. Since both induced and uninduced cells have been grown in high glucose media, there was a possibility of even uninduced cells to make insulin since control cells also stained with insulin (shown earlier). Also once the cells were differentiated into insulin producing cells, we could not grow them in culture for too long. So, we were not sure whether we should inject mice with differentiated cells or give them MSCs grown in high glucose media for long enough. So, we set up five sets of experiments, one where the cells were induced to make insulin producing cells, and others where passage 9, 10, 11, 12 and 13 cells grown in high glucose media were injected in mice at 9th week or at 6th week. In the set 62 where differentiated insulin producing cells were given to mice at the 9th week 60% remained non-diabetic at the end of 28 weeks compared to 50% in the control group. Similarly 75% of the NOD mice in both control group and the groups treated with passage 9 and 10 cells remained non-diabetic at the end of 29 weeks. However, in the groups where passage 11, 12 and 13 MSCs were given to NOD mice, 100% in the treated group and 40-50% in the control groups remained non-diabetic (data not shown). These studies are being repeated to check for reproducibility.
B. Vitiligo
Validation of Microarray results Enrichment of several pathways connected with cell cycle in the IFN-γ and IL-17A and IFN-γ combination treatment set of normal human keratinocytes (NHK), prompted us to validate these results using real time PCR. Cluster analysis and heatmap analysis was done on 42 gene targets involved in the cell cycle in both the data from the microarray and real time PCR for the same gene set. It was observed that the real time PCR data reproduced the microarray data well, where cell cycle genes were downregulated in keratinocytes treated with a combination of IFN-γ and IL-17A (Figure 2a) and CDK inhibitors were upregulated (Figure 2b). Comparison of the cell cycle profile of normal human keratinocytes using propidium iodide after 48 h treatment with IFN-γ alone, IL-17A alone or a combination of both cytokines against an untreated control revealed a significant decrease in number of cells in the S phase of the cell cycle in the keratinocytes treated with IFN-γ and IFN-γ IL-17A combined treatment. However, a concomitant increase in the number of cells in the G2+M phase was also observed. No alterations were observed in the G1 phase indicating that cell cycle arrest has occurred both at the G1/S transition, and at the G2/M transition. This data is consistent with the stark down-regulation of cyclin B, A and E and CDK1 and CDK2 at the mRNA level. a.
Control IFN-γ IL-17A Combination 5
4
3
2 Fold Change Fold 1
0 2 K4 NA2 NB1 C C CNB CNE1 CDK1 CDK2 CD C C C CCND1 CCND2 C
63 b.
NC 80 IFN 60 IL-17 Combination 40
20
6 Fold Change Fold 4
2
0
A B 2 2 F1 N N 2 K K HEK1 HEK2 E E2F4 C C CDKN1A CDKN1B CD CD
Figure 2: a. Real time PCR for cyclin and CDK genes showing downregulation of cyclins CCNA2, CCNB1, CCNB2 CCNE and their catalytic partners CDK1 and CDK2 and CDK4 with combined treatment of IFN-γ and IL-17A. (N=3). b. IFN-γ and combination treatment of IFN-γ and IL-17A upon keratinocytes resulted in the up- regulation of CDK inhibitors CDKN1A, CDKN1B, CDKN2A, CDKN2B, while cell cycle check point genes CHEK1, and CHEK2 were down-regulated, and E2F1 S-phase gene was down-regulated as well. Horizontal lines at 1.5 and 0.66 fold indicate significant up- and down-regulation respectively.
Keratinocyte differentiation and epidermal development were other pathways that were synergistically affected by IFN-γ and IL-17A treatments according to DAVID analysis of GO process terms of the genes. To validate these results, real time PCR analysis was performed (Figure 3). Basal keratinocyte marker K5 (KRT5) and K14 (KRT14) were down-regulated by IFN-γ and IL-17A alone as well as synergistically by both cytokines indicating that the keratinocyte are moving towards the terminal differentiation program. Involucrin (IVL), a marker for keratinocytes undergoing cornification and K16 (KRT16) which has been reported to be upregulated in abnormal differentiation of keratinocytes, were upregulated. Integrin alpha 2 (ITGA2) and integrin beta 1 (ITGB1) were both upregulated. While they are normally basal cell markers they have been reported to be involved in wound healing in suprabasal layers of the skin and also in abnormally induced differentiation of keratinocytes in-vitro. Filaggrin was down-regulated by IL-17Aas reported earlier, and we observed a synergistic trend in its regulation as well (Figure 3).
NC 10 IFN-γ 8 IL-17A 6 Combination 4 2 1.5
1.0 Fold Change Fold 0.5
0.0 4 5 6 7 8 0 L T1 T1 T1 T7 T8 IV R R FLG KRT5 ITGA2 K KR KR KRT19 KRT3 K KR
64 Figure 3: Alteration in keratins and keratinocyte differentiation markers by IFN-γ and IL-17A: Real time PCR for keratin and keratinocyte differentiation markers after NHK treatment with IFN-γ and IL-17A for 48 hrs. Basal keratinocyte markers KRT5 and KRT14 are down-regulated, while abnormal keratinocyte differentiation marker KRT16 and ITGA2 and cornification marker IVL are up-regulated.
At translational level, vitiligo patients were transplanted with autologous epidermal cells and pure melanocyte cultures bilaterally in 30 patients and the results show significantly better pigmentation in the sides treated with pure melanocyte cultures. This work was done in collaboration with Dr. Kar from RMLH.
Future Plans We would further like to explore different stem cell treatment strategies for prophylactic and therapeutic effect of differentiated and undifferentiated MSCs in NOD mice with a view to be translated in humans with cord blood derived MSCs. We would like to explore the possibility of using two-pronged approach for treatment of Type 1 diabetes, where peptides encapsulated in microparticles and mesenchymal stem cells will be used for treatment of diabetes in NOD mice. We would like to see whether this two-pronged approach can be used to inhibit autoimmunity in non-obese diabetic mice before the onset of the disease. For this purpose, we would treat the diabetic mice with mesenchymal stem cells / differentiated MSCs with or without peptide encapsulated micro particles and study the efficacy of these treatment regimens on manifestations of diabetes. For vitiligo confirmation of pathways regulated in the micro-array data after treatment of epidermal cells with different cytokines will be done at protein level and analyses will be to understand their role in manifestation of vitiligo.
Action taken on RAP/SAC recommendations Queries raised by the RAP/SAC members were satisfactorily answered and their recommendations have been followed.
Publications Original peer-reviewed articles
1. Natarajan VT, Ganju P, Singh A, Vijayan V, Kirty K, Yadav S, Puntambekar S, Bajaj S, Dani P, Kar HK, Gadgil CJ, Natarajan K, Rani R, Gokhale RS* (2014) IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation. Proc. Natl Aca Sci, USA 11: 2301-2306. #2. Saini C, Prasad HK, Rani R, Murtaza A, Misra N, Shankar Narayan NP, Nath I* (2013). Lsr 2 of Mycobacterium leprae and its synthetic peptides elicit restitution of in vitro T cell responses in erythema nodosum leprosum and reversal reactions in lepromatous leprosy patients. Clin Vaccine Immunol 20: 673-682.
Review/Proceedings/Etc. 1. Rani R*, Singh A (2014) Functional implications of MHC associations in autoimmune diseases with special reference to Type1 diabetes, Vitiligo and Hypoparathyroidism. Accepted
65 for publication in the book "HLA & Associated Human Diseases", InTech Open Access Publishers, Janeza Trdine 9, Rijeka, Croatia (In press). 2. Rani R* (2013) MHC Applications. In W. Dubitzky, O. Wolkenhauer, K. Cho & H. Yokota (eds.), Encyclopedia of Systems Biology, Springer New York. 3: 1158-1162.
*Corresponding author #In press, published since last year
66 Study of immunotherapeutic potential of Mycobacterium indicus pranii (MIP) and the underlying mechanisms in animal models of tuberculosis & tumor
Investigator Sangeeta Bhaskar
Ph.D. Students Pawan Kumar Mohd. Saqib Bindu Singh Ananya
Research Assistant Rahul Khatri
Collaborator Pramod K. Upadhyay, NII
Theme of research
Whole bacterial vaccines rely on multiple antigens and built-in-adjuvanticity. Mycobacterial strains which share cross-reactive antigens with M. tuberculosis are being considered as alternatives to M. bovis for vaccine use. MIP shares antigens with M. tuberculosis and initial studies had shown that vaccination with killed MIP induces protection against tuberculosis. Hence, we further studied the protective potential of MIP and the underlying immune responses. Generation of antitumor immunity is often difficult in the tumor-bearing host because of various negative regulatory mechanisms which can be overcome by activation of innate and Th1 immune response. There were indications from different clinical studies also that MIP could be useful as an immunomodulatory adjunct in some cancers. In animal model of tuberculosis we had found that MIP induces Th1 response which is also important for antitumor activity. Hence, we had started this study to evaluate the immunotherapeutic activity of MIP in mouse syngeneic tumor models.
Objectives
1. To investigate the protective efficacy of MIP immunisation in live or killed forms, through parenteral route as well as by aerosol immunization, against subsequent infection with M.tuberculosis in animal models. Study of immune response to M.tb in animals immunised with MIP as compared to those immunized with BCG. 2. Evaluation of immunotherapeutic efficacy of MIP along with chemotherapy in animal infection models. 3. To evaluate Immunoprophylactic and Immunotherapeutic activity of MIP in mouse syngeneic tumor model. Study of MIP as an adjunct to chemotherapy in combination with commercial anti cancer drugs in tumor bearing mice and simultaneous study of mechanism of MIP mediated host immune activation.
67 Work reported in 2012 -2013
A. Protective efficacy of MIP against tuberculosis and mechanistic insights as compared to BCG
Modulation of immune response was analyzed in the lungs of MIP / BCG immunised / Control group at different time points after infection with M.tb. Immune cell infiltration in the infected lungs and also inside the granuloma were analysed in the MIP / BCG-immunized groups. Reduced bacterial load and improved pathology was observed in the MIP-immunized group as compared to BCG-immunized group. Potential of MIP immunotherapy as an adjunct to standard chemotherapy was evaluated in guinea pig model of tuberculosis. In M.tb-infected guinea pigs, accumulation of immune cells in the lungs and expression of different cytokine genes was analysed. Accelerated bacterial clearance and balanced Th1 immune response was observed in the ‘Drug + Immunotherapy’ group as compared to ‘Only Drug’ group. As we observed higher protection with live MIP as compared to its killed form and BCG, we further investigated the mechanism by which antigen presenting cells recognize and respond to stimulation with live / killed MIP/ BCG. Live MIP induced significantly higher level of proinflammatory cytokines and costimulatory molecules as compared to BCG. Role of different TLRs in the recognition of live vs killed MIP and the underlying pathway was also investigated. To understand the role of TLRs, we analysed the immune response in the presence of specific pharmacological inhibitors of TLRs. It was observed that MIP mediates its effect through TLR2, TLR4 as well as through intracellular TLRs but major contribution of TLR2 was observed. We further delineated the TLR2 signaling by MIP and BCG using HEK293 cells expressing murine TLR2 alone or in combination with TLR1 or TLR6. MIP-Live showed higher level of TLR2/2, TLR2/1 and TLR2/6 agonistic activity as compared with MIP-killed and BCG. Quantitatively all the above immune responses were found to be significantly high with live MIP and thus possibly explain the higher protective efficacy observed with live MIP as compared to killed MIP. Evaluation of immunostimulatory activity of different fractions of MIP We further investigated differential immunostimulatory activity of cellular fractions of MIP viz. cell wall, cytosolic fraction and DNA. These cellular fractions were evaluated for their relative immunostimulatory potency in terms of macrophage, dendritic cell and splenocyte activation. Among all the cellular fractions, immune stimulating property of the cell wall was found to be the highest.
B. Immunotherapeutic potential of MIP and the underlying mechanisms in mouse tumor model In mouse model of tumor, immune cell composition in tumor microenvironment was analysed in MIP treated mice as compared to untreated control mice. Higher frequency, upregulation of activation markers and enhanced functional activity was observed in the macrophages and T cells isolated from tumor of MIP treated mice. An interesting finding of this study was that along with activation of antigen presenting cells, NK cells and T cells, significantly less number of regulatory T cells were observed in the tumor of MIP treated mice.
68
Progress of work during the current reporting year (2013-2014)
A. Immunotherapeutic potential of MIP and the underlying mechanisms in mouse tumor model
Ex-vivo studies with macrophages and dendritic cells showed that TLRs has major role in MIP mediated activation of these cells. We then examined whether signaling through these TLRs is responsible for MIP mediated protection against tumor. Tumors were implanted in MyD88 knockout and wild type mice and MIP treatment was given by peritumor injections. While in wild type mice treated with MIP about 40-50% mice had no visible tumor or very small tumor, MIP treated MyD88 knockout mice had tumors comparable to PBS treated mice. These results provide evidence of major role of TLRs in MIP mediated protection against tumor. Further studies with TLR knockout mice strains showed that TLR2 has important role in MIP mediated protection. TLR2 knockout mice treated with MIP showed no reduction in tumor volume whereas MIP treated TLR4 knockout mice had tumor volumes comparable to wild-type MIP treated mice. Cytokine profile of tumor microenvironment was studied by analyzing the mRNA expression of cytokine genes. RNA was extracted from tumor of control PBS treated, MIP treated, cyclophosphamide treated, and MIP + cyclophosphamide treated mice and analyzed by RT PCR. Interestingly, it was observed that MIP treatment resulted in increased IFN-γ expression in tumor milieu whereas MIP + cyclophosphamide treatment induced type-I Interferons IFN-α and IFN-β. In the group treated with ‘only drug’ expression of type-I interferons was not observed. These results suggest that drug + MIP therapy modulates the immune status of tumor microenvironment.
B. Protective efficacy of MIP against tuberculosis and mechanistic insights as compared to BCG. Evaluation of immunostimulatory activity of different fractions of MIP Differential immunostimulatory activity of cellular fractions of MIP was investigated. Cell wall fraction showed highest immune stimulating activity. It was further fractionated into aqueous soluble and lipid soluble parts and determined their immune stimulatory activity in terms of macrophage and T cell activation. Aqueous extract of MIP cell wall was found to be very potent immune stimulator. Mycobacterial cell walls have potent TLR agonistic activity hence, to analyse the TLR agonistic activity of MIP cell wall fraction, immune response in the presence of specific pharmacological inhibitors of TLRs was studied. About 20% reduction in secretion of major proinflammatory cytokine was observed with TLR4 inhibitor, which further increased to almost 50% when a combined TLR2 and TLR4 inhibitor was added to MIP cell wall treated macrophages. As chemical inhibitors are not 100% efficient for blocking the signal transduction through a particular receptor, we repeated the experiments with macrophages isolated from TLR2 and TLR4 knock out mice. About 75% reduction in secretion of major proinflammatory cytokine was observed in TLR2 knock out mice as compared to the wild type. Reduction in cytokine secretion was about 30% in TLR4 knock out mice. This data provide evidence of strong TLR2 agonistic activity and moderate TLR4 stimulating property of MIP cell wall fraction.
C. Efficacy of MIP as a booster to BCG: Immunogenicity, protection and safety study when given by aerosol route in animal models. Efficacy of BCG remains controversial, particularly against pulmonary TB in young adults. BCG is effective against severe form of childhood tuberculosis however, effective protection
69 from TB in adults is still a challenge which indicates that there is a need to boost the protective immune response against M.tb. However, paradoxically, multiple doses of BCG have resulted in reduced protection and poor survival in the susceptible animal model. Initial studies showed that vaccination with MIP gives protection against TB in both BCG responder & non-responder strains of mice. In large scale human trials (28,948 people) in 272 villages in Kanpur, India, protective efficacy of MIP against TB was observed. The vaccine/placebo was given to healthy contacts of leprosy patients who had no evidence of tuberculosis. This area is endemic for tuberculosis. After 13 years, a significant difference was observed in the occurrence of TB cases in the vaccinated and placebo groups. Further higher protective efficacy of MIP was observed in the persons who had earlier received BCG in their childhood. MIP has also shown higher protective efficacy as compared to BCG in animal models through subcutaneous and aerosol routes. Hence, it was proposed that a booster with MIP may enhance the protective immunity in animals primed with BCG. The intranasal route is proposed to induce both local and systemic immunity and provides a non-invasive delivery system intended to target lung. Hence, it is aimed to evaluate the efficacy of MIP administered through aerosol as booster to BCG vaccine in animal model. Initial results have shown higher protection in terms of lung and spleen bacterial load in guinea pigs given MIP booster, two months after BCG priming as compared to only BCG vaccinated animals. Further experiments to confirm the findings are underway. We are also analyzing the lung immune response in the above groups of animals.
Future Plans
MIP cell wall fraction will be further characterized and compared with BCG. The role of MIP/ BCG treated antigen presenting cells in modulating the profile of effector immune cells will be delineated. M.tb challenge experiments will be completed to evaluate and compare the long term protective efficacy of the group given MIP booster to BCG primed animals vs only BCG immunized animals. M.tb specific immune response will be further analysed and compared in the above groups.
Action taken on the RAP/SAC 2012 recommendations
The scientific and technical clarifications sought during the interactive session were provided.
Publication Original peer-reviewed article
#1. Roy A, Singh M, Upadhyay P*, Bhaskar S* (2013) Nanoparticle mediated co-delivery of Paclitaxel and a TLR-4 agonist leads to tumor regression and enhanced immune response in the tumor microenvironment of mice. Int J Pharm 445: 171-180.
*Corresponding authors #In press last year, since published
70 Analysis of antigen processing and presentation
Principal investigator Satyajit Rath
Research Associate Savit Prabhu
Ph.D. students Rupali Gund (till June 2013) Shalini Tanwar Jasneet Kaur Khalsa Renu Balyan Amanpreet Singh Chawla Atika Dhar (from Jan 2014)
Research Fellow Bahadur Singh Gurjar
Collaborators Vineeta Bal Anna George Subeer Majumdar Shinjini Bhatnagar, PBC-THSTI, Gurgaon Satyajit Mayor, NCBS, Bangalore Apurva Sarin, NCBS, Bangalore B Ravindran, RMRC, Bhubaneswar Tushar Vaidya, CCMB, Hyderabad Jeannine M Durdik, Univ Arkansas, Fayetteville, USA
Theme of research The aim of the ongoing programmes in this group is to examine the generation and activation of T, B and antigen-presenting myeloid cells using multiple interlinked experimental systems.
Objectives
A variety of experimental approaches are taken to address the theme issues. The approaches in current use examine APCs and pathways involved in antigen presentation to MHC class I and class II-restricted T cells, and analyse the consequences of intracellular signal transduction modulation for both development and responses of B cells, T cells and macrophages using genetic as well as pharmacological tools.
Work reported in 2012-2013 A. Cellular aging of CD8 T cells Based on preliminary data that CD8 T cells from aged mice are CD8lo, we examined CD8hi and CD8lo CD8 T cells from young mice and observed that, like CD8 T cells from aged mice, CD8lo naïve CD8 T cells respond poorly compared to CD8hi cells. To extend these in vitro studies further, we developed an in vivo aging model for naïve CD8 T cells of adoptive transfer of MHC class I (MHCI)-restricted T cell receptor (TCR)-OT-I-transgenic T cells into young congenic recipients for varying periods. As naive CD8 T cells are parked for increasing time, they show progressive decline in responsiveness and in CD8 levels. OT-I CD8 T cells
71 parked in MHCI-deficient recipients showed higher cell-surface CD8 levels, were of smaller sizes and showed reduced surface levels of the negative regulatory molecule CD5 as compared to cells parked in wild-type (WT) recipients. These data on modulation of the cell- surface phenotype indicate that naïve CD8 T cells may begin to accumulate age-related alterations due to tonic signaling via self-pMHC I ligands in the periphery. B. Bruton's tyrosine kinase (Btk) in B cell development Btk plays a crucial signaling role in B cell development, such that a lack of its functioning in patients of XLA (X-linked agammaglobulinemia) results in greatly reduced numbers of mature circulating B cells, severe agammaglobulinemia and bone marrow B cell development arrest. However, the defect manifested in the murine XID or even the Btk-deficient Btk-null mice is subtle, with a reduction in circulating mature B cells to half the normal numbers, and relatively normal bone marrow B cell development. However, introducing the XID defect into a T cell-deficient genotype leads to a major deficit in peripheral B cells. XID mice show normal numbers of splenic T1 and T2 stage transitional B cells, but show radical reductions in subsequent stages such as T3 or follicular B cells. In XID-TCRß-null mice, this peripheral maturation block is moved upstream; while they have near-normal numbers of cells at the T1 stage, they show substantial reduction in cell numbers at the T2 stage itself, unlike in XID mice. Thus, in the absence of functional Btk, T cells provide a part-redundant signal to the T1- stage B cells in the periphery to regulate their developmental transition.
Progress of work during the current reporting year (2013-2014) A. The role of ligand density in controlling outcome of CD8 T cell activation CD8 T cells can be activated within 1-3 h of exposure to peptide-MHC class I (MHCI) ligand on antigen-presenting cells (APCs). Even if these activated CD8 T cells are then separated from the APCs, they undergo activation, proliferation and differentiation on their own. As summarized in previous annual reports, we have been using CD8 T cells from mice transgenic for T cell receptors (TCRs) specific for peptide-MHCI complexes to address the regulation of the quantitative parameters of CD8 T cell responses. We have tracked the acquisition of early and late-arising cell-surface activation markers such as CD69, CD25, and CD44. Proliferation has been followed by CFSE dilution and by tracking cell numbers. Differentiation has been measured by the ability of the T cells to secrete interferon-gamma (IFNγ) upon re-stimulation, and by their ability to kill peptide-MHCI-bearing target cells. We have mainly used the OT-I TCR-transgenic mouse strain; OT-I CD8 cells recognize a chicken ovalbumin-derived peptide bound to H-2K-b. In the previously reported preliminary analyses, we observed that variation in ligand density led to a dose-response relationship with the frequency of CD8 cells showing induction of CD69. However, the intensity of CD69 levels on individual activated CD8 cells was not different between cells activated by different ligand densities. Thus, induction of CD69 expression by individual naïve CD8 T cells is a binary decision made regardless of ligand density. However, the frequency of responding T cells in the population is dependent on ligand density. These data indicated that heterogeneity in an apparently homogenous population is responsible for the dose-response relationship of a CD8 T cell response. Our earlier data also indicated that more complex components of the activation response by naïve CD8 T cells, namely, the proliferation programme of 4-6 successive cell divisions in ~60 h, could also be a binary decision made by individual cells. To test this, we purified CD69+ naïve OT-I CD8 T cells after 3 h of exposure to APCs pulsed with differing SIINFEKL peptide concentrations. When put into culture subsequently, these activated CD8 T cells underwent the same numbers of successive rounds of proliferation, and yielded 72 comparable cell numbers as a result. These data suggested that the binary, ligand density- independent nature of the activation response of the individual T cell was not confined to induction of CD69 expression but to more complex outcomes such as cell division as well. However, when ligand density was titrated down exponentially (from 1000 nM to 1 nM peptide concentrations), we found that the CD69+ CD8 OT-I T cells purified from low ligand density-stimulated cultures took longer to enter the first cell division than those purified from high ligand density-triggered cultures. This difference was due to a lag, rather than any differential induction of cell death in these cultures. This indicated that individual T cells were indeed able to identify and respond differentially to very low levels of ligand density. We examined the basis of this delayed entry into cell cycle, and found that low ligand density- triggered cells showed slower degradation of the cell cycle repressor protein p27kip1. In order to examine the basis for this ligand density-dependent difference in the behaviour of ligand-triggered naïve CD8 T cells showing comparable induction of CD69, we examined the degree TCR internalization on these cells at the 3 h time point. It is known that after engagement with cognate pMHC, TCRs are rapidly internalized, and that the extent of internalization depends on the level of TCR occupancy. We found that, over the ligand density range used here, OT-I CD8 T cells that had been successfully triggered to the extent of expressing CD69 fell into two categories; one category also showed loss of cell-surface TCR, while the other did not. The TCR internalization decision was not made in any T cells that had not begun to express CD69, and therefore was made downstream from that outcome of early activation. In cultures with high ligand density, most CD69+ CD8 cells showed evidence of TCR internalization, while in very low ligand density-activated situations, most CD69+ CD8 cells underwent no TCR internalization regardless of the time of exposure. Thus, individual naïve CD8 T cells that had made the decision to become activated (as evidenced by CD69 expression), appear to be making a second decision, namely, to internalise cell-surface TCRs. Both decisions were ligand density-independent at the level of the individual cell, but were ligand density-dependent at the level of the cell population, since frequencies of cells showing these outcomes varied over a range of ligand densities. We therefore tested the hypothesis that the decision to internalize TCRs determined the length of the lag phase before cell cycle entry in responding CD8 T cells. After 3 h of exposure to differing ligand densities, we sorted CD69+TCRlo and CD69+TCRhi OT-I cells from each ligand density and cultured them separately to track their proliferation. Regardless of ligand density, all CD69+TCRlo OT-I cells proliferated to the same extent, and did so faster than all CD69+TCRhi OT-I cells which, again, were comparable to each other regardless of ligand density. We next tested the effect of change in ligand affinity on these responses of naïve CD8 T cells. A variant peptide, SIIQFEKL (Q4), has been shown to have unchanged affinity for H-2-K-b, but the Q4-MHCI complex has a much lower affinity for the OT-I TCR than the wild-type (WT) SIINFEKL-MHCI complex does. As expected, the dose-response curve of ligand density versus frequency of CD69 induction was shifted to the right for Q4-containing ligand. However, the intensity of CD69 expression on those OT-I T cells that did express it was no different between high- and low-affinity ligands, underlining the ligand density/strength- independent nature of the primary activation decision. However, at no concentration of the Q4-MHCI complex did OT-I cells show any TCR internalization at all, indicating that the TCR internalization decision, like the CD69 expression decision, can be modulated by ligand affinity. Further, the CD69+TCRhi OT-I cells triggered by even the highest tested concentrations of low-affinity ligand consistently showed delay before the first cell division as compared to the CD69+TCRlo OT-I cells triggered by far lower concentrations of high-affinity
73 ligand, supporting a causal relationship between TCR internalization and reduction on the time to first cell division. These results demonstrate that T cells make two serial decisions upon encounter with cognate pMHC complexes, whether to be activated at all (as measured by the decision to express CD69) or not, and then a second one regarding whether to internalize TCRs or not. The combination of these two relatively early decisions helps decide the number of precursor cells recruited and when they enter cycle for the first time. At the level of the individual naïve CD8 T cell, these decisions appear to be insensitive to ligand density and/or affinity. Instead, the precise relationship between ligand density/affinity and response magnitude/rapidity is determined by heterogeneity in the response-capable T cell population. The genesis, selection, variation and basis of this population heterogeneity are thus major new questions of relevance to understanding T cell responses.
Future plans The re-engineering of metabolic pathways during lymphocyte development, as suggested by our previous data on the apoptosis-inducing factor (Aif), is being further dissected. The basis of T cell population heterogeneity will also be pursued further in light of our new data. Work in ongoing on delineating the precise role of Btk-dependent and Btk-independent signals in the regulation of peripheral maturation of B cells. These plans are based on the current state of work, but it is difficult to predict the areas in which the next year will see most notable progress. Therefore, these plans are only rough guides to the intended efforts over the next future, and may be carried over for a variety of reasons.
Action taken on the RAP/SAC 2013 recommendations While the RAP-SAC had extensively discussed the data shown last year, no specific suggestions had been made for changes.
Publications Original peer-reviewed articles
1. Sinha A, Gulati A, Saini S, Blanc C, Gupta A, Gurjar BS, Saini H, Kotresh ST, Ali U, Bhatia D, Ohri A, Kumar M, Agarwal I, Gulati S, Anand K, Vijayakumar M, Sinha R, Sethi S, Salmona M, George A, Bal V, Singh G, Dinda AK, Hari P, Rath S, Dragon-Durey MA, Bagga A* (2013) Prompt plasma exchanges and immunosuppressive treatment improves the outcomes of anti-factor H autoantibody-associated hemolytic uremic syndrome in children. Kidney Int (doi: 10.1038/ki.2013.373) 2. Upadhyay M, Priya GK, Ramesh P, Madhavi MB, Rath S, Bal V, George A, Vaidya T* (2014) CD40 signaling drives B lymphocytes into an intermediate memory-like state poised between naïve and plasma cells. J Cell Physiol (doi: 0.1002/jcp.24572). 3. Saini AS, Shenoy GN, Rath S*, Bal V*, George A* (2014) Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells. Nat Immunol 15: 275-282.
74 Reviews 1. Salam N*, Rane S, Das R, Faulkner M, Gund R, Kandpal U, Lewis V, Mattoo H, Prabhu S, Ranganathan V, Durdik J, George A, Rath S, Bal V* (2013) T cell ageing: Effects of age on development, survival & function. Indian J Med Res 138: 595-608. 2. Prabhu SB*, Khalsa JK, Banerjee H, Das A, Srivastava S, Mattoo HR, Thyagarajan K, Tanwar S, Das DS, Majumdar SS, George A, Bal V, Durdik JM, Rath S* (2013) Role of apoptosis-inducing factor (Aif) in the T cell lineage. Indian J Med Res 138: 577-590.
*Corresponding author(s)
75 Fine tuning of NF-κB signaling
Principal Investigator Soumen Basak
Research Associate Pramod Kumar Kushawaha
Ph.D. Students Balaji Banoth Payel Roy Tapas Mukherjee Sachendra Singh Bais
Project Fellows Bharath Vijayaraghavan Budhaditya Chatterjee
Theme of research
In physiological settings, cells receive multiple signals from its microenvironment those within a cell-network impinge upon pathogen responsive circuit. Indeed, such cell autonomous crosstalks are thought to engage “apparently” distinct signlaing pathways in a synergistic or antagonistic relationship involving a variety of mechanisms to regulate immune responses. Here, we have focused on cell-differentiating LTβR for its ability to augment TLR induced inflammatory response through signaling crosstalk. In a multidisciplinary research program that combines biochemistry, genetics and mathematical biology, we have considered the NF-κB system for its ability to mediate signaling crosstalk to fine-tune TLR induced inflammatory response.
Objectives
A. Exploring the signaling crosstalk between developmental LTβR and inflammatory TLRs: In a multidisciplinary research program, we will examine the ability of lymph node inducing LTβR to fine-tune the inflammatory immune responses to pro-inflammatory cytokines or pathogenic cues. We will investigate the plausible engagement of NF-κB signaling system as a mediator of crosstalk between developmental and inflammatory signaling. Effects of crosstalk control at the level of inflammatory gene-expression will be evaluated in cell culture settings. Finally, physiological relevance of crosstalk in defining innate immune response to pathogens will be tested in a murine infection model.
B. Exploring cross-regulations of TLR response through PTMs: Using bioinformatics tools, we will predict novel post-translational modification (PTM) sites on NF-κB/RelA subunit. By expressing NF-κB/RelA proteins mutated at these newly identified sites, in both over-expression as well as stable cell-line based systems, we will examine the potential role of the predicted sites in inflammatory gene-expression downstream of various TLRs. Next, we will address if PTMs modulate inflammatory response at the level of NF-κB activation in crosstalk settings. 76 Work reported in 2012-2013
A. Exploring the signaling crosstalk between developmental LT R and inflammatory TLRs: We have earlier reported the presence of a “duration threshold filter” that selectively engages long duration NEMO-IKK2 and NIK-IKK1 activities into signaling crosstalk to amplify the resolution phase RelA/NF-κB responses in the LTβR-TLR4 co-treatment regime as compared to cell activation through individual receptors. We have also analysed the gene- effects of crosstalk. Our mathematical analyses predicted possible engagement of NF-κB/IκB mediators in crosstalk.
B. Exploring cross-regulations of TLR response through PTMs: Previously, we have reported that threonine-54-alanine or threonine-54-aspertate mutations on RelA/NF-κB abrogates in transcriptional activity downstream of TLR4.
Progress of work during the current reporting year (2013-2014)
A. Exploring the signaling crosstalk between developmental LTβR and inflammatory TLRs:
Figure1: Gene Set Enrichment Analyses revealing enrichment of NF-κB targets among crosstalk controlled genes identified in microarray. Average crosstalk score (bottom panel), which reflects synergistic gene activation for a positive value, was determined to rank LPS up-regulated genes in the expression data set. Using an experimentally validated list of RelA/NF-κB response genes, GSEA demonstrated statistically significant enrichment of NF-κB targets (top), with enrichment score 0.49, p<0.001 and false discovery rate<25%, among genes synergistically controlled through crosstalk. Bars in the middle panel indicate NF-κB response genes.
a) Global gene expression analyses We compared global gene expressions in MEFs activated through TLR4 or LTβR or both at 24h post-stimulation using microarray. In addition to the negative feedback by IκBs, active proteasomal degradation of promoter bound RelA (Natoli et al., 2005) terminates canonical NF-κB transcriptional responses (Saccani and Natoli, 2002). Not surprisingly, Match 1.0 analysis identified kappaB sites in the promoter of only ~ 8.5% of the 409 genes up-regulated in response to LPS at 24h. Next, we analyzed genes for their average crosstalk scores, which reflect synergistic gene activation for a positive value, to reveal that a similar select set of 41
77 genes induced by LPS at the resolution phase are further up-regulated in the co-treatment regime (bottom panel, Figure 1). Intriguingly, we were able to demonstrate an enrichment of RelA/NF-κB targets among genes positively controlled through crosstalk (middle and top, Figure 1) in gene set enrichment analysis (Subramanian et al., 2005) using a pre-determined set of 290 NF-κB response genes. We have also noted repression of several LPS induced genes in the co-treatment regime that appears to involve an NF-κB independent mechanism (Figure 1).
Nevertheless, our quantitative RT-PCR analyses confirmed that the late expression of multiple NF-κB response genes, those encoding important cytokines and chemokines IL-1β, RANTES are hyper-induced in the co-treatment regime (not shown). Previous studies have articulated a role of RelB in modulating inflammatory gene-expression (Natoli et al., 2005). Intact hyper- induction of IL-1β and RANTES in the co-treatment regime in relb-/- cells (not shown) further ascertained a pre-dominant RelA dependent control mechanism that selectively amplifies resolution phase pro-inflammatory gene-expression downstream of TLR4.
Figure 2: (A) Immunoblot revealing cellular abundance of IκBα/p100 and p52 (top panel) as well as IκBα (middle) during signaling. An immunoblot of actin (bottom) served as a loading control. (B) Supershift analysis revealing the composition of the RelA dimers induced upon various cell treatments at 24h post-stimulation. Enhanced accumulation of both RelA:p50 and RelA:p52 dimers were detected in the co-treatment regime.
b) Biochemical characterization of molecular determinant of crosstalk Our prior mathematical analyses predicted important role of gene products encoded by nfkb2 and iκba in crosstalk. By analyzing transcripts and proteins, we could reveal a reciprocal control of signaling crosstalk by nfkb2 and ikba encoded regulators. Our immunoblot analysis indicated that concomitant LTβR signaling not only targets IκBδ/p100 for degradation but also utilize LPS induced, newly synthesized monomer p100 to potently generate p52 (Figure 2A). Limited transcriptional up regulation of nfkb2 restrained p52 and RelA:p52 production to a modest level during solitary LTβR ligation (Figure 2A). Such cross-regulatory mechanism would concurrently release RelA from IκBδ/p100 inhibited complex and enhance RelA:p52 association in the co-treatment regime. In supershift analysis, indeed, we could experimentally confirm augmented nuclear accumulation of both RelA:p50 as well as RelA:p52 dimers upon co-stimulation (Figure 2B). The levels of IκBα, however, followed similar temporal profiles in LPS and co-treatment regimes with comparable signal induced degradation and NF-κB dependent resynthesis (Figure 2A).
78 c) Genetic analysis of crosstalk control How does IκBα control crosstalk? Our modeling studies suggested coordinated and compensating functioning of IκBδ and IκBα downstream of TLR4. By quantitatively comparing respective early and late DNA binding activities induced by LPS, we could demonstrate only modestly elevated resolution phase RelA responses in iκbα-/- or nfkb2-/- as compared wild type MEFs. In crosstalk settings, however, the model simulated an inverse relationship, where, the lack of ikba expression strengthened and nfkb2 abrogated crosstalk. Consistently, compensatory negative feedback mediated by IκBδ/p100, which restricted the resolution phase LPS response in iκbα-/- MEFs, was entirely ablated by non-canonical signals in the co-treatment regime to elicit an unabated RelA activity in IκBα-deficient cells. Conversely, compensating IκBα not only withered LPS induced late activity in nfkb2-/- MEFs but also prevented synergistic RelA activation during co-stimulation due to its insensitivity to NIK-IKK1 signals (data not shown).
Figure 3: A graphical depiction of the proposed crosstalk motif that is composed of two negative feedback loops, one of which is converted into a positive feed-forward loop through crosstalk to generate synergy at the level of RelA/NF-κB activation.
d) A crosstalk motif In sum, we have elucidated a novel crosstalk motif hardwired within the NF-κB system that discriminates between solitary TLR4 engagement and concomitant cell activation through TLR4 and LTβR (Figure 3). Negative feedback loops mediated by IκBδ/p100 and IκBα coordinately terminate canonical TLR4 response. But the gene products encoded by nfkb2 function pro-synergistically during crosstalk; inhibitory IκBδ by undergoing degradation and monomer p100 by initiating RelA:p52 mediated positive feed-forward loop amplifies RelA/NF-κB response in the co-treatment regime in comparison to cell activation by LPS. Indeed, the feedback mediated by nfkb2 could be either negative or positive, as it encodes chimeric p100, which is capable of acting as an inhibitor or as a precursor of NF-κB function. In contrast, IκBα negatively controlled crosstalk by inversely regulating the effective feedback strength of IκBδ/p100 ανδ by sequestering RelA:p52 dimers generated de novo upon co-stimulation.
79 C. Exploring cross-regulations of TLR response through PTMs
Our previous analysis suggested similar abrogation of RelA mediated gene-activation in both
phospho-mimicking RelAT54D and phospho-null RelAT54A mutants. One could argue that aspartate and alanine might not be bone fide phospho-defective substitutions for threonine. To address this question, now we have generated RelAT54E RelAT54V mutants those represent possible phospho-mimicking and phospho-null RelA mutants, respectively. To address the potential functional redundancies between serine and threonine at amino acid 54 of RelA, we
have further generated RelAT54S mutant.
Future plans
A. Exploring the signaling crosstalk between developmental LTβR and inflammatory TLRs: As we have identified the molecules underlying crosstalk control, we will use our insight to understand the mechanistic basis of duration threshold filter, which generates stimulus specificity in crosstalk regulations. Also, we will explore plausible cell-type specificity of crosstalk. Finally, we will develop murine infection model to understand physiological relevance of crosstalk in vivo. B. Exploring cross-regulations of TLR response through PTMs: In this project, we will
further test phospho-mimicking RelAT54D, phospho-null RelAT54A and RelAT54S mutants by reconstructing rela-/- MEFs. The gene activation potential of these mutants will be compared to that of wild type RelA in TLR4 signaling regime.
Action taken on the RAP/SAC 2013 recommendations
Both the projects described in (A) as well as (B) was discussed with the RAP-SAC members in April 2013. In general, the members lauded the duration threshold hypothesis as novel and elegant. It was advised that the laboratory focuses on project (A) and attempts to delineate the mechanism underlying duration control. To this end, we have made significant advancements and the laboratory is currently testing the physiological relevance of the proposed mechanism in in vivo infection model. Subscribing to the RAP-SAC panel recommendations, lesser efforts were given on project (B) in the last one-year to focus on project (A).
80 Biology of Japanese encephalitis virus
Principal Investigator Sudhanshu Vrati Ph.D. Students Jayita Thakur Richa Jalodia
Theme of research Japanese Encephalitis virus (JEV) is a member of the Flaviviridae family of animal viruses that contains several other medically important viruses such as Dengue and Yellow fever. JEV is a major cause of human encephalitis and is responsible for considerable mortality and morbidity in India. Frequent epidemics of Japanese encephalitis (JE) are being reported from various parts of India and JEV has become endemic in several parts of the country. We are studying the biology of JEV replication with a view to develop novel anti-virals.
Objectives
JEV genome is a plus-sense single-stranded RNA of ~11 kilo bases. A minus-sense RNA template is generated during virus replication, which is then copied to produce a large number of plus-sense genomic RNA molecules. Based on the amino acid sequence homologies with other replicases, NS3 and NS5 viral non-structural proteins have been speculated to be involved in replication of the JEV genome. However, we do not know if any of the cellular proteins also are needed for viral replication. We are, therefore, studying cellular proteins that interact with JEV genome sequences, which are likely to be involved in viral replication. Besides, viral non-structural proteins may interact with viral genome or host proteins during replicaion. We wish to study these interactions.
Work reported in 2012-2013 Replication of JEV requires several host proteins and previously we identified two such proteins as Mov34 and La. These were identified based on their specific binding to the non- coding regions (NCRs) of JEV RNA which are thought to be involved in genome replication. We used electrophoretic mobility shift assay (EMSA), UV-cross linking, North-western analysis and super shift assay to demonstrate that the 55 kDa PTB interacted with both 5’- NCR and 3’-stem loop of 3’-NCR of the viral genomic RNA, albeit with different affinities. The site of proten-RNA interaction was further delineated by RNA toe-printing assay. Protein over-expression and knock-down studies showed that PTB regulated JEV replication in a negative manner.
Progress of work during the current reporting year (2013-2014) We have studied interaction of JEV non-structural proteins with host proteins. The proteins under study are: NS5 which has the RNA-dependent RNA polymerase activity, and NS2A whose function is yet unknown. Yeast-two-hybrid system was used to fish out potential interacting partners with these two proteins.
81
The Yeast-2-hybrid system idnerified Rbm12b, Aldolase A, Rogdi, Itm2C and Cccd91 as potential interacting partners of NS5 protein. Interaction of these preotins with NS5 was further verified using co-immunoprecipitation assays. These assays indicated that Aldolase A and Rbm12b proteins interacted with NS5 in cultured cells infected with JEV. This was further explored by studying co-localization of these two proteins within the virus-infected cells using confocal microscopy. While no co-localization was observed for NS5 and Alodase A during the early phase of infection (4-16 h post infection), these two proteins co-localized in the cytoplasm at the later phase of infection (24-36 h post infection). No significant interaction with NS5 was observed in cells transfected with Myc-Rbm12b followed by infection with JEV. Thus Aldolase A appears to interact with NS5 in a time-dependent manner during the later phase of JEV replication. A number of putative interacting partners for NS2A protein have been identified. These are under various stages of validation using pull down assys and confocal microscopy.
Future plans We will like to further characterize the NS5 interactions with Aldolase A with respect to the amino acid residues involved. We will also investigate the role of NS5 interaction with Aldolase A in virus replication by protein over-expression or knock-down experiments.
Action taken on the RAP/SAC 2013 recommendations No specific recommendations were made.
Publications Original peer-reviewed articles
1. Bhattacharya S, Sen U, Vrati S* (2013) The RIDD pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication. J Gen Virol 95: 71-79.
2. Appaiahgari MB, Glass R, Singh S, Taneja S, Rongsen-Chandola T, Bhandari N, Mishra S, Vrati S* (2014) Transplacental rotavirus IgG interferes with immune response to live oral rotavirus vaccine ORV-116E in Indian infants. Vaccine 32: 651-656.
Review
1. Bharati K, Vrati S* (2013) DNA vaccines: Getting closer to becoming a reality. Indian J Med Res 137 : 1027-1028.
*Corresponding author
82 Biology of T lymphocytes
Principal Investigator Vineeta Bal
Ph.D. Students Arundhoti Das Neelam Oswal Nidhi Jain Parna Kanodia Sanket Rane
Collaborators Anna George, NII, New Delhi Arvind Bagga & his team, AIIMS, New Delhi B. Ravindran, ILS, Bhubaneswar Guruprasad Medigeshi, THSTI, Gurgaon Jeannine M. Durdik, University of Arkansas, USA Nitya Wadhwa, THSTI, Gurgaon Satyajit Rath, NII, Delhi Shailaja Sopory, THSTI, Gurgaon Shinjini Bhatnagar, THSTI, Gurgaon Sudhanshu Vrati, NII/THSTI, Gurgaon UCM Natchu, THSTI, Gurgaon
Theme of research
Two major areas are under investigation. Firstly, role of infection and inflammation in immune response and secondly studies in T cell fate decisions. While in the first part renal dysfunction and proteinuria as well as role of T cells in Japanese encephalitis infection is studied, in the second part CD4 T cell aging, survival, proliferation and differentiation are analysed in different systems.
Objectives
1. To study mechanisms associated with renal dysfunction and proteinuria. 2. To study the role of T cells in Japanese encephalitis infection in mouse model. 3. To characterise the effects of in vivo aging on CD4 T cell function.
Work reported in 2012-2013
To understand the regulatory mechanisms involved in cell intrinsic differentiation of CD4 cells in Th1 and Th2
Using 2 mouse strains with identical MHC but different background genes, Balb.b and C57Bl6 (B6), we reported that sorted naïve CD4 T cells (NCD4) activated in vitro produced both IFNγ and IL-4/-13 from Balb.b cells whereas only IFNγ from B6 cells. This was in contrast to activated CD4 cells sorted ex vivo where cells from both strains produced IFNγ and IL-4/IL-13. These data indicated differences in the intrinsic potential of NCD4 cells from 2 strains to differentiate when stimulated in non-polarised fashion. Interestingly, we observed
83 that a large proportion of NCD4 cells activated in vitro from either of the strains showed simultaneous presence of T-bet and Gata-3 transcription factors reported to be exclusively expressed in polarised Th1 and Th2 cells respectively. Thus our data indicated plasticity of expression of transcription factors. However, when the same cells were analysed for their ability to secrete cytokines, hardly any cells were capable of secreting both IFNγ and IL-13 simultaneously. These data indicate complexities in the regulation of transcription factors and cytokines when NCD4 cells are activated in non-polarising conditions as commonly happens in vivo.
To characterise the effects of in vivo aging on CD4 T cell function and phenotypic features.
In our effort to study cellular ageing in NCD4 cells, we showed that tonic signals provided by the presence of MHC II to naïve CD4 T cells in vivo are critical. Using MHC-II-deficient mice and wild type (WT) MHC-II sufficient mice we reported that transfer of congeneic naïve CD4 T cells in WT mice results in lowering of CD4 levels, decrease in the ability to respond to TCR mediated signal and accumulation of ROS in donor cells as compared to those transferred in MHC-II-deficient mice. A less dramatic but similar pattern as observed in MHC-II-deficient mice is also observed in invariant chain-deficient mice.
To study mechanisms associated with renal dysfunction and proteinuria.
Last year using lipopolysaccharide (LPS) induced proteinuria in mice as a model for minimal change nephrotic syndrome commonly observed in children, we reported that expression of CD80 on podocytes, and not bone marrow (BM) derived cells, is necessary for the induction of proteinuria in response to LPS.
Progress of work during the current reporting year (2013-2014) To study mechanisms associated with renal dysfunction and proteinuria In continuation of the work in mouse model of minimal change nephrotic syndrome, we checked whether ligation of other TLRs would lead to proteinuria. B6 mice treated with Pam3CysK4 and poly(I:C), TLR2 and TLR3 ligands respectively, resulted in proteinuria which was milder than that observed with LPS, however this underscored the role of TLR family in the pathology. For further characterisation, we used C3H/HeJ mice which have a mutation in TLR4 making it non-functional with C3H/OuJ as the WT counterpart. LPS treatment of C3H/HeJ mice did not enhance proteinuria, whereas WT mice showed enhancement. However, C3H/HeJ mice did respond to TLR2 and TLR3 ligands with proteinuria. In order to elucidate the role of MyD88 in downstream signaling leading to proteinuria we used MyD88-null mice. TLR3 mediated signals are known to be independent of MyD88 and hence we compared TLR3 and TLR4 ligands for their ability to induce proteinuria. While WT B6 mice showed proteinuria in response to both poly(I:C) and LPS, MyD88-null mice showed proteinuria only in response to poly(I:C). These data suggest that though LPS is known to signal in MyD-dependent and independent pathway, for inducing proteinuria the inflammatory activation caused by LPS via MyD-dependent pathway is critical. Podocytes express mRNA for many TLRs. How robust is their expression at protein level is not known. But in order to evaluate whether podocyte TLRs are critical in regulating proteinuria or not we made BM chimeras following lethal irradiation and reconstitution. Thus, B6 and MyD88-null mice were lethally irradiated and BM cells from B6 or MyD88-null mice were transferred for reconstitution making 4 way chimeras (B6 to MyD88-null, MyD88-null to MyD88-null, MyD88-null to B6 and B6 to B6). Mice were challenged with LPS ~10-12 84 weeks post irradiation. The data showed that presence of TLRs on BM derived cells, not on podocytes, is necessary for mediation of proteinuria. These data, along with findings on CD80-null mice reported last year, clearly indicate that ligation of TLRs on BM derived cells by pro-inflammatory ligands leads to proteinuria for which expression of CD80 on podocytes is necessary. In the past few years it has been shown that different TLR ligands can induce M1 (pro- inflammatory) or M2 (anti-inflammatory) differentiation and cytokine secretion of macrophages. While LPS induces M1 activation chitohexaose (Chx) via TLR4 is reported to induce M2 activation of macrophages. We tested the effect of Chx on LPS induce proteinuria. Pretreatment or parallel treatment of mice with LPS and Chx results in decrease in proteinuria as compared to LPS alone. Chx treatment does not induce proteinuria. This can be attributed to direct competition between LPS and Chx because both of them work via TLR4. However Chx given along with poly(I:C) also inhibits poly(I:C) induced proteinuria in B6 mice suggesting that Chx induced anti-inflammatory activation of BM-derived cells may be dominant and might help in prevention of proteinuria triggered by pro-inflammatory activation of TLR ligands. Along with presence of albumin in the urine we measured creatinine and CD80 in different experiments. While levels of creatinine do not change with pro-inflammatory stimuli like LPS, CD80 excretion in urine goes up significantly. Treatment with Chx completely abrogates CD80 excretion in urine, suggesting that inflammatory condition induced by presence of LPS or poly(I:C) may trigger upregulation of CD80 on podocytes followed by its urinary excretion. Ability of Chx to prevent proteinuria and CD80 excretion seems to offer a potential therapeutic tool to prevent and/or control proteinuria. Preliminary results done in collaborator’s lab on podocyte cell lines suggest TNF as a potential candidate responsible for the induction of CD80 on podocytes. To study the role of T cells in Japanese encephalitis infection in mouse model Japanese encephalitis causes acute infection in humans with both B cell and T cells contributing to the immune response. However, a good model to study the course of infection and the role of T cells in adult mice is not available. We showed that adult T cell receptor (TCR) β chain deficient mice which do not have CD4 or CD8 T cells bearing α/β TCR can be infected i.v. and succumb to infection over 16-20 day period. In addition to mortality we also scored weight loss and clinical score in these mice. About 80% of JE infected TCRβ-null mice die in ~18 days whereas <20% of B6 mice die, and the surviving B6 mice gain weight over this period. Viral load in JE infected TCRβ-null mice is many logs higher than in WT mice by day 7. Higher levels of TNFα and IL-1β mRNA from day 7 onwards are detectable by real time PCR in these mice along with a breech in the blood brain barrier by day 12 post-infection. These data suggest that possibly absence of T cells leads to higher levels of the virus in the brain, pro-inflammatory response and breech in the blood brain barrier. We further tried to identify cell types which may provide protection from morbidity and mortality in TCR -null mice. Adoptive transfer of ~5x106 naïve T (CD4+CD8) cells prior to infection provided partial protection with mortality decreasing to around 40-50% instead of 80%. This provided scope to compare mortality with different kinds of cell transfer. We used T cells from MHC-II-null or TAP-null mice which primarily provided CD8 and CD4 cells respectively. TCRβ-null mice receiving T cells from MHC-II-null showed protection comparable to total T cell transfer whereas transfer of T cells from TAP-null mice provided practically no protection. Thus survival of mice receiving T cells from TAP-null mice was as poor as TCRβ-null mice themselves. These data clearly indicated that CD4 T cells from TAP-
85 null mice did not provide any protection whereas CD8 T cells from MHC-II-null mice did, underscoring a major role for CD8 T cells in protection from JE infection. Beige mice have LYST mutation resulting in a defect in granular exocytosis amongst other defects. Because of this defect both NK and CD8 T cells in beige mice show poor functionality. When T cells from beige mice were transferred in TCRβ-null mice, on JE infection recipient mice died earlier and also showed >90% mortality further confirming a significant role for functional CD8 T cells in providing protection. To characterise the effects of in vivo aging on CD4 T cell function and phenotypic features. We have been working on this aspect for a long time and some of the recent work is to try and understand potential molecular components responsible for in vivo aging. Recently published work showed that levels of micro-RNA181a in naïve T cells from elderly humans are lower than those from young individuals because of malfunctioning of dual specificity phosphatase (DUSP)-6. We looked at miRNA181a levels by real time PCR in naïve cells from young and aged mice and found that naïve CD4 (NCD4) T cells from aged (ANCD4) mice have indeed lower levels. The same was tested in NCD4 T cells sorted for highest (NCD4hi) and lowest (NCD4lo) CD4 levels from young mice. NCD4lo cells in this case also resemble ANCD4 cells, similar to many other parameteres which have been reported in the past. We also showed that inhibition of suppressive activity of DUSP6 by a pharmacological agent BCI [(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one] could bring about better proliferation in NCD4lo and ANCD4 cells, at least partially reversing aging effect.
Future plans 1. Following are the best possible directions of research on various themes as perceived today. Depending upon the progress made and results obtained they may change over a period of time. Using histopathology and fluorescence imaging of kidney, independent confirmation of findings from proteinuria work reported above will be obtained. 2. Histopathology, fluorescence imaging and possibly electron microscopy will be used for kinetics of cellular localisation in JE infected brain. 3. Effect of in vivo aging of NCD4 T cells in their differentiation will be studied, in addition to looking at the role of background genes in Th1/Th2 differentiation. 4. While some work done on follow up of nephritic syndrome patients to understand the role of Th1 and Th2 differentiated cells in vivo and their contribution to pathology is going on, it is expected that the work will be completed in the coming year.
Action taken on the RAP/SAC 2013 recommendations There were no specific suggestions conveyed to change the proposed course of investigations, though a lot of discussion had taken place.
Publications Original peer-reviewed articles
1. Sinha A, Gulati A, Saini S, Blanc C, Gupta A, Gurjar BS, Saini H, Kotresh ST, Ali U, Bhatia D, Ohri A, Kumar M, Agarwal I, Gulati S, Anand K, Vijaykumar M, Sinha R, Sethi S, Saloma M, George A, Bal V, Singh G, Dinda AK, Hari P, Rath S, Dragon-Durey M-A, Bagga 86 A*. (2013) Prompt plasma exchanges and immunosuppressive treatment improves the outcomes of anti-factor H autoantibody-associated hemolytic uremic syndrome in children. Kidney Int (doi: 10.1038/ki.2013.373). 2. Saini S, Shenoy G, Rath S*, Bal V*, George A* (2014) Inducible nitric oxide synthase is a major intermediate in signaling pathways for the survival of plasma cells. Nat Immunol 15: 275-282. 3. Upadhyay M, Priya GK, Ramesh P, Madhavi MB, Rath S, Bal V, George A and Vaidya T*. (2014) CD40 signaling drives B lymphocytes into an intermediate memory-like state, poised between naïve and plasma cells. J Cell Physiol (doi: 10.1002/jcp.24572). Reviews / proceedings 1. Salam N*, Rane S, Das R, Faulkner M, Gund R, Kandpal U, Lewis V, Mattoo H, Prabhu S, Ranganathan V, Durdik J, George A, Rath S, Bal V* (2013) T cell ageing: Effects of age on development, survival & function. Indian J Med Res 138: 595-608. 2. Prabhu SB*, Khalsa JK, Banerjee H, Das A, Srivastava S, Mattoo HR, Thyagarajan K, Tanwar S, Das DS, Majumdar SS, George A, Bal A, Durdik JM, Rath S* (2013) Role of apoptosis-inducing factor (Aif) in the T cell lineage. Indian J Med Res 138: 577-590.
*Corresponding author(s)
87 Cellular and molecular biology of human cancer
Principal Investigator Anil Suri
Staff Scientist III Nirmala Jagadish
Ph.D. Students Namita Gupta Swarnendra Singh Vikash Kumar
Research Fellows Sumit Agarwal Deepak Parashar Deepak Verma Rahul Dwivedi Nainee Goyal Amrita Arora
Collaborators Rajive Kumar, Dr. B.R.A. Institute of Rotary Cancer Hospital, AIIMS, New Delhi P.K. Julka, Dr. BRAIRCH, AIIMS, New Delhi G.K. Rath, Dr. BRAIRCH, AIIMS, New Delhi D. Pandey, Dr. BRAIRCH, AIIMS, New Delhi Amlesh Seth, AIIMS, New Delhi Alok Thakar, AIIMS, New Delhi Govind Makharia, AIIMS, New Delhi Vaishali Suri, AIIMS, New Delhi Ashish Suri, AIIMS, New Delhi Arvind Chaturvedi, AIIMS, New Delhi Anju Gupta, VIMHANS Hospital, New Delhi Amar Bhatnagar, Safdarjung Hospital and Vardhman Mahavir Medical College, New Delhi Aruna Batra, Safdarjung Hospital and Vardhman Mahavir Medical College, New Delhi T. C. Sadasukhi, Mahatma Gandhi Medical College and Hospital, Jaipur P. N. K. Lohiya, Centre for Advanced Studies, University of Rajasthan, Jaipur T. Rajkumar, Cancer Institute (WIA), Chennai
Theme of research
Over the last three decades, knowledge on the molecular biology of human cancers has vastly expanded. A host of genes and proteins involved in cancer development and progression have been identified and many mechanisms at the molecular, cellular and even tissue level have been, at least partly, elucidated. In fact, cancer research has now reached a critical stage, in which the accumulated knowledge on molecular mechanisms needs to be translated into improved prevention, diagnosis, and treatment. Understanding the mechanisms involved in tumorigenesis has wide ranging implications for targeting the treatment of cancer. Tumor specific antigens (TSA) represent a unique
88 class of tumor antigens, which are expressed in a variety of cancerous tissues and are silent in normal tissues. Cancer testis (CT) antigens represent a unique class of tumor antigens under this category, which are expressed in a variety of cancerous tissues and are silent in normal tissues, except for the testis. A characteristic commonly shared by cancer testis antigens is, aside from the highly tissue‐restricted expression profile, their likely correlation with tumor progression and immunogenicity in cancer patients. Also the differential expression of germ cell specific genes in various cancer tissues reveals the important link between the two complementary disciplines of cell survival i.e. developmental and cancer biology.
Objectives
Numerous candidate cancer associated genes have been identified to date. However, for the vast majority of these genes, neither the expression pattern of the protein product, nor its localization and function in the tumor tissues has been investigated. The identification of specific genetic markers that are associated with tumor progression and aggressiveness may prove to be useful to assess the progression of disease. We are focusing on tumor associated proteins for the assessment of disease risk, early detection of disease, therapeutic prognosis and response to treatment as well as disease recurrence. The application of such gene products (biomarkers) to cancer will lead the way because of the unique association of genomic changes in cancer cells with the disease process. Most importantly, cancer biomarkers for prognostic, prediction and pharmacodynamics may aid in the rational development of anti-cancer drugs. In addition, our goal is to delineate in greater detail the gene-expression pathways involved in cellular growth, cell migration, and invasion for the treatment of cancer.
Work reported in 2012-2013
Breast cancer is the second leading cause of cancer related deaths in women worldwide. Reports about the early diagnosis of breast cancer are suggestive of an improved clinical outcome and overall survival rate in cancer patients. Therefore, cancer screening biomarker for early detection and diagnosis is urgently required for timely treatment and better cancer management. In this context, we investigated an association of cancer testis antigen, A-Kinase anchor protein 4 (AKAP4) with breast carcinoma.
AKAP4 gene and protein expression was investigated in breast cancer patient’s tissue specimens by employing in situ RNA hybridization and IHC. Our results revealed that AKAP4 gene expression was detected in 85% (77/91) of breast cancer patients. Among the various histotypes, AKAP4 gene expression was detected in 100 % DCIS (4/4), 83% IDC (69/83) and 100 ILC (4/4) specimens. Validation of AKAP4 protein expression was carried on serial sections of breast tissue specimens used for in situ RNA hybridization by employing immunohistochemistry (IHC). Our results distinctly revealed cytoplasmic localization of AKAP4 in 85% (77/91) of breast cancer patients.
To explore the role of AKAP4 in tumorigenesis, AKAP4 gene expression in human normal mammary epithelial cells and breast cancer cells of two different origins namely adenocarcinoma (MCF7, MDA-MB-231 and SK-BR3), and ductal carcinoma (BT474) was investigated by RT-PCR. Our results revealed AKAP4 gene expression in all four breast cancer cell lines (MCF7, MDA-MB-231, SK-BR3 and BT474), but not in normal mammary epithelial cells. Subsequently, we also investigated the AKAP4 surface expression in live 89 breast cancer cells by flow cytometry which revealed a distinct surface localization of AKAP4 protein suggesting that it may be a potential target candidate for therapeutic use in breast cancer patients.
Progress of work during the current reporting year (2013-2014) Breast cancer is the most common cause of cancer-related deaths among women worldwide, with the highest mortality incidence in developing countries. Breast cancer is a complex disease which has different histotypes and molecular subtypes based on molecular profiling with different prognostic and therapeutic implications. Recent studies have documented that breast cancer disease is a resultant of accumulation of genomic and epigenomic alterations resulting in reduced apoptosis, unchecked proliferation, increased motility and invasion abilities and metastasis in various other distant sites. In this regard, understanding the underlying mechanisms involved in such process would eventually reveal the novel target molecules involved in the disease progression and may help in cancer treatment. Recently, we reported an association of sperm-associated antigen 9 (SPAG9) expression, a new member of CT antigen family, in early grades of breast cancer patients. Collectively, our data suggested that SPAG9 could be playing a potential role in various malignant properties of breast tumorigenesis.
Metastasis is a complex process involving multiple steps including epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET) resulting in migration, invasion, colony forming abilities and subsequently tumor growth at distant sites. In this context, it is important to investigate gene and gene products involved in early spread, tumor progression and metastasis. In clinical practice, breast cancer treatment modalities are based on the specific proteins that are expressed in cancerous tissue specimen. Majority of the breast cancer patients express proteins such as estrogen receptor (ER) and progesterone receptor (PR) for which targeted hormone therapy is available with better clinical outcome. In addition, around 15-20% patients express human epidermal growth factor receptor 2 (HER2) protein, for which effective trastuzumab therapy is available with good prognosis. In contrast, around 15% of diagnosed breast cancers are designated as triple-negative and are characterized as ER negative (ER-), PR negative (PR-) and HER2 negative (HER2-). Triple-negative breast cancers patients represent an important clinical challenge because these patients do not respond to endocrine therapy or any other available targeted agents. Therefore, it is necessary to investigate and characterize target molecules in triple-negative breast cancers for better cancer management. In the present study, we investigated the SPAG9 expression in four breast cancer cell lines of various subtypes, harbouring different hormone receptors, such as MCF-7 (luminal-A, ER+PR+Her2-), BT-474 (luminal-B, ER+ PR+ Her2+), SK-BR-3 (HER2 overexpressing, ER-PR-Her2+) and MDA-MB-231 (highly metastatic basal, triple-negative ER-PR-Her2-).
Our RT-PCR and Western blotting analysis revealed that SPAG9 expression was found in all breast cancer cell line models used in the present study [MCF-7 (ER+/PR+/Her2- luminal-A subtype), SKBR- 3 (ER-/PR-/Her2+ ERBB2 associated subtype), BT- 474 (ER+/PR+/Her2+ triple-positive luminal-B subtype) and MDA-MB-231 (ER-/PR-/Her2- triple-negative basal subtype)]. In addition all four cancer cells revealed surface localization by flow cytometry analysis (Figure 1). Further, involvement of SPAG9 was investigated for in vitro and in vivo on various malignant properties [cellular proliferation, colony forming ability, migration and invasion] in triple-negative MDA-MB-231 cells, employing plasmid-based small interfering RNA (siRNA) approach.
90
Figure 1: SPAG9 expression in various breast cancer cell lines
Gene silencing of SPAG9 inhibits cellular proliferation and colony forming ability of MDA-MB-231 cells
Small interfering RNA mediated gene silencing approach was used to selectively knock down SPAG9 to study its role in cellular proliferation and colony forming ability. Highly aggressive triple-negative basal subtype MDA-MB-231 cells were used for in vitro gene silencing studies. SPAG9 siRNA construct transfected in MDA-MB-231 cells revealed ablation of SPAG9 protein as compared to control siRNA transfected cells as detected in Western blot analysis. However, residual SPAG9 protein expression was also detected in SPAG9 siRNA transfected cells. Subsequently, MDA-MB-231 cells transfected with SPAG9 siRNA revealed significant reduction in cellular growth (p< 0.01) as compared to control siRNA transfected cells. Cell growth was reduced by 32% post 72 h of treatment. Interestingly, colony forming ability was also significantly reduced by 56% (p< 0.001) for various cell numbers seeded for MDA-MB-231 cells transfected with SPAG9 siRNA but not in cells transfected with control siRNA. These results indicated that siRNA based knockdown of SPAG9 resulted in significant reduction in cellular growth and colony forming ability of triple-negative MDA-MB-231 cells
(Figure 2).
91
Figure 2: Effect of SPAG9 gene silencing on various properties of malignant cells
Knockdown of SPAG9 inhibits migration and invasion abilities ofMDA-MB-231 cells
SPAG9 association with migratory and invasive abilities of MDA-MB-231 cells was further investigated. Our results showed a significant inhibition of 52.5% in migrating ability of MDA-MB-231 cells transfected with SPAG9 siRNA(p< 0.005) as compared to control siRNA. Invasive ability of MDA-MB-231 cells was investigated using a reconstituted basement membrane barrier (Matrigel). Our results revealed a significant reduction of invasive ability (62.5%;p< 0.005) with SPAG9 siRNA as compared to control siRNA. Our gene silencing studies collectively suggests that SPAG9 may be involved in migration and invasion of MDA-MB-231 cells.
Gene silencing of SPAG9 significantly reduces cellular motility
The important feature of metastasis process is the spread of tumor cells from the primary site to distant organs by cellular motility process. In order to investigate the role of SPAG9 in cellular motility, an in vitro wound healing assay was carried out. The motility of MDA-MB- 231 cells was found to be significantly retarded when transfected with SPAG9 siRNA as compared to cells transfected with control siRNA. Our results revealed a closure of wound within 12 h in control siRNA transfected cells, while MDA-MB-231 cells transfected with SPAG9 siRNA failed to close the wound scratch even after 48 h. This data clearly indicated that SPAG9 is involved in cellular motility and early spread of breast cancer cells, suggesting that SPAG9 may
SPAG9 depletion reduced tumor growth in vivo
Our in vitro data indicated that ablation of SPAG9 expression by SPAG9 siRNA significantly reduced colony formation which led us to investigate its effect on human breast xenograft tumor growth in nude mice in vivo. To determine the effect of SPAG9 siRNA or control siRNA on tumor growth, mice were treated with control siRNA or 92 Figure 3: Effect of SPAG9 gene silencing on human breast cancer xenograft
SPAG9 siRNA and were observed for 42 days. A representative photograph shows reduced tumor growth in SPAG9 siRNA treated group compared with control siRNA treated group (Figure 3a). The tumor volume of mice injected with SPAG9 siRNA showed a significant reduction in tumor growth as compared to mice administered with control siRNA (Figure 3b; P< 0.001). Furthermore, in order to investigate whether the reduction of tumor growth is a result of ablation of SPAG9 expression, the xenograft tumors were excised and processed for immunohistochemical staining of SPAG9 protein expression. The SPAG9 protein was ablated in SPAG9 siRNA treated mice compared with mice treated with control siRNA (Figure 3c). Furthermore to investigate whether SPAG9 siRNA treated animals which showed reduced tumor growth was associated with reduced cellular proliferation, serial tumor sections were probed for PCNA expression. Our data revealed that there was significant reduction of PCNA expression (Figure 3d; 72%; P<0.0001) in tumors treated with SPAG9 siRNA treated compared with control siRNA. These results suggest that SPAG9 may be a molecular target for novel cancer treatment modalities.
In conclusion, to the best of our knowledge, this is the first report where we have put forth an evidence of potential role of SPAG9 in cellular growth, migration, invasion and colony forming ability in highly aggressive triple-negative MDA-MB-231 breast cancer cells. In addition, in vivo xenograft studies further strengthen the role of SPAG9 in breast cancer. Our study provides an association between SPAG9 expression and its potential role in breast cancer, and thus lays a foundation for developing a promising therapeutic target for triple- negative breast cancer. 93 Future plans
1. Novel cancer associated candidate discovery platform for cancer therapeutics approaches 2. Investigate the immunotherapeutic modalities for cancer treatment. 3. Study the effect of gene silencing and discovery of novel candidate targets to understand the underlying mechanisms and signaling pathways involved in various malignant properties of cancer cells.
Action taken on the recommendations of RAP/SAC
As suggested in the previous RAP/SAC, the progress has been achieved as reflected in the present year reporting.
Publications Original peerresearch articles
1. Kanojia D, Garg M, Saini S, Seth A, Bhatnagar A, Gupta A, Kumar R, Lohiya NK, Suri A* (2013) Expression of cancer testis antigen, SPAG9 in bladder transitional cell carcinoma is associated with disease progression and plays role in cellular proliferation, migration and invasion of cancer cells. PLoS ONE 8: e81348.
2. Sinha A, Agarwal S, Parashar D, Verma A, Sinha A, Jagadish N, Ansari AS, Lohiya NK, Suri A* (2013) Down regulation of SPAG9 reduces growth and invasive potential of triple- negative breast cancer cells: possible implications in targeted therapy. J Exp Clin Can Res 32: 69 1-11.
3. Saini S, Agarwal S, Sinha A, Verma A, Parashar D, Gupta N, Ansari AS, Lohiya NK, Jagadish N, Suri* A (2013) Gene silencing of A-kinase anchor protein 4 inhibits cervical cancer growth in vitro and in vivo. Can Gene The 20: 413-420
4. Agarwal S, Saini S, Parashar D, Verma A, Sinha A, Jagadish N, Batra A, Suri S, Gupta A, Ansari AS, Lohiya NK, Suri* A (2013) The novel cancer testis antigen A-kinase anchor protein 4 (AKAP4) is a potential target for immunotherapy of ovarian serous carcinoma. Oncoimmunol 2: e24270.
#5. Agarwal S, Saini S, Parashar D, Verma A, Jagadish N, Batra A, Suri S, Bhatnagar A, Gupta A, Ansari AS, Lohiya NK, A Suri* (2013) Expression and humoral response of a- kinase anchor protein 4 in cervical cancer. Int J Gynecol Can 23: 650-658.
Patent
1. Suri A (2013) siRNA useful in inhibiting cellular growth/proliferation of cancerous tissues. India Patent No.257434 granted on October 1, 2013.
*Corresponding author
# In press last year, since published
94
Study on expansion and plasticity of bone marrow stem cells
Principal Investigator Asok Mukhopadhyay
Research Associates Prakash Baligar Suender K. Sharawat (from July 2013)
PhD Students Amit Kumar (till July 2013) Abinaya Sundari T Veena Kochat Zaffar Iqubal
Research Fellows Sushmita Roy Neety Sahu (till August 2013) Saborni Chattopadhyay (till July 2013) Snehasis Mukherjee (from September 2013) Shyam Krishna M (from November 2013)
Collaborators Shiv K. Sarin, ILBS, New Delhi Nirupama Threhan Pati, ILBS, New Delhi Sanjeev Mathur, AIIMS, New Delhi Rashmi Mathur, AIIMS, New Delhi Subhas C. Kundu, IIT-KGP, Kharagpur Perumal Nagarajan, NII
Theme of research
Bone marrow (BM) niche controls self-renewal and differentiation of HSCs. To understand the regulation of hematopoiesis and the effect of aging on stem cells, it is necessary to decipher stem cell niche in different age groups of mice. A comprehensive knowledge on hematopoietic niche will help to mimic an ex vivo expansion culture in which stem-ness and engraftability of cells can be restored. It is now known that hematopoietic cells are involved in regeneration of many non-hematopoietic organs. Ex vivo cultured cells seem to facilitate transplantation for hematological reconstitution as well as treating other diseased organs. The overall themes of our research involve the study of stem cell niches, plasticity of BM-derived stem cells, and the role of BM-derived cells in progression of cancer.
Objectives We intend to dissect HSCs niche to elucidate its function in marrow regeneration, as well as their role in various pathological conditions of solid organs, and plasticity of adult stem cells. The objectives are as follows: 1. Molecular control of self-renewal and engraftability of HSCs in mice. 2. To understand liver regeneration by BM-derived cells and to elucidate the mechanism of hepatic differentiation. 3. Role of BM cells in stem-ness and cancer progression. 4. Mechanistic insight in the regeneration of neurons by MSC-derived precursor cells. 5. Study of molecular interplay during fibrosis and normal regeneration of tissue.
95 Work reported in 2012 -2013 A. Hematopoietic stem cells niche and marrow regeneration Preliminary results showed that HSCs undergo apoptosis in the absence of BM stromal cells- secreted Areg, a ligand of ErbB pathway, when compared to vector control cells. These results hypothesize the involvement of ErbB signaling pathway for conferring protection to HSCs from undergoing apoptosis. B. Plasticity of BM-derived cells
Last year we have shown that FL-derived MSCs can be differentiated into dopaminergic neurons as confirmed by genes and proteins expression. When these differentiated cells were tested electrophysiologically, only K+ current was observed. Further, we mentioned that differentiated neurons are transplanted in PD mouse to examine the therapeutic effect.
In transplantation of allogeneic BM cells, we reported that allo-antigen specific Treg cells suppress CD4+ T cells-mediated DTH response. This suppression improved trans- differentiation potential of uncommitted BM cells into hepatocytes, sinusoidal endothelial and Kupffer cells of liver for the synthesis of FVIII protein, thereby protected hemophilia A mice from death due to blood loss.
+ We also reported that CD45 BM cells are able to resolve CCl4-induced liver fibrosis in mice by secretion of MMPs and deactivation of activated hepatic stellate cells (HpSC). Besides these, our results showed that BM-derived cells involved in liver regeneration. Interesting, we observed that liver injury by CCl4 led to improvement of hematopoietic chimerism with respect to the donor cells, possibly through the activation of cell cycle.
C. Ovarian cancer
We confirmed in two independent cancer models (ovarian and lung carcinoma), the tumor cells got fused with hematopoietic cells, especially macrophages. This fusion led to expression of chemokine receptor (CXCR4), which confers a superior migratory property of the cells when compared to the crude tumor cells or lab grown culture.
D. Muscle regeneration and fibrosis We observed that in chronic muscle injury model there was reduction of satellite cell population at the early phase of regeneration due to over expression of cell cycle regulators and myogenic genes. In later time points, even though satellite cells were activated, normal regeneration process was largely inhibited.
Progress of work during the current reporting year (2013-14) A. Hematopoietic stem cells niche and marrow regeneration The functional role of Areg gene was studied by silencing its expression in stromal cell line using shRNA technology. Murine bone marrow HSCs were cultured in the presence of Areg+ and Areg-stromal cell lines to evaluate the functional role of this gene. The results showed that Areg-stromal cells did not support HSCs in culture rather cells underwent apoptotic death as compared to wild-type (Areg+) stroma-dependent culture. Within 2 days, 96 the number of apoptotic HSCs was found to be 6-fold higher in Areg- stroma-supported culture. The apoptosis of cells could not be reversed even in the presence of anti-apoptotic SCF. Interestingly, HSCs were partly protected from apoptosis by supplementing culture with the conditioned medium of wild-type stromal cells. Areg is the ligand of ErbB pathway, which mediates the activation of Stat5 and control synthesis of anti-apoptotic factors. We presumed that in Areg- stromal-dependent culture, due to reduced Areg synthesis, Stat5 did not activate, which led to apoptosis and less survival of HSCs. To prove that, we checked Stat5 phosphorylation status in HSCs in both the culture conditions (Areg+ and Areg- stroma- supported culture). As a control of ErbB pathway inhibition, HSCs were also cultured in wild- type stroma but in the presence of pimozide, a potent inhibitor of Stat5 phosphorylation. Immunocytochemical analyses confirmed that Stat5 was activated in almost 80% HSCs in case of wild-type stroma-dependent culture, whereas in Areg- stroma or in the presence of pimozide the activation was significantly suppressed. Altogether, these results implicate an active role of ErbB pathway for conferring protection to HSCs from apoptosis. Downstream gene expression analyses are now under progress.
B. Plasticity in BM cells
Transdifferentiation of MSC into dopaminergic (DA) neurons In past one year we have analyzed functional recovery of Parkinson’s diseased (PD) mice following transplantation of FL-MSCs derived DA neurons. Mice were examined on the basis of four criteria: (a) apomorphin-induced contra-lateral rotation, (b) motor neuron function by rotaroid test, (c) engraftment and differentiation of donor cells on striatum by IHC, and (d) dopamine secretion by the damaged striatum. The first two functional studies confirmed significant improvement in 80% of recipient mice in motor neuron activities. The recovered mice were sacrificed after 4 months of transplantation. Immunohistochemical analyses showed the presence of donor-derived neuronal cells in the damaged striatum. Further, we determined tissue level expression of DA by LC/MS in the damaged striatum where donor cells were transplanted. The results suggested about 2-fold increase of DA as compared to PD mice stratum, suggesting that additional DA was synthesized by donor-derived cells.
Therapeutic effect of allogeneic BM cells in hemophilia A mouse Earlier, we have shown that allo-antigen sensitized Treg cells (sTregs) prevented rejection of allogeneic bone marrow cells, improved hepatic differentiation, increased plasma factor FVIII level and protected mice from death due to excess blood loss. In this reporting year, we compared the regulatory potential of naïve Tregs (nTregs) and sTregs in terms of inhibition of + - hi CD4 CD25 T cell proliferation, expression of Foxp3 in CD25 fraction of Tregs, CTLA4 expression and IL-10 secretion. The results clearly suggested that allo-antigen sensitized Tregs are more potent for immune suppression than Tregs.
To investigate cellular fusion between donor and recipient cells, we analyzed the resultant BM-derived hepatic cells by quantitative PCR for sry gene and XY-FISH. As donors (female) and recipients (male) were sex mismatched, we studied the presence of X and Y chromosomes to check for fusion. We performed absolute quantification of Y chromosome marker Sry gene in purified recipient and donor-derived hepatocytes. Y-chr+ cells in GFP+ (donor) and GFP- cells were 1.85 ± 0.86% and 97.9 ± 15.9%, respectively. Further, inter-phase FISH analyses confirmed that fusion heterokaryons (XXXXXY and XXXY) in the bone marrow-derived hepatocytes were only 1.8% of the total neo hepatocytes. Further, we confirmed that these directly differentiated hepatocytes were lacking CD45 expression.
We then isolated GFP+ donor hepatocytes by flow sorting and performed time course gene expression analyses after 1 month and 5 months of transplantation. We found that the donor 97 cells gained the expression of liver enriched transcription factors (GATA4, GATA6, HNF3α, HNF3β, HNF1α, HNF1β, HNF4α, HNF6, OC-2, CEBPα and CEBPβ), liver specific proteins (TDO, Albumin. FVIII, CK18, E-cadherin and CYP1A2) and gradually lost hematopoietic stem cell gene (GATA2, CD45, Sca-1 and c-Kit) expression. Further analysis of the expression of various chromatin modifying enzymes (DNMT3A, DNMT3B, EZH2, SETDB1, MLL, p300) suggested significant (p < 0.05) changes in the donor-derived neo hepatocytes when compared to Lin- BMCs.
Lin- BM cell therapy in PiZ transgenic mice In past two years, we are working on development of a novel therapeutic strategy in mouse model expressing mutant human alpha1-antitrypsin (AAT). The hallmark of the AAT deficiency is formation of diastase resistant PAS-stained globules in hepatocytes. In order to examine any effect of BM stem cells on loading of these globules, a group of mice were transplanted with single dose of cells. The mice were sacrificed in three cohorts after 1, 3 and 6 months of transplantation. Interestingly, the loading of PAS-stained globules was significantly (p < 0.05 – 0.01) reduced in all three times of the transplantation, indicating overall improvement in the disease pathology. It has been found that the accumulation of misfolded AAT is associated with dysfunction of many cellular pathways. Glycogen storage and its metabolism is one such dysfunction in mouse model of AAT deficiency. The glycogen storage of wild type, PiZ and PiZ (transplantation) were compared after 6 months of transplantation. The results suggested a significant drop of storage glycogen level in PiZ transgenic mice liver as compared to wild type mice. The glycogen storage level was significantly (p < 0.05) improved after transplantation of BM-derived cells. To know whether the donor cells are engrafted in the liver, and if so, their lineage commitment, serial liver sections were analyzed by immunohistochemistry. The donor cells expressed albumin, thus confirming that hepatic cells were generated. As in AAT deficient mice chronic liver injury tends to develop fibrosis followed by cirrhosis, so the effect of transplantation on fibrosis was determined. The liver sections were stained with picrosirius red, which stains total collagen. Deposition of collagen, the hallmark of fibrosis, was determined in terms of collagen proportionate area (CPA). No collagen deposition was observed in wild-type mice, whereas in 6 months PiZ it was increased up to 6%, interestingly in transplanted mice collagen deposition was significantly declined.
C. Ovarian cancer
Following our earlier results of ascitic samples of mouse and human ovarian carcinoma, we extended the work and conducted phenotypic analysis of EpCAM-expressing human tumor cells. We divided samples into two groups: without chemo-therapy and post-chemotherapy with combination of carboplatin, paclitaxel and cisplatin treatment. Out of 12 samples analyzed, irrespective of the patients were treated or not, about 70% (range 52.9 – 94%) of EpCAM-expressing tumor cells also co-expressed pan hematopoietic marker CD45. Surprisingly matching with the mouse data, about 60% of EpCAM CD45+ cells also expressed CD14, indicating that these cells might have acquired properties of monocytes/macrophages. Further analyses of these cells suggest that this phenotype was quite stable in culture. We have tried to decipher whether this new phenotype had appeared after fusion with hematpoietic cells, as found in mouse model, by ploidy and inter-phase X-FISH. However, the results were inconclusive. In order to compare differential gene expression and functional gain of these hemato-epithelial cells with respect to EpCAM-expressing tumor cells, we sorted these two compartments in three samples for microarray analyses.
98 D. Fibrosis and molecular interplay
The results of the early phase of muscle regeneration suggest that satellite cells (SCs) were activated until day7 post injury; there after activation pattern was changed between day7 and day21, indicating imbalance regeneration. The trend of gene expression was corresponds to histopathological evidence, where there was a decrease of SCs in the early phase but increased during the later stage. The quiescent SCs were identified by staining muscle sections with Pax7 and P57, whereas myofibroblasts (MFs) by staining with αSMA and collagen. High transcription of TNF and reduced expression of IL1 in milieu of the fibrotic tissue together with significant expression of myostatin by day14 indicated hindrance of SCs proliferation by day21. To confirm satellite cells being pushed into differentiation, we did western blot to analyze the levels of MyoD with respect to nuclear localization of RelA (p65). This will be further confirmed by in vitro experiments that the inhibition of NF- B indeed brings about proliferation of SCs.
To establish the pattern of interactions between SCs and MFs, cells were cultured in conditioned media (CM) obtained from either. CM of satellite cells was used to grow MFs and vise versa. The pilot studies show shorter activation lag phase and better myotubes formation of the SCs when grown in MFs CM as compared to the control, which was grown in normal serum containing media.
Future plans 1. Epigenetic analysis of BM-derived hepatocytes isolated from recipient liver. 2. Gene expression and functional analyses of EpCAM+CD45+ and EpCAM+ cells of human ovarian tumor samples. 3. Deciphering cues in fibrotic muscle tissue impede natural muscle generation.
Action taken on the RAP/SAC 2013 recommendations Scientific and technical queries raised by the members were answered. No specific recommendation was made.
Publications Original peer-reviewed article
1. Ramakrishnan M, Mathur SR, Mukhopadhyay A* (2013) Fusion-Derived Epithelial Cancer Cells Express Hematopoietic Markers and Contribute to Stem Cell and Migratory Phenotype in Ovarian Carcinoma. Cancer Res 73: 5360-5370.
Reviews #1. Mukhopadhyay A* (2013) Perspective on liver regeneration by bone marrow-derived stem cells: A scientific realization or a paradox. Cytotherapy 15: 881-892 2. Kochat V, Baligar P, Maiwall R, Mukhopadhyay A* (2013) Bone marrow stem-cell therapy for genetic and chronic liver diseases. Hepatol Int (DOI 10.1007/s12072-013-9499-z).
99 Patent application 1. Baligar P, Teckman J, Mukhopadhyay A. Bone marrow-derived cells ameliorates the pathological consequences of the liver in case of alpha1-antitrypsin deficiency. UP Patent Application # 14/109212 (17/12/2013).
*Corresponding author #In press last year, since published
100 Cell death regulation
Principal Investigator Chandrima Shaha
Research Associate Rajeev Pandey
Ph. D Students Abhishek Aich Dipankar Ash Radhika Mathur Ashish Kumar Sagnik Giri Durgesh Manohar Pitale
Collaborator Karl Werbovetz, Ohio State University, Ohio
Theme of Research
The overall theme of the research program is to elucidate the processes that influence cell death programs under varying physiological conditions in diverse model systems.
Objectives
Regulatory networks driving cell fate decisions are important to investigate in the context of understanding diseases. Broadly, our research programme explores the underlying mechanisms of cell survival and death in diverse intracellular and extracellular conditions. The model systems used by us are a lower eukaryotic cell, the Leishmania parasite and the mammalian embryonal carcinoma cell as a model of a higher eukaryotic cell. Study of both the cellular models is expected to help us understand how complex eukaryotic regulatory systems evolved because some of the cellular pathways may be universal features of eukaryotic cells. Using different experimental approaches in these two models, we explore the precise mechanisms by which cells die, how these processes are regulated by diverse signaling pathways, inter-relationship between the various death processes and the evolutionary significance of cell death in a lower eukaryote.
Work reported in 2012 – 2013
A. Cell death in protozoan parasites
As mentioned above, one of our experimental model systems for cell death studies is a protozoan parasite that branched early during eukaryotic evolution surviving in disparate biological environments during its life cycle in an insect vector and in a mammalian host. It provides an interesting system for studying cellular pathways leading to death in contrasting environmental milieu. In the last reporting period, we described the mechanism of the transport of a defensive enzyme, the mitochondrial tryparedoxin peroxidase (mTXNPx) of Leishmania. Defensive enzymes are essential for survival of the parasite and we have shown the importance of the tryparedoxin peroxidases of the Leishmania on cell survival in the past. During this reporting period, we validated that a 30 amino-acid region at the N-terminus actually serves as a MTS in vivo through deletion studies. Also, we showed that in the 101 kinetoplastid parasites the signal is cleaved after translocation to the mitochondria. The MTS contained a CaM binding site generating a hypothesis that CaM may be essential for mitochondrial translocation. Mutation studies with the CaM binding site indicated that substitution of 5 amino acids in the CaM binding site did interfere with transport to the mitochondria while substitution of 2-3 amino acids resulted in translocation with decreased efficiency. In addition to the above, we reported sterol changes in Leishmania in response to ROS as a possible defense mechanism of the cell.
B. Mechanisms underlying cell death in cancer cells
The primary purpose of this programme is to understand how the balance between apoptosis and autophagy change cell fate decisions. Our earlier studies established that embryonal carcinoma (EC) cells are sensitive to cisplatin and undergo death by apoptosis at certain doses. We reported that in cells expressing the wild-type p53 (wtp53), apoptosis was preceded by increased autophagy after cisplatin exposure, however, this autophagy was not required for cell death because inhibition of autophagy was unable to stop apoptosis. Lowering of p53 levels resulted in resistance to apoptosis and increase in Beclin-1. The high Beclin-1 expression in p53 lowered cells correlated with the higher basal autophagy. In wtp53 cells, Beclin-1 was elevated with increase in cisplatin dose but in p53 downregulated conditions, cisplatin treatment did not significantly influence either Beclin-1 expression or autophagic vacuole formation. Xenografts in nude mice showed the growth of much larger tumors in 60 days with p53 downregulated cells as compared to cells with normal complement of p53. Presence of wortmannin, the autophagy inhibitor during cisplatin treatment induced significant reduction in tumor size.
Progress of work during the current reporting year (2013-2014)
A. Cell death in protozoan parasites
Defensive enzymes and cell death The Leishmania parasites, not being naturally endowed with any canonical peroxidases such as catalase or selenium type glutathione peroxidase rely on the trypanothione dependent tryparedoxin peroxidase system for defense against reactive oxygen species. Our earlier work showed various functional aspects of this enzyme in the parasite. In continuation to our work reported last year on the mitochondrial transport of this enzyme, we have further shown using reconstituted import assays, that biochemical inhibition of calmodulin (CaM), absence of CaM or Ca2+ prevents mitochondrial translocation of the mTXNPx both in vitro and in vivo. This supported the requirement of CaM activity for mitochondrial transport as shown by the earlier mutation studies. We also demonstrate the prevention of temperature driven mTXNPx aggregation in the presence of CaM. Based on the aggregation and unfolding assays it can be speculated that CaM is responsible for maintaining the translocation competence of mTXNPx from cytosol to the mitochondria in the Leishmania parasite. These findings further established the idea that CaM is required for the efficient transport of the protein to mitochondria.
Ageing in culture and crowding stress increases the levels of mTXNPx in the parasites suggesting that stress conditions induce the enzyme. To see if mTXNPx was absolutely necessary for survival, we tried to generate parasites with compromised mTXNPx levels through gene replacement by homologous recombination. Even haplo-insufficient parasites for mTXNPx gene did not survive, suggesting the essential nature of the enzyme. To further confirm the critical nature of the mTXNPx, studies are in progress with site directed 102 mutagenesis studies to see if inactivation of the mTXNPx enzyme alters development and survival of the parasite.
Sterol function in the Leishmania parasite Sterol composition of some Leishmania species are known, however, sterol composition of Leishmania donovani, the parasite causing Kala-azar is not known. We show that the major sterol in L. donovani is ergosterol which is similar to data shown in L. major. Our observations show that when anti-leishmanial drug antimony is given, the ergosterol level changes suggesting an effect of the drug on parasite sterols. The increase in ergosterol was preceded by an increase in ROS and when ROS was scavenged this increase was abrogated. Sterol biosynthesis inhibition induced parasite death suggesting the importance of the sterol increase in cell death. These observations were confirmed by enhanced susceptibility of sterol deficient parasites to ROS and drugs. Further investigations are being carried out on the role of the sterols in survival of these ancient eukaryotes.
B. Mechanisms underlying cell death in cancer
All our studies with the embryonal carcinoma cells are directed towards the understanding of mechanistic aspects of how the changes in balance between apoptosis and autophagy determine cell fate. In continuation to our studies reported last year on the outcome of changes in apoptosis and autophagy in embryonal carcinoma cells when chemotherapy was given, we now demonstrate a new observation that p53 interacts with Beclin-1, an event that plays an important role in the determination of cell fate. Complexes formed between molecules from apoptosis and autophagy pathways present potential targets for chemotherapeutics design, as disruption of such complexes could alter cell survival. We find that Beclin-1 levels are regulated by p53 through ubiquitination. Immunoprecipitates from cells expressing wild type p53 and cells where p53 levels were lowered by shRNA mediated interference, were probed with both anti-p53 and anti-Beclin-1 antibody. Beclin-1 could be immunoprecipitated by anti- p53 antibody and p53 could be immunoprecipitated using anti-Beclin-1 antibody. The ability of both antibodies to pull-down p53 and Beclin-1 together suggested an existing interaction between the two molecules. Further, the site of interaction was identified as the cytosol. Analysis of possible binding site of p53 to Beclin-1 with cells expressing flag-tagged domains of the Beclin-1 (Bcl-2-BD-flag, CCD-flag, and ECD-flag) subjected to FLAG-IP using anti- flag antibody showed reactivity in the region of 1-150 amino acid of Beclin-1. This region harboring the BH3 domain is essential for binding to other BH3 domain proteins. Therefore, the kinetics of binding of different proteins to this site is important for deciding how the cell will behave during changes in intracellular environments. Cells transfected with constructs expressing His tagged ubiquitin and Beclin-1 showed lesser ubiquitination of Beclin-1 in p53 downregulated cells as compared to wtp53 cells. The p53 downregulated cells concurrently transfected with GFP-p53 constructs to compensate for the low p53 and a plasmid containing a HA tagged ubiquitin protein (HA-Ub) showed higher Beclin-1 ubiquitination in p53 downregulated cells as compared to cells with vector only control. The experiments clearly showed that p53 levels are important for Beclin-1 ubiquitination. Since ubiquitination occurs at specific lysine residues, we tested if Beclin-1 ubiquitination was K48 linked because K48 linked ubiquitination is related to proteasome mediated degradation. Experiments showed that Beclin-1 was undergoing K-48 linked ubiquitination. The above observations support the hypothesis that p53 is necessary for Beclin-1 ubiquitination and in the absence of p53, Beclin- 1 accumulates. In the above studies, we demonstrate a complex interplay between two proteins of the apoptosis and autophagy pathway where apoptosis was preceded by autophagy but autophagy was not required for apoptotic death. Autophagy inhibition pushed the cells towards apoptosis that was measurable in reduced tumor sizes in vivo and increased apoptosis
103 of cells in vitro. Therefore, autophagy inhibition during drug treatment can potentially improve efficacy of the treatment.
The mTOR which integrates upstream signals and also senses cellular nutrient, oxygen, and energy levels consists of two complexes, the mTORC1 and C2. In our studies looking at the balance between the two complexes in determining cell fate, we find that both mTORC1 and C2 components change in response to drugs to define the routes of survival or death primarily involving apoptosis and autophagy. Detailed studies are being conducted to establish how mTORC1 and C2 operate in determining the cellular destiny in a changing intracellular environment due to drug exposure in embryonal carcinoma cells.
Future Plans
We plan to pursue our in depth studies on the functioning of mTXNPx with a view to analyze the binding sites on mTXNPx for its substrates to yield inhibition and check the outcome of such inhibitions. The findings in vitro will be expanded to in vivo to check if parasite inhibition can be achieved. Further studies on the effect of defective translocation of mTXNPx with the consequences of such events will be continued. Sterol and fatty acids in Leishmania survival will be further evaluated with a view to provide sufficient information on the role of these components in parasite function. Decoding the mechanistic aspects of cellular changes involving the death or survival pathways under chemotherapy is an area where lot of interest exists worldwide; therefore, we would like to continue looking at the related events in in vivo models.
Action taken on the RAP/SAC 2013 recommendations
Various suggestions were offered after in-depth discussions in the last RAP/SAC meetings and specific studies were carried out during the year keeping the suggestions in view.
Publications Original peer-reviewed articles
1. Aich A, Shaha C* (2013) Novel role of calmodulin in regulating protein transport to mitochondria in a unicellular eukaryote. Mol Cell Biol 33: 4579-4593.
Reviews/Proceedings/Etc.
1. Shaha C* (2013) Vesicle traffic wins the Nobel. Curr Sci 105: 1651-1652.
*Corresponding author
104 Cellular and molecular aspects of reproduction and viral infections
Principal Investigator Satish Kumar Gupta
Research Associates Nachiket Shembekar (since July 2013) Ananta Prasad Arukha (since July 2013) Pankaj Singh (since August 2013) Nutan (up to June 2013) Ashok Kumar Saini (up to August 2013)
Ph. D. Students Abhinav Shrestha Sudha Saryu Malhotra Ankita Malik Piyush Chaudhary Sonam Verma (since January 20
Research Fellows Vidisha Minhas Ajay Kesharwani (since Feb 2014) Neha Gupta (up to July 2013) Anita Goyala (up to July 2013) Sonali Singh (up to December 2013) Prachi Kumar (up to January 2014)
Collaborators Amulya K. Panda, NII Wan-Xiang Xu, Shanghai Institute of Planned Parenthood Research, P. R. China Swadesh Malhotra, NBRC, Lucknow Raghavan Varadarajan, IISc, Bangalore R.K. Singh, University of Allahabad, Allahabad Rajeev Dhere, Serum Institute, Pune Sujata Bhatt, Mumbai University, Mumbai Deepak N. Modi, NIRRH, Mumbai Pankaj Suman, Amity University, Noida, UP
Theme of research
One of the areas of scientific pursuit in our lab is to design and evaluate the contraceptive potential of the gamete (egg/spermatozoon) specific recombinant immunogens. To understand implantation biology during pregnancy, the regulatory mechanisms associated with cytokines, growth factors and hormones mediated trophoblast cell invasion and differentiation is being investigated. Other areas of studies include identification of compounds for anti-HIV-1 activity and generation & characterization of monoclonal antibodies against influenza/bird flu virus.
Objectives 1. To develop contraceptive vaccine 2. To understand the molecular mechanisms associated with invasion and differentiation of trophoblast or trophoblast derived cancer cells 105 3. To discover molecules with anti-HIV activity for their application as potential microbicide 4. To develop influenza/bird flu virus neutralizing monoclonal antibodies
Work reported in 2012-2013 A. Development of contraceptive vaccine
Towards our efforts in developing contraceptive vaccine, previous year we reported the contraceptive potential in female mice of E. coli-expressed recombinant protein encompassing promiscuous T cell epitope of tetanus toxoid (TT) and dog zona pellucida glycoprotein 3 (dZP3) separated by di-lysine linker (TT-KK-dZP3) and fusion protein encompassing promiscuous T cell epitope of TT followed by di-lysine linker, dog ZP3 (aa residues 165-346), tri-glycine spacer and sperm specific protein, Izumo (aa residues 165-250) (TT-KK-dZP3- GGG-dIz).
B. Invasion and differentiation of trophoblast cells
The role of Pappalysin 1 in Leukemia Inhibitory Factor (LIF) mediated invasion of HTR- 8/SVneo cells was presented. By using ‘BeWo’ cells wherein either α or β subunits of human chorionic gonadotropin (hCG) were knocked-down by shRNA, the relevance of hCG in forskolin mediated cell fusion was demonstrated.
C. Anti-HIV compounds
We reported anti-HIV activity and pre-clinical safety of the extracts prepared from Rhus pariflora. Further, anti-HIV-1 activity and inhibition of HIV-1 integrase by synthetic Labdane analogs was reported.
D. Bio-neutralizing monoclonal antibodies against influenza/bird flu virus
Humanization of murine monoclonal antibody (MAb) with potent neutralization of the pandemic H1N1 virus (A/California/07/2009) and generation of additional MAbs against pandemic and seasonal HA protein of H1N1 influenza virus was reported in the previous year.
Progress of work during the current reporting year (2013-14)
A. Development of contraceptive vaccine
Towards our efforts in developing contraceptive vaccine for street dogs, last year, upscaled production of tag-free recombinant TT-KK-dZP3 in fermentor was reported. In addition, contraceptive efficacy in female mice of a chimeric recombinant fusion protein comprising of dog ZP3 and spermatozoa-specific dog Izumo was reported. This year, immunogenicity and contraceptive efficacy of TT-KK-dZP3 was investigated by immunizing female mice with 25, 50 and 125 µg of recombinant protein supplemented with 200 µg alum/mouse/injection. Control group showed 100% pregnancy with 8.4 ± 0.76 pups/mated female. Animals immunized with 25 and 50 µg of recombinant TT-KK-dZP3 showed 70% curtailment in conception with mean antibody titres on day 56 being 58.4 ± 6.64 and 70.7 ± 8.35 AU X 103 and number of pups/mated female being 2.2 ± 1.27 and 1.6 ± 0.95, respectively. Group of mice immunized with 125 µg recombinant TT-KK-dZP3 showed mean antibody titre of 103.1 ± 8.30 AU X 103 and 90% curtailment in pregnancy with 0.6 ± 0.60 pups/mated female. Significant correlation in the antibody titres and contraceptive efficacy was observed in these 106 studies. Further, to reduce the number of injections required to achieve infertility, experiments were initiated with one and two injection schedule with various doses. The highest curtailment in conception (60%) was observed in group of mice administered two injections each of 125 µg recombinant TT-KK-dZP3. The experiments with recombinant TT-KK-dZP3 administered along with CpG DNA or incorporated in poly-lactide (PLA) microspheres are undergoing to enhance contraceptive efficacy with reduced number of injections.
Further, sperm specific proteins SP17 and Equatorin have been cloned in various combinations, expressed in E. coli and recombinant proteins purified by Ni-NTA affinity chromatography. Their immunogenicity and contraceptive efficacy have been evaluated in female mice. Following recombinant proteins were prepared: (i) TT-KK-Sp17(N-terminal)- GGG-Equatorin: encompassing T cell epitope of TT followed by dilysine linker, mouse Sp17 N-terminal (aa residues 1- 80), triglycine spacer and mouse Equatorin (aa residues 21-185); (ii) TT-KK-Sp17(C-terminal)-GGG-Equatorin: comprising of T cell epitope of TT followed by dilysine linker, mouse Sp17 C-terminal (aa residues 76- 126), tri-glycine spacer and mouse Equatorin; (iii) bRNase-KK-Equatorin: encompassing T cell epitope of bovine RNase (bRNase; aa residues 120- 131) followed by dilysine linker and mouse Equatorin; (iv) TT- KK-Sp17(N-terminal); (v) TT-KK-Sp17(C-terminal). Immunization of female FvB/J mice with the above recombinant proteins led to variable degree of contraceptive efficacy.
B. Molecular mechanisms associated with trophoblast invasion and differentiation
LIF-mediated trophoblastic JEG-3 cell invasion: cross-talk between STAT3 and ERK1/2 activation To investigate the relative importance of STAT3 and ERK1/2 activation during invasion, JEG-3 cells were treated with LIF. LIF treatment led to activation of STAT3 and ERK1/2 signaling pathways which was followed by changes in the expression of several invasion- associated molecules like mucin 1 (MUC1), Fos, Jun etc. Abrogation of either STAT3 or ERK1/2 signaling reduced (p<0.05) the LIF mediated invasion of JEG-3 cells. It was associated with a significant reduction in the expression of both MUC1 and Fos; suggesting a common denominator in LIF-STAT3/ERK1/2 signaling. To this effect, we observed a decrease in LIF mediated p-STAT3 ser727 upon blocking either STAT3 or ERK1/2 signaling. Thus, ERK1/2 as well as JAK-STAT mediated STAT3 ser727 phosphorylation play an important role in LIF mediated JEG-3 trophoblastic cell invasion and gene expression.
107
Schematic representation of the significance of STAT3 and ERK1/2 mediated signaling in gene expression and invasion of JEG-3 cells: Panel I shows the scheme of LIF mediated signaling when both STAT3 and ERK1/2 mediated signaling pathways are active. Panel II shows the scheme of LIF mediated signaling when STAT3 expression is silenced by siRNA and ERK1/2 signaling pathway works normal. Panel III shows the scheme of LIF mediated signaling when STAT3 tyr705 phosphorylation is normal but, STAT3 ser727 phosphorylation gets compromised due to inhibition of ERK1/2 activation.
Molecular mechanisms underlying the trophoblastic BeWo cell fusion During the year of reporting, we investigated, if hCG supplementation will restore cell fusion in α- or β-hCG knocked-down BeWo cells to the level as observed in normal BeWo cells. Supplementation of hCG led to a dose dependent increase in cell fusion, which was however, lower in both α- as well as β-hCG knock-down cells as compared to control cells. Thus, hCG could partially restore cell fusion in the α- and β-hCG knock-down cells. To delineate downstream signaling pathways associated with the decrease in cell fusion of α- or β-hCG knock-down BeWo cells, we observed a decreased activation of p-PKA, p-CREB and p-β Catenin in the knock-down cells upon treatment with hCG/forskolin as compared to normal BeWo cells. These results demonstrate conclusively the importance of hCG during syncytilization using BeWo cells as an experimental model.
108 C. Evaluation of anti-HIV activity and pre-clinical safety of medicinal plants and semi- synthetic compounds
Acacia catechu (Mimosa family) stem bark extracts have been used traditionally as dietary supplement as well as a folk medicine with its reported anti-inflammatory, immunomodulatory, hepatoprotective, antioxidant, anti-microbial and anti-tumor activities. The present study was undertaken to evaluate the anti-HIV-1 activity of the extracts from stem
bark of A. catechu. The aqueous and 50% ethanolic extracts of A. catechu showed IC50 values of 1.8 µg/ml and 3.6 µg/ml respectively, in cell-free virus based assay using TZM-bl cells and
HIV-1NL4.3 (X-4 tropic). Further fractionation yielded petroleum ether, chloroform and n- butanol soluble fractions, among which n-butanol fraction exhibited anti-HIV-1 activity with
an IC50 of 1.7 µg/ml, using the above mentioned assay. The n-butanol fraction showed a dose- dependent inhibition against HIV-1NL4.3 infection in peripheral blood lymphocytes and HIV- 1BaL infection in TZM-bl cells. In the HIV-1 protease assay, the n-butanol fraction showed good inhibitory activity (IC50 = 12.9 µg/ml), whereas no inhibitory effect was observed against HIV-1 reverse transcriptase and integrase activity up to 50 µg/ml. The n-butanol fraction of A. catechu interfered Tat-Long Terminal Repeat transactivation mediated HIV-1 transcription as observed by qRT-PCR and electrophoretic mobility shift assay (EMSA). The n-butanol fraction did not lead to any increase in pro-inflammatory cytokines secretion by Vk2/E6E7 as well as was non-deleterious to the intact monolayer formed by Caco-2 and HEC-1A epithelial cells. These investigations demonstrate the potential of A. catechu to prepare a herbal microbicide formulation for prevention of sexual transmission of HIV-1. Using synthetic/natural compounds, potent anti-HIV-1 activity of prostratin, rosmarinic acid and epigallocatechin gallate has also been demonstrated.
D. Neutralizing monoclonal antibodies (MAbs) against influenza virus
Avian influenza viruses, H5N1 and H7N9, normally infect avian populations but some variants are known to have the ability to infect humans resulting in high mortality. Antiviral drug resistance is reported for avian influenza viruses and there will likely to be a lag between the emergence of H5N1/H7N9 human epidemic and bulk production of the vaccine against the causative virus strains. In such a scenario, passive immunotherapy based on neutralizing antibodies may be one of the option. Specific antibodies generated against H5N1/H7N9 can also be used in the development of in-vitro diagnostic kits. In this direction, 6 murine MAb’s against recombinant HA protein of H5N1 (A/turkey/Turkey/1/2005) have been generated. Out of the these, 5 MAbs reacted to variable extent with HA protein of H5N1 (A/turkey/Turkey/1/2005; A/Hongkong/213/2003; A/Barheaded goose/Qinghai/14/2008 and A/Common magpie/Hongkong/2256/2006) and also A/Turkey and A/Vietnam whole virus in ELISA. Interestingly, one of the MAb, MA-12 showed broad reactivity against H1N1, H2N2, H3N2 and H7N9 in addition to H5N1. The initial results using pseudovirus assay system showed that MA-5 neutralize H5N1 A/Turkey virus. In addition, 10 MAb’s against recombinant HA protein of H7N9 (A/Anhui/1/2013) virus have also been generated. The reactivity profile in ELISA with HA protein from different strains of viruses revealed that 8 were specific to H7N9 and 2 MAbs in addition reacted with H1N1, H2N2 and H3N2.
109 Future plans
In the coming year, new contraceptive immunogens will be designed and attempts will be made to reduce the number of injections to achieve desired contraceptive efficacy. The role of transcription factors, micro RNA and other non-coding RNA in the invasion and differentiaon of trophoblastic cells will be investivated. Attempts will be made to make herbal formulation for prevention of HIV infection through sexual route.
Action taken on the RAP/SAC 2013 recommendations There were no specific comments, which required special action.
Publications Original peer-reviewed articles
1. Suman P, Gupta SK* (2014) STAT3 and ERK1/2 cross-talk in leukemia inhibitory facter mediated trophoblastic JEG-3 cell invasion and expression of Mucin 1 and Fos. Am J Reprod Immunol (doi:10.1111/aji.12248).
2. Pawar R, Das T, Mishra S, Nutan, Pancholi B, Gupta SK*, Bhat SV* (2014) Synthesis, anti-HIV activity, integrase enzyme inhibition and molecular modeling of catechol, hydroquinone and quinol labdane analogs. Bioorg Med Chem Lett 24: 302-307.
3. Shrestha A, Wadhwa N, Gupta SK* (2014) Evaluation of recombinant fusion protein comprising dog zona pellucida glycoprotein-3 and Izumo and individual fragments as immunogens for contraception. Vaccine 32: 564-571.
4. Nutan, Modi M, Dezutti CS, Kulshreshtha S, Rawat AKS, Srivastava SK, Verma A, Ranga U, Malhotra S and Gupta SK* (2013) Extracts from Acacia catechu suppress HIV-1 replication by inhibiting the activities of the viral protease and Tat. Virology J 10: 309.
5. Singh UP, Bhat HR, Verma A, Kumawat MK, Kaur R, Gupta SK, Singh RK* (2013) Phenyl hydrazone bearing pyrazole and primidine scaffolds: Design and discovery of novel class of Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) against HIV-1 and their antibacterial properties. RSC Adv 3: 17335-17348.
6. Modi M, Nutan, Pancholi B, Kulshrestha S, Rawat AKS, Malhotra S*, Gupta SK* (2013) Anti-HIV-1 activity, protease inhibition and safety profile of extracts prepared from Rhus pariflora. BMC Complement Altern Med 13: 158.
7. Nutan, Modi M, Goel T, Das T, Malik S, Suri S, Rawat AKS, Srivastava SK, Tuli R, Malhotra S*, Gupta SK* (2013) Ellagic acid and gallic acid from Lagerstroemia speciosa L. inhibit HIV-1 infection through inhibition of HIV-1 protease and reverse transcriptase activity. Indian J Med Res 137: 540-548.
#7. Gupta N, Chakrabarti K, Prakash K, Wadhwa N, Gupta T, Gupta SK* (2013) Immunogenicity and contraceptive efficacy of E. coli-expressed recombinant porcine zona pellucida proteins. Am J Reprod Immunol 70: 139-152.
110 #8. Gupta N, Shrestha A, Panda AK, Gupta SK* (2013) Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development. Mol Biotech 54: 853-862.
Reviews/Proceedings
1. Gupta SK*, Shrestha A, Minhas V (2014) Milestones in contraceptive vaccine development and hurdles in their applications. Hum Vaccin Immunother (doi: org/10.4161/hu.27202).
2. Gupta SK*, Nutan (2013) Clinical use of vaginal or rectally applied microbicides in patients suffering from HIV/AIDS. HIV AIDS (Auckl) 5: 295-307.
3. Suman P, Malhotra SS, Gupta SK* (2013) LIF-STAT signaling and trophoblast biology. JAK-STAT 2: e25155.
4. Suman P, Gupta SK* (2013) Regulators of early invasion of trophoblast cells. In Immunology of Pregnancy-2013 (Eds. Chaouat G, Sandra O and Ledee N). Bentham e-book, Bentham Science Publishers, pp 199-217.
5. Gupta SK* (2013) Contraceptive vaccines: past, present and future. In Immunology of Pregnancy-2013 (Eds Chaouat G, Sandra O and Ledee N). Bentham e-book, Bentham Science Publishers, pp 100-134.
6. Nutan, Gupta SK* (2013). Medicinal plants with anti-HIV activity. In Emerging Frontiers and Challenges in HIV/AIDS Research (Eds Bandivdekar A and Puri CP). National Institute for Research in Reproductive Health, Mumbai, India. pp 91-110.
Patent
1. Gupta SK, Gupta N, Chakrabarti K, Prakash K, Wadhwa N and Gupta T (2013) Recombinant zona pellucida (ZP) proteins, vaccine composition and method of producing said vaccines. (1210/DEL/2013 Indian patent filed on 25/04/2013).
*Corresponding author(s) #In press last year, since published
111 Studies of sertoli cells and spermatogonial stem cells of the testis and other endocrinology related research
Principal Investigator Subeer S. Majumdar
Ph.D. students Satyapal Arya Kamal Mandal
Project Fellows Sayon Basu Hironmoy Sarkar Rajesh Sarkar Bhola Shankar Pradhan
Collaborators Umesh Rai, University of Delhi Samir Bhattacharya, Vishwabharti, Shantiniketan
Theme of research
We use testis as an organ of multiple research interest 1) exploiting spermatogonial stem cells for propagation of transgene; i.e. for generation of transgenic animals, 2) analyzing differential gene expression by Sertoli cells (during active vs. inactive phase of spermatogenesis) to identify factors regulating germ cell division and differentiation with an intent to divulge unknown (inborn or environmentally induced) non hormonal causes of idiopathic male infertility and 3) undertaking germ cell transplantation studies to restore fertility upon chemotherapy. In addition, we also participate in other endocrinological research as collaborators.
Objectives 1. To exploit spermatogonial stem cells of testis for insertion and propagation of transgene through several generations in an attempt to over express or knock down specific genes. 2. To undertake gene expression studies of rat, mice and monkey Sertoli cells to identify factors important for induction of spermatogonial stem cell division and differentiation in the testis. 3. To study biology of spermatogonial stem cells and to use germ cell transplantation technique for restoration of fertility following chemotherapy. 4. To study paracrine and endocrine modulation of signal transduction in target cells of the endocrine system.
Work reported in 2012-2013
Functional geniomic studies of genes selected from studies of differential genomics by DNA microarray using mRNA from rhesus monkey Sertoli cells
Sperm quality has been declining globally over last 50 years (from 165 million to 60 million), which could lead to an increased prevalence of infertility in the world in coming 20-30 years. Testicular Sertoli cells (Sc) in males produce several factors generating functional niche for germ cell (Gc) division and differentiation at puberty, failure of which may cause infertility. We compared genes expressed by infant (spermatogenically inactive) and pubertal 112 (spermatogenetically active) monkey Sc using DNA microarray. Important genes were used to generate transgenic mice to evaluate their effects in vivo using functional genomics.
Wnt3: Wnt3, a morphogen of Wnt/β-catenin signalling pathway which is crucial for cellular differentiation, was found to be highly expressed in pubertal Sc as compared to infant Sc. Over expression of Wnt3 during infancy resulted in Gc overcrowding and precocious initiation of lumen formation within seminiferous tubules. Conversely, knocking down of Wnt3 during puberty resulted in decreased sperm count and testicular weight, along with Gc sloughing and disrupted Sc-Gc association. Wnt3 overexpression led to activation of classical β-catenin mediated signal transduction whereas knockdown of Wnt3 lead to increased destabilisation of β-catenin and consequently inhibited downstream transcriptional targets. Interestingly, knockdown of Wnt3 led to decreased Conexin-43 (Cx43) level which is known to be associated with disrupted spermatogenesis. CD30: CD30, a member of the TNF receptor super family involved in cytokine mediated processes within the body, was overexpressed by Sc during puberty in monkey as well as mice. The evidence from knockdown of CD30 in Sc of mice during puberty suggested CD30 as an important factor in processes leading to gain of spermatogenic function by Sc at puberty. Aberration in CD30 expression may be one of the causes of non-hormonal idiopathic male infertility. Endocrine signaling Sertoli cell: Our studies suggested that intracellular post FSHR downstream signaling cascades were operational in infant Sc although there is restricted FSH responsiveness during infancy. Adipose tissue: Free fatty acids (FFAs) augment adipose tissue inflammation through the TLR4 pathway, causing insulin resistance. FFA-induced proinflammatory cytokine expression in adipocytes occurred only in the presence of both FetA and TLR4; removing any of them limited FFA-induced insulin resistance. The data suggested that modulation of FetA may help in managing insulin resistance.
Progress of work during the current reporting year (2013-2014) Functional genomic studies of genes selected from studies of differential genomics by DNA microarray using mRNA from Rat Sertoli cells. We cultured rat Sertoli cells (Sc) from 5 days and 12 days old rat testes to compare genes expressed during spermatogenically inactive and active phase of the testis, respectively. Earlier, we have undertaken such studies using Monkey Sc and after using tools of bioinformatics, selected genes and made transgenic mice (different species). Now, we used rat Sc as a source of information and generated transgenic rats to study functional genomics. In rats, in spite of sufficient level of circulating hormone, since birth, the division and differentiation of spermatogonia do not occur until, 11-12 days of age. In our study, we evaluated the differential gene expression of Sc cultured from infant and pubertal rat. In view of this hypothesis, we choose microarray technique to study the differential transcriptome related to robust onset of spermatogenesis. Sc were cultured from infant (5 day old) as well as pubertal (12 day old) rats and RNA from such Sc was further used for DNA microarray experiment utilizing the Agilent platform. Three sets of hormone treated infant and pubertal Sc were hybridised independently to account for culture to culture variation. DNA microarray 113 reads were normalised and processed by Genespring Gx software to generate comparative transcriptome profile of hormone treated infant and pubertal Sc. Three such data sets, representing independent microarray analysis of hormone treated infant and pubertal Sc, were combined to generate a general transcriptomic profile wherein genes which were over expressed in Sc during infancy as well as genes which were over expressed in pubertal Sc were adequately represented. Specific signalling pathways and biological processes that were differentially expressed in infant and pubertal rat Sc were identified by in silico analysis of the combined microarray analysis. Differential gene expression was observed in various biological processes. However, our study was aimed at evaluating few genes which may have relevance to Sc mediated process of initiation of Gc division and differentiation. To this end we analyzed differentially expressed pathways which are known to influence cell division and differentiation. We found 2003 genes as up regulated in 12 day old rat Sc as compared to 5 day old rat Sc and 2205 genes as down regulated in 12 day old rat Sc as compared to 5 day old rat Sc. However, to be certain that selected genes were truly up or down regulated at the transcript level, RT PCR of Sc mRNA was performed. We found that certain genes like Ell Associated Factor (eaf2), Ninjurin 2 (ninj2), Neuramedin (nmu), Nuclear receptor 4A3 (Nr4a3) were up regulated in pubertal rat Sc as compared to infant rat Sc. Sostdc1, Angiotensin type 2 receptor (Agtr 2), Extracellular growth factor ligand 3 (Egfl3), Tetraspanin 8 (Tspn8) were found to be down regulated in pubertal rat Sc as compared to infant rat Sc. Eaf2 is positive regulator of RNA Pol II, Ninj2 is a cell- cell adhesion molecule, Nmu is involved in energy homeostasis, Nr4a3 is a orphan nuclear receptor, Sostdc1 is a Wnt and BMP inhibitor, Agtr 2 is a receptor for apoptosis, Tspn 8 is an inhibitor of differentiation whereas Egfl3 is a ligand for EGF pathway. To undertake functional genomics study of the differentially expressed genes obtained from rat microarray, transgenic rat models are being generated. We wish to over express specific genes at an age when they have limited or no expression, naturally. We have made constructs using murine PEM proximal promoter (a kind gift from M. Wilkinson) because its expression occurs in Sc of the pubertal testis only, but Pem expression is low or absent in the infant testis. Agtr 2, Tspn8 and Sostdc1 naturally having low expression in Sc during puberty were cloned with a flag tag at their c- terminal region downstream of PEM promoter to make transgenic rats for evaluating regulation of spermatogenesis by these genes. Endocrine signaling
Sertoli cell: We have demonstrated that unlike pubertal testicular Sertoli cells (Sc), infant Sc fail to produce substantial amount of cAMP upon FSH treatment. In an effort to understand why cAMP production is restricted in the infant Sc, we attempted to divulge molecular basis of this deficiency in the signal transduction. In this study, we have compared the FSH-R mediated signaling events in Sc of infant and pubertal rhesus monkeys. We found that expression of GαS and its activator, Ric8b are very low in infant Sc. This may be responsible for suboptimal cAMP generation and insufficient expression of spermatogenically relevant genes by infant Sc, in spite of sufficient expression of FSHR, circulating levels of FSH and their binding to Sc. We also found that levels of Gαi increased upon FSH treatment in infant Sc unlike pubertal Sc. Our experimental observations suggested that infant Sc are as competent as pubertal Sc for transcriptional events related to spermatogenesis, when sufficient amount of cAMP is generated inside these cells by treatment with pharmacological agents like forskolin and 8Bromo-cAMP.
This observation in primates, generates a remarkable scope for generating advanced Gc, in vitro, using a fraction of seminiferous tubules from infertile individuals displaying FSH 114 resistance. Intracellular cAMP in Sc of such tubules may be augmented by treatment with forskolin or supplementation with 8Bromo-cAMP which may lead to robust transcription of genes conducive to generation of advanced Gc which may be used for assisted reproduction. This may become a new approach to enable an infertile individual to father a child from his own genome, when hormonal therapy fails. A set of new information generated about some signal transduction mediator molecules of primate Sc by us may provide basis for diagnosis and treatment of certain forms of idiopathic male infertility.
Adipose tissue: We have shown earlier that Fetuin A is necessary for Free fatty acids (FFAs) mediated augmentation of adipose tissue inflammation through the TLR4 pathway which causes insulin resistance. Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. Studies in Prof Bhattacharya’s lab with our collaboration demonstrated that excess lipid in adipose tissue ambience may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet (HFD) fed mice and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels compared to their controls; partially hepatectomized HFD mice did not show noticeable alteration indicating adipose tissue to be its source. In adipocytes, fatty acid (FA) induces FetA gene and protein expressions resulting in its copious release. We found that FetA could act as chemoattractant for macrophages. To simulate lipid induced inflammatory condition when proinflammatory adipose tissue and macrophages create a niche of altered microenvironment, we set up transculture system of macrophages and adipocytes; addition of FA to adipocytes released FetA into the medium which polarized M2 macrophages to M1. Taken together, lipid induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings opened a new dimension for understanding obesity induced inflammation. Germ Cell transplantation We have generated a new technique for evacuating testicular germ cell niche without compromising bone marrow, for studies related to germ cell transplantation for restoration of fertility, post chemotherapy. Using this model, we have started doing homologous germ cell transplantation post in vitro expansion of spermatogonial stem cells.
Action taken on RAP/SAC 2013 recommendations No specific recommendation was made by the Committee.
Future plans 1. Functional genomics of more differentially expressed genes (from microarray analysis) by the testicular Sertoli cells (Sc) of rat and monkeys will be undertaken. 2. Testicular transgenesis will be used to insert constructs for destroying Y chromosome bearing sperm in the testis so that only X chromosome bearing sperm are generated for obtaining only female offspring. 3. Cell-cell interaction studies in the testis will be continued and collaborative research in the field of endocrinology will be undertaken. Publications Original peer-reviewed articles
115
1. Chatterjee P, Seal S, Mukherjee S, Kundu R, Mukherjee S, Ray S, Mukhopadhyay S, Majumdar SS, Bhattacharya S* (2013) Adipocyte Fetuin-A Contributes to Macrophage Migration into Adipose Tissue and Polarization of Macrophages. J Biol Chem 288: 28324– 28330.
2. Nagarajan P, Arindkar S., Singh S, Majumdar SS* (2013) Effect of long-term castration on serum biochemistry in rhesus monkeys. J Med Primatol (doi:10.1111/jmp.12046).
3. Das DS, Wadhwa N, Dubey N, Pradhan B, Majumdar SS* (2013) Dickkopf homolog 3 (DKK3) plays a crucial role upstream of WNT beta CATENIN signaling for Sertoli cell mediated regulation of spermatogenesis. PLoS ONE 8: e63603.
4. Majumdar SS*, Bhattacharya I (2013) Genomic and post-genomic leads toward regulation of spermatogenesis. Prog Biophys Mol Biol 113: 409-422
Reviews/Proceedings
1. Majumdar SS*, Bhattacharya I, Khan M (2013) Pharmacogenomics and Personalized Medicine in Infertility D. Barh et al. (eds.), Omics for Personalized Medicine, pp. 743-760. Springer, Germany.
*Corresponding author
116 Molecular mechanism of enzymatic reactions and enzyme-ligand interactions
Principal Investigator Apurba K. Sau
Research Associates Maskoor Alam Safikur Rahman Saurabha Srivastava
Ph.D. Students Esha Pandita (upto April, 13) Shiv Kumar (upto July, 13) Sudeepa Rajan Nikunj Harilal Raninga Vineet Sadarangani (since Jan, 14) Ankita Dutta (since Jan, 14)
Research Fellow Ginto George (since, Nov 13)
Collaborator Shashank Deep, IIT Delhi, New Delhi
Theme of research
The aim of this project is to understand molecular mechanism of different classes of GTPases induced by immunomodulatory cytokine interferon-γ (IFN-γ) and to compare the mechanistic similarities and differences with other GTPases within the same as well as different classes. The study has been currently focused on human guanylate binding protein-1 (hGBP-1) and other proteins in the same family. The mechanism along with the structural data may provide an insight to design drug candidates on novel GTPases and their effectors involved in the disease.
A. IFN-γ induced GTP-binding proteins and their mechanism of GTP hydrolysis
Objectives: To study the regulation of GTP hydrolysis in IFN-γ induced guanylate binding proteins p67 (hGBP-1 and hGBP-2) and to understand their similarities and differences within the same family as well as same and different classes.
B. Understanding the function of arginine metabolic enzymes in Helicobacter pylori
Objectives: The aim is to investigate a detailed molecular mechanism of two arginine metabolic enzymes arginase and ADC in H. pylori. The mechanism along with structural data from other organisms may provide a novel strategy to develop new inhibitors with greater efficiency against H. pylori infection.
117 Work reported in 2012 – 2013
By temperature dependent kinetic studies our data showed that GDP formation occurs through the dissociation of GDP-bound enzyme dimer. This was also supported by the biochemical assays where addition of external GDP reduces GDP formation of hGBP1 catalyzed GTP hydrolysis. We also provided thermodynamic insight into difference of the product formation at different temperature. The role of SSEHA motif in the stability of H. pylori arginase was investigated by temperature induced unfolding studies.
Progress during the current reporting year (2013-2014)
Stability of wthGBP1 and its mutants in the presence of the substrate analogue and biochemical assays
To investigate whether the stability of the protein upon binding with the substrate is related to the product formation, kinetic assays of the wild type and mutant proteins were carried out. In the x-ray crystal structure of the full-length hGBP1 with GppNHp, Ser157 (globular domain) and Glu313 (helical domain) are found to interact at a distance of ≤ 3.5 Å and have been mutated to Ala. These mutants were used to check whether the activity can be correlated to the stability of the proteins upon binding with the substrate. The catalytic efficiency for GDP formation in Glu313Ala is decreased by ~ 2 fold compared to wild type, but for GMP it is increased by ~ 1.5 fold. The decrease and increase in the catalytic efficiency for GDP and GMP respectively for Glu313Ala compared to wild type is consistent with a model which was proposed earlier, where after the first hydrolysis there is a competition between the dissociation of GDP-bound enzyme dimer and second hydrolysis. The decrease in the catalytic efficiency for GDP in Glu313Ala compared to wild type suggests that Glu313 plays a role in the dissociation of GDP-bound enzyme dimer and thus increases GDP. But for Ser157Ala, the catalytic efficiency for GDP is increased by ~ 1.5 fold compared to wild type suggesting that Ser157 is important in preventing the dissociation of GDP-bound enzyme dimer. To understand whether the difference in the product formation can be correlated to the stability, we carried out heat-induced unfolding studies of the wild type and mutant proteins separately with or without GppNHp. Like wild type the binding of the analogue increases the stability of the mutant proteins. The relative difference in the ΔGD between the analogue free and bound proteins for Ser157Ala is lower than wild type (1.7 kcal/mol vs 0.9 kcal/mol for wt vs Ser15Ala respectively) but for Glu313Ala it is higher than wild type (2.4 kcal versus 1.7 kcal/mol for Glu313Ala versus wt respectively). This suggests that the difference in ΔGD between the analogue free and bound proteins plays an important role in the product formation; larger difference (higher stability) favors the second hydrolysis i.e. GMP formation, but smaller (lower stability) favors dissociation of GDP-bound enzyme dimer resulting more GDP. This is in agreement with the kinetic assays, where Glu313Ala shows higher catalytic efficiency for GMP (consequently lower catalytic efficiency for GDP) compared to wild type but Ser157Ala shows lower for GMP (consequently higher catalytic efficiency for GDP).
118 Mutational studies on the sequence motif of H. pylori arginase
H. pylori arginase has a distinct sequence motif 153ESEEKAWQKLCSL165, which is absent in the analogous enzymes. To examine the role of the sequence, each residue was individually mutated with alanine and the kinetic assays were carried out. Among the mutants, Glu155Ala, Trp159Ala and Cys163Ala showed remarkable results. Glu155Ala and Trp159Ala resulted in complete loss of the catalytic activity. Cys163Ala showed a decrease in the catalytic efficiency by about 16 fold compared to the wild type in the presence of Co2+. However, with Mn2+ Cys163Ala failed to show detectable activity indicating that Cys163 is also critically important for catalysis. The catalytic efficiency for Glu156Ala decreased by ~ 5 fold than the wild type, suggesting that Glu156 is also important for the function of the protein. With Mn2+ Glu156Ala did not exhibit detectable activity, further suggesting the role of Glu156 in the function. The mutant proteins exhibit cooperativity similar to wild type, except Cys163Ala for Co2+ (n ~1.2), and Leu162Ala (n ~ 1.1) and Ser164Ala (n ~ 1.1) for Mn2+, suggesting that the regulation of activity is not primarily altered for the most of the mutations. Since, Cys163Ala did not show appreciable cooperativity and detectable activity with Co2+ and Mn2+ respectively, implying that Cys163 may play a role in oligomerization of the protein. To investigate this, analytical gel-filtration assays of wild type and Cys163Ala were carried out. Unlike wild type, Cys163Ala showed mainly as a monomer with dimer being a smaller amount, indicating that Cys163 plays a critical role in dimerization of the protein. Thus, the kinetic assays in combination with the gel-filtration analysis demonstrate that the motif is extremely critical for the function of the protein.
Simulations studies and location of the sequence motif
The model structure of H. pylori arginase with respect to its closest homologue Bacillus caldovelox enzyme suggested that the sequence motif is far from the active-site and exposed to the solvent. To get a structural insight into how the motif is important for the function of the protein, we have done MD simulations to refine the reported model structure of H. pylori arginase. The protein backbone RMSD values showed equilibrium structures after 400 ns of simulations. A snapshot of the simulated structure at 445.7 ns shows that the sequence consists of largely a loop with a small helix in Lys161-Ser164. Trp159 moved closer to the metal binding site and is buried in the protein. The positioning of the motif near the active-site is due to the hydrogen bonding interaction of Trp159 with Asp126 (distance ~ 2.65 Å). This interaction was formed at around 120 ns and remained intact during rest of the simulations, implying that it may have an impact on the structure and function of the protein. To verify this, Asp126 was mutated to Ala and the kinetic assay was done. Interestingly, this mutant resulted in complete loss of the catalytic activity, indicating that Asp126 is vital for the function. As shown in the mutational study, Trp159Ala failed to show detectable activity. The data further suggest that the interaction of Trp159 with Asp126 appears to be critical in positioning the motif near the active site for the function of the protein. Thus, the MD simulations results show that the sequence motif is located near the active-site with a loop-cum-helix structure, suggesting its role in both structure and function of the protein.
119 Deletion studies on the sequence motif
Our studies so far show that Glu155, Trp159, Cys163 and Asp126 are critical for the function of the protein. To examine the role of the whole sequence motif in the activity, a truncated H. pylori arginase was constructed, where the residues 153-165 have been deleted. Unlike wild type and mutant proteins, the GST-tag could not be cleaved in the truncated protein using caspase-6 indicating that the cleavage site may be buried inside the fused protein. The activity assay of the GST-tagged truncated protein was carried out. As expected, the protein failed to display activity, further implying that the sequence motif is critical for the function of the protein. It is to be noted that the wild type protein is found to show similar activity with and without the tag.
Evaluation of the metal binding upon mutations in the sequence motif
Arginases are known to have an intact bimetallic center at the active-site for catalytic activity. Like wild type, all mutants except Trp159Ala exhibit similar metal binding (~ 2 metals per unit monomer) in the presence of either Co2+ or Mn2+. Trp159Ala exhibits one metal per monomer, suggesting that Trp159 is crucial for retaining a metal ion. To verify this, Trp159 was mutated to Phe. Like Trp159Ala, Trp159Phe also showed no detectable activity and contains one metal ion, further verifying that Trp159 is critical for retaining a metal ion and thus in the activity. Interestingly, Asp126Ala also showed one metal ion, indicating that Asp126 is crucial for retaining a metal ion. These studies demonstrate that both Trp159 and Asp126 are close to the metal binding/active-site and critical for retaining the bimetallic center, thus for the function of the protein, which is consistent with the kinetic assays and MD simulations.
Future plans
A detailed study into the regulation of GTPase activity by a higher order assembly of hGBP-1 and hGBP-2 will be investigated. The impact of the substrate binding, hydrolysis and interdomain interaction in the formation of a higher order assembly will also be studied. The structure and stability of the nonconserved motif in H. pylori arginase will be examined. The positioning of the motif near the active-site will also be investigated.
Action taken on the RAP/SAC 2012-2013 recommendations
The scientific and technical clarifications sought during the presentation were provided.
120 Structural-function relationship studies of mycobacterial proteins
Principal Investigator Bichitra K. Biswal
Research Associate Avishek Anant
PhD students Nazia Nasir Mohd. Saeed Ahangar Deepak Chandra Saroj Khundrakpam Herojit Singh Abhisek Dwivedi Bhavya Jha
Theme of research
We have been pursuing two major projects on proteins from Mycobacterium tuberculosis (Mtb). In the first project, we aim at addressing molecular mechanisms underlying the actions of the enzymes involved in the Histidine biosynthesis. The second project deals with the structural and biochemical studies of membrane associated proteases (MAPs).
Objectives
1. Determine the 3D structures of native and ligand-bound forms of target enzymes primarily using X-ray crystallographic technique, deduce the mechanisms underlying their action and perform biochemical study. 2. Identify the plausible physiological substrates of MAPs through a proteomics approach. 3. Examine whether MAPs are involved in Mtb pathogenesis through in vivo studies. 4. Design inhibitors against these targets through a structure-based-inhibitor design approach and examine the efficacy of these inhibitors in Mtb infect macrophages 5. Elucidate the 3D structures of enzyme/inhibitor complexes, analyse the interactions between these, and use this information to design more potent inhibitors.
Work reported in 2012-2013
In the previous year, we have reported data pertaining to detailed structural and kinetics studies of HisB, which catalyses the sixth step in histidine biosynthesis pathway, conversion of imidazole glycerol phosphate (IGP) to imidazole acetol phosphate (IAP). The detailed interactions of HisB/IGP and HisB/3-Amino-1,2,4-triazole (ATZ) were mapped. Briefly, it was established that HisB is a 24-mer in crystal as well as in solution and that it indeed possesses enzymatic activity as a dehydratase with a Km value of about 120 µM. It was also shown that ATZ inhibits HisB competitively with a Ki value of 310 µM. HisB. In the other project, we have reported that Rv2224c-encoding membrane protein deprived of N-terminal
121 30 amino acids was purified and that the enzyme possesses esterase activity. Needle-like crystals of Rv2224c were grown.
Progress of work during the current reporting year 2013-2014
A. Structural and biochemical studies of HisC (Rv1600)
Previously we have determined 3D structures of HisB and HisC2 and have characterized these enzymes biochemically. During the past one year, we have determined structures of free and ligand-bound forms of another enzyme HisC, a PLP-dependent histidinol-phosphate aminotransferase involved in the catalysis of seventh step, conversion of imidazole acetol phosphate to L-histidinol phosphate. The biological functional unit of HisC consists of a symmetric dimer (Figure 1). Like HisC2, each protomer of HisC consists of two domains, a larger PLP-binding domain having an α/β/α topology and a smaller domain (Figure 1). The PLP-binding domain contains a seven-stranded β-sheet sandwiched between four α-helices. Superimposition of ligand-bound and free forms suggests that the N-terminal arm consisting about 40 amino acids undergoes a huge conformational change upon ligand binding. Briefly the loop comes closer to the ligand binding region and a few residues of the loop make hydrogen and van der waals interactions with PLP and the substrate. The active site is situated in the dimeric interface and residues that line the active site region include Tyr25A, Asn37A, Thr38A, Asn39A, Gly101A, Ser102A, Asn103A, Tyr127A, Met129A, His130A, Ala172A, Asn176A, Asp201A, Ala203A, Tyr204A, Thr229A, Ser231A, Lys232A, Arg240A, Leu241A, Gly242A, Arg337A, Arg346A, Tyr67B, Pro260B, and Tyr261B. The active-site cavity possesses a surface area of about 1950 Å2 and a volume of about 5500 Å3. Kinetic studies with various amino acid residues show that Rv1600 is a PLP-dependent histidinol-phosphate aminotransferase (Figure 2).
122 Figure 1: (A) Stereoview of cartoon representation of the quaternary (dimeric) structure of HisC (Rv1600). Also shown in the figure is the two fold axis perpendicular to the plane of the paper that relates the two monomers. The location center-of-mass of each protomer is shown by a magenta sphere.
Figure 2: Relative activity of Rv1600 for different substrates. The standard aminotransferase enzymatic assay in the presence of cofactor PLP and amino group acceptor α-ketoglutarate was carried out. The formation of glutamate by the aminotransferase was measured, using a coupled enzymatic reaction involving the conversion of NAD to NADH, at 340nm. All reactions were performed in triplicates.
B. Crystallogenesis of Rv2224c-encoded membrane protein
The membrane protein (encoded by Rv2224c) was over-expressed in Mycobacterium smegmatis and was purified using affinity and gel-filtration chromatography. Extensive crystallization trial experiments using various commercially available and custom made screen solutions were set up. Diffraction quality crystals, after several rounds of optimization, were grown in a drop containing 30% PEG 3350 (Figure 3). Native and anomalous X-ray diffraction data have been collected. Attempts to determine the structure is underway.
Figure 3: Crystals of Rv2224c grown in a drop containing PEG 3300 as the precipitant.
123 Future plans
1. Our efforts to crystallize, elucidate the 3D structures, and carry out biochemical of the remaining histidine pathway enzymes will continue. In addition, efforts to design new inhibitors for these enzymes through a structure-guided approach will be made. 2. We are planning to test the inhibitory potential of an inhibitor 3-Amino-1,2,4-triazole of (HisB) imidazole glycerol phosphate dehydratase, a histidine pathway enzyme whose native, substrate-bound and inhibitor-bound structures have been elucidated by us, in Mtb growth at cell culture level. 3. Ongoing efforts to grow diffraction quality crystals of MAPs in the presence of various detergents and solve their 3D structures will continue.
Action taken on the RAP/SAC 2013 recommendations
The recommendations made by the RAP/SAC committee were incorporated to their satisfactions.
Publication Original peer-reviewed article
1. Ahangar MS, Vyas R, Nasir N, Biswal BK* (2013) Structures of native, substrate-bound and inhibited forms of Mycobacterium tuberculosis imidazoleglycerol-phosphate dehydratase. Acta Cryst. Section D69: 2461-2467.
*Corresponding author
124 Molecular modelling of proteins and protein-ligand complexes using knowledge-based approaches and all atom simulations
Principal Investigator Debasisa Mohanty
Research Associate A. Madhumalar
Ph.D. Students Shradha Chopra Deepak Chhaya Dhiman Neetu Sain Mansi Grover
Research Fellow Nikhil P. Damle
Collaborators Rajesh S. Gokhale, NII Sagar Sengupta, NII Pushkar Sharma, NII Vinay Nandicoori, NII
Theme of research
The main theme of the research project is to understand the structural principles that govern folding of peptides/proteins to stable conformations and binding of various ligands to proteins, and use these principles for developing novel computational approaches for prediction of the structures of peptides/proteins and specificities of protein-ligand complexes. Theses prediction approaches for structure and substrate specificity are being used to assign functions to proteins in the various genomes for identifying novel biosynthetic and protein interaction networks.
Objectives
The specific objectives of the various projects are to investigate, whether the combination of knowledge-based and ab initio approaches can be used for predicting:
1. Substrate specificity of proteins involved in biosynthesis of secondary metabolites, 2. Substrate specificity of various peptide recognition modules (PRMs) like MHCs, kinases, PTB, PDZ and WW domains etc, 3. Structure and dynamics of miRNA-protein complexes.
125 Work reported in 2012-2013
A. In silico analysis of enzymes associated with novel post-translational modifications and biosynthesis of lantipeptides
We have carried out sequence and structural analysis of enzymes associated with five different types of novel post-translational modifications (PTMs), namely, AMPylation, eliminylation, hydroxylation, sulfation and deamidation. Based on a variety of analysis of these sequence and structural information, whole sequences as well as active site residue profiles have been built for enzymes involved in each of these PTMs. Our benchmarking studies on completely independent dataset indicate that, these profiles can predict the presence of these 5 PTM catalyzing domains with high sensitivity and specificity.
We have also carried out a comprehensive analysis of LanM like enzymes involved in class II pathways for biosynthesis of lantipeptides. Fold prediction as well as profile to profile comparisons have been carried out for identifying possible presence of kinase or phosphothreonine lyase domains in LanM.
B. Analysis of interaction networks involving kinases and other PRMs
Development of computational method for prediction of protein phosphorylation networks The correlation between kinase families and PFAM domains of their substrate was successfully utilized to develop an automated computational tool called PhosNetConstruct for distinguishing cognate kinase-substrate pairs from all other non-cognate combinations. PhosNetConstruct will be a useful computational tool for generating potential list of cognate kinase-substrate pairs by filtering out large number of non-cognate kinase-substrate pairs and subsequently exact phosphosites can be identified using phosphosite predictors like GPS and NetPhosK.
In silico identification of small molecule modulators of PDZ-peptide interactions An attempt was also made to benchmark a computational protocol for identification of small molecule or peptidomimetic modulators of PRMs using PDZ domain as test case. A set of 38 small molecules with known Ki/Kd values for PDZ2 and PDZ3 domain of PSD-95 protein were selected for the benchmarking study. The affinity scores predicted by docking and the MM-PB/SA binding energy values calculated after MD simulations were compared with the experimental binding energy values.
C. Structure and dynamics of miRNA-protein complexes
Explicit solvent molecular dynamics (MD) simulations have been carried out on the crystal structures of miRNAs in complex with Argonaute and Lin28. The MD trajectories have been analyzed to identify the crucial tertiary structural features of miRNA and key specificity determining residues of the interacting proteins, which govern the specificity of recognition. To better understand the role of Argonaute protein and the structural features involved in miRNA target recognition, modelled Argonaute-miRNA-mRNA ternary complex has been refined MD simulations.
126
Progress of work during the current reporting year (2013-2014)
A. In silico analysis of enzymes associated with biosynthesis of secondary metabolites and lantipeptides
Development of computational method for retro-biosynthetic enumeration of enzymatic reactions leading to a given secondary metabolite With the availability of huge number of experimentally characterized secondary metabolites, a retro approach of identifying genes associated with known metabolites would be relatively easier compared to forward approach for identifying the unknown metabolite biosynthesized by a given set of genes. The retro-biosynthetic or reverse approach would involve enumeration of various chemical transformations or enzymatic reactions which would generate a given chemical moiety and identifying the enzymes that can catalyze the given biochemical transformation. The assembly line mechanism of biosynthesis of polyketides involves various chemical transformations like decarboxylative condensation of two acyl groups by ketosynthase (KS), optional reduction of the ketide group formed by ketoreductase (KR), dehydratase (DH) and enoylreductase (ER). The polyketide is finally released either by thioesterase (TE) mediated simple hydrolysis, macrolactonization, macrolactamization or claisen condensation or product template (PT) mediated C-C cyclization. These reactions were stored as generic reaction rules explaining the reaction mechanism. The reaction rules are constructed based on the sub-structural changes that occurring in the reaction. In addition to the above mentioned reactions, rules were stored for post PKS reactions like O-, N- and C- glycosyltransfer, O- and N- methyltransfer, N- N dimethylation, transamidation, cyclic carbamoylation, carbamolation, acylation and epoxidation. Spontaneous cyclic reactions have also been included in the reaction rule database. Corresponding to each generic reaction functional group information of product are stored in a separate database as SMARTS. Functional group here refers to the substructure where the change occurs. Obgrep tool of Open Babel is first used to locate a functional groups present in the given chemical compound. Then using Reactor module of Chem Axon's JChem technology corresponding generic reaction is used to transform given chemical moiety into its precursor molecule. This precursor molecule then becomes target for another functional group search and hence transformation. This process is continued till no other functional group is detected in the compound. In retro approach the order of reaction enumeration is reversed compared to natural biosynthetic order. Therefore in this approach first reaction catalyzed by tailoring enzymes are enumerated, followed by cyclization or chain release and then the condensation and reductive reactions. This process allows us to enumerate the reactions involved in the biosynthesis of a given polyketide. The next step would be link the biosynthetic reactions to their respective genes using our earlier developed databases of PKSs and NRPSs.
Threading analysis for class II lanthioninesynthetase LanM is the class II lanthioninesynthetase whose N-terminal part performs dehydration of Ser/Thr into Dha/Dhb respectively and C-terminal part catalyzes the cyclization of the peptide by forming lathionine structure. However, LanM does not show sequence similarity with protein kinases or any elimination domains. Threading analysis of N-terminal part of LanM indicate the possible structural similarity with PI3-kinase. However, residues known to be
127 important for elimination of phosphate were also found in the sequence stretch aligning with kinase domain. It is possible that the kinase like domain is catalyzing both phosphorylation and eliminylation by conformational rearrangements in the loop regions.
B. Analysis of interaction networks involving kinases and other PRMs
Structure based analysis of disease associated nsSNPs on kinases We have investigated plausible structural rationale for some of these disease associated nsSNPs which are present on catalytic domains of protein kinases in an enriched manner. We have systematically mapped these nsSNPs from dbSNP onto the individual kinase catalytic domains for which high confidence homologous crystal structures are available in PDB. Such mapping is helpful to understand effect of these mutations in a kinase-specific manner. Interestingly, many of the residues harbouring mutations were found to be conserved across different kinases indicating their possible common mode of action. Therefore, we have mapped these conserved residues onto catalytic domain of CDK2. The novel aspect of our analysis is investigation of the role of nsSNPs in regulation of conformational transitions in kinase catalytic domains. We have explored their role in inducing conformational transitions from inactive to active state by analyzing trajectories obtained from previous MD simulations on CDK2. Comparative analysis of MD trajectories with respect to the network of contacts involving these residues indicates that there are certain crucial contacts which are differentially maintained in active and inactive forms. Mutations in these conserved residues might destabilize the kinase catalytic core and shift the conformational equilibrium towards catalytically inactive state thus providing structural rationale for some of these disease associated mutations. In fact, we have proposed structural roles for various disease associated mutations based on the MD studies and classified them based on their roles in maintaining structural integrity, catalytic function, interaction with other downstream macromolecules and regulation of catalysis. Mapping of disease associated nsSNPs onto individual kinase structures would also help in rational design of kinase inhibitors and generation of experimentally testable hypotheses.
Benchmarking of multi-scale approach on human PDZ domains The structure-based multi-scale approach developed earlier for predicting binding partners of PDZ domains has been benchmarked on human PDZ using phage display data. This benchmarking analysis was carried out on the 20 human PDZ domains for which the phage display data, artificial negative interaction data and structural information on PDZ-peptide complexes were available. The ROC analysis of pair potential based predictions showed AUC values 0.761 when all the 100 non-binders available for each domain were used in benchmarking. We also carried out predictions using the binder peptides for a given PDZ domain as non-binder peptides for other domains. However, this resulted in lower AUC values for several PDZ domains. This suggests possible presence of PDZ domains with overlapping specificities. We analyzed the PDZ domain specificities in terms of similarity between the binding pocket residues and similarity between the phage display peptides for any two domains. Apart from pair potential based prediction of PDZ binding peptides, we have also performed MD simulation on complexes involving internal and C-terminus peptides for Par6 PDZ domain. Our simulations could reproduce the subtle conformational changes which are required for recognition of internal peptides by PDZ domains. The specificity determining
128 residues for internal peptide recognition could be incorporated into our pair potential based prediction method.
C. Structure and dynamics of miRNA-protein complexes
The binding of the miRNA to the target mRNA takes place in the RNA induced silencing complex (RISC) which consists of a ternary complex involving Argonaute (AGO) protein, miRNA and mRNA. Therefore, it is necessary to investigate the role of structural features of miRNA/mRNA and their interactions with AGO for better understanding of the mechanisms of miRNA target recognition. The recent high-throughput data from HITS-CLIP and CLASH experiments have reported multiple exceptions to the canonical rules of miRNA target recognition. We have attempted to decipher the role of AGO in miRNA:mRNA interactions involving such non-canonical interactions like G:A pairing and G bulge etc. Explicit solvent molecular dynamics simulations have been carried out on AGO-miRNA binary complex (PDB ID: 4F3T), free AGO and AGO-miRNA-mRNA ternary complex. Since, there are no available structures for an argonaute protein in association with a microRNA-mRNA complex we have modeled an AGO-miRNA-mRNA ternary complex. The results of the simulations suggest that the free AGO is highly flexible as compared to binary and ternary complexes. However, the flexibility of the binary and the ternary complex is more or less comparable. This indicates that binding of RNA stabilizes the AGO protein. Also, out of the four AGO domains, PAZ and N domains are highly dynamic while the PIWI and the MID domains are comparatively rigid. The inter domain movements could also be correlated to the opening and closing of the nucleic acid binding channel in the presence and absence of miRNA/miRNA-mRNA duplex. The 500 ns simulations also revealed readjustments in the miRNA-mRNA interactions in the ternary complex, involving bulging out of one nucleotide (U5) at the miRNA side and formation of a non watson-crick G:A base-pairing. Interestingly, in simulations on the miRNA- mRNA duplex in isolation i.e. in absence of the AGO protein, no such readjustments involving non-canonical interactions were observed. Therefore, our simulations highlight the role of AGO in determining specificity of miRNA target recognition involving non-canonical interactions.
Future plans
Systematic comparison of site specific kinase substrate relationships (ssKSRs) with domain specific kinase substrate relationship (dsKSRs) will be carried out for improved prediction of phosphorylation network. Role of co-occurrence of other interacting domains in substrate recognition by kinase domains will be investigated. Attempt will be made to identify small molecules which can potentially disrupt protein-protein interactions involving other PRMs like WW, SH2 and SH3 domains. Genome informatics studies will be carried out for analysis of biosynthetic pathways in which novel peptide natural products are made by post-translational modifications of ribosomally synthesized polypeptides. Analysis of transcription factor (TF)- miRNA network will be carried out to decipher conserved regulatory modules.
129 Action taken on the RAP/SAC 2013 recommendations
Scientific and technical queries raised by the members during presentation were answered. No specific suggestions were given by RAP/SAC members.
Publications Original peer-reviewed articles
1. Tiwari G, Mohanty D* (2013) An In Silico analysis of the binding modes and binding affinities of small molecule modulators of PDZ-Peptide interactions. PLoS ONE 8: e71340.
2. Damle NP, Mohanty D* (2014) Deciphering kinase-substrate relationships by analysis of domain specific phosphorylation network. Bioinformatics (DOI: 10.1093/ bioinformatics /btu112).
3. Tiwari G, Mohanty D* (2014) Structure Based Multi-Scale Approach for Identification of Interaction Partners of PDZ Domains. J Chem Inf Model (DOI: 10.1021/ci400627y).
*Corresponding author
130 Structure, interaction and design studies involving regulatory peptides and proteins
Principal Investigator Dinakar M. Salunke
Ph.D. Students Ashish Kumar Sharad Vashisht
Research Fellow Sonam Roy
Collaborator Kanwaljeet Kaur
Theme of research
The structural aspects of molecular recognition and its applications in analyzing the mechanisms associated with specific regulatory events and in rational molecular design.
Objectives
1. Understanding the protein architecture and the structural biology of various regulatory events. 2. Analysis of the structural principles of immune recognition and molecular mimicry. 3. Rational molecular design studies based on the above.
Work reported in 2012-2013
The hallmark of acquired immune system is the remarkable specificity in its recognition repertoire that not only counters the invading pathogen but also ensures self-nonself discrimination. Although immune system has evolved to discriminate finer differences between the molecules, degeneracy in immune response is often been observed. Studies towards understanding specificity/degeneracy against an invading pathogen were further extended to Influenza A Virus. The influenza hemagglutinin’s antigenic regions were previously been mapped. A seven amino acid (WTGVTQN) epitope from an unusual protruding loop near the receptor binding region which has been previously shown to have neutralizing effect was selected. The influenza research database (flu database) had 46 analogues of this epitope of which 9 were selected based on the maximum variation present. Polyclonal humoral response showed differential degenerate response against different analogues. When the booster was given the degenerate profile was retained. Two monoclonal antibodies 1A5 (KWTGVTQN- TT) and 4B1 (KWTGVTQN-KLH) were generated and characterized after purification. These two antibodies have shown differential cross-reactivity profile as was indicated in the polyclonal responses.
131 The seeds of a medicinally important plant, Mucuna Pruniens, were known for pharmacological properties such as anti-Parkinson, antineoplasty, antioxidant, antidiabetic and antivenom activity. Structure of a 21 kDa protein, MP-4, determined and refined by itterative molecular replacement approch to a resolution of 2.6A with Rcryst of 26% and Rfree 28%. Towards functional characterization of MP-4, the protein was subjected to antivenom activity. Different sets of balb/c mice were immunized with MP-4 protein and non-lethal dose of Echis carinatus whole venom protein and sera collected. The polyclonal antibody raised in mice against MP-4 protein reacts with one of Echis carinus venom proteins, phospholipase A2 (PLA2). Further, the observed sequence homology of MP-4 with serine protease inhibitor from Delonix regia, led to screening the inhibitory activity and binding efficiency with trypsin and chymotrypsin. Competition ELISA and binding experiments were carried out. MP-4 showed IC50 values of 1.96uM and 1.03uM for trypsin and chymotrypsin, respectively. Thus, Inhibitory efficiency of MP-4 was found to be less as compared to commercially available soybean trypsin inhibitor (STI) and chymotrypsin inhibitor (chymostatin). Binding efficiency of MP-4 with trypsin and chymotrypsin was analysed indicating that MP-4 binds with the KD values 2.65e-6 and 3.39e-6, respectively, which is comparatively poor as compared to other known Kunitz type inhibitors.
Progress of work during the current reporting year (2013-2014)
Although immune system is shown to be highly specific, degenerate specificity in immune recognition is often been observed. We have been working words understanding degenerate specificity of antibodies using a peptide (DVFYPYPYASGS) and a sugar (methyl α D mannopyranoside) as model antigens. Both, carbohydrate antigen and the corresponding mimicking peptide, were shown to be equivalent in polyclonal responses as well as in the monoclonal antibodies that were generated, in which an anti-sugar affinity matured antibody (2D10) was able to recognize both sugar and the peptide with equivalent affinities. Thermodynamic analysis of antigen-antibody (2D10) interaction had suggested that the flexibility in the antigen combining site may possibly help in the manifestation of molecular mimicry. However, when the structures of 2D10 in apo and the antigen-bound forms (with the sugar as well as the peptide) were determined, it was evident that no conformational flexibility in CDRs of 2D10 existed, instead it was evident that the plasticity in the interaction had helped in the manifestation of molecular mimicry. One interesting aspect of the study as pointed out by authors was that even if the potential for flexibility existed, it was not been utilized while recognizing both ligands.
Therefore, in order to address this conundrum, we began looking for other sugars and peptides which can bind to the 2D10 antibody with comparable affinities. Crystallographic analyses of 2D10 bound to five different sugars (methyl α D glucopyranoside, α D lactose, α1-3- Mannobiose, α1-6-Mannobiose, α1-3, α1-6-Mannotriose) carried out at 2.5 Å, 2.5A, 2.1Å, 2.75 A and 1.7 Å, respectively have given interesting insights underlying the basics of specificity in molecular recognition. Comparison of the structures has shown that the antigen combining site for sugars is constituted of CDR H3, L1 and L3 only. All the five sugars have an overlapping primary binding site (equivalent to the methyl α D mannopyranoside interacting region). This primary sugar binding site have been shown to accommodate same/similar as well as dissimilar sugars by utilizing plasticity in the interacting residues available in the antigen combining site.
132 The second sugars of the similar disaccharides (α1-3-Mannobiose, α1-6-Mannobiose) have been adjusted in the same direction but with utilizing different sets of interacting residues of the antibody paratope. However, the second sugar of dissimilar disaccharide (lactose in comparison to α1-3-Mannobiose, α1-6-Mannobiose) exploits different paratope space altogether. The trisaccharide (α1-3, α1-6-Mannotriose) was accommodated in the same site by differential positioning of the second and third sugar rings in the antibody paratope (in comparison to all disaccharides) as well as by utilizing conformational flexibility in the paratope region (mainly in CDR L1). This study had demonstrated that an affinity matured antibody can utilize atleast three different strategies in order to accommodate structurally similar/dissimilar sugars.
The structural proteomics of allergy seed proteome of eggplant (Solenum melongena), was explored. A 45 kDa protein, SM80.1 showing weak homology with other 7S vicilins for which preliminary crystallographic studies were presented earlier, was further refined building almost entire model of the protein. The model was refined at 1.5 Å to an Rfree of 0.22 and Rwork of 0.21. The overall crystal structure of SM80.1 indicates that it is a homotrimer consisting of 393 residues in each monomer of which only residues 274-293 are structurally disordered. A monomer subunit is composed of two similar domains further subdivided into a core and a loop sub domain. Each domain consists of 2 elements, a compact eight-stranded beta barrel having the “swiss roll” topology and an extended flexible fragment containing several short alpha helices. This is the first native structure in this family of proteins with only one disordered region. Each domain of SM80.1 forms a central cavity to facilitate ligand binding. It was found that N terminal β-barrel domain has a pyroglutamate molecule whereas an acetate molecule was present in the C-terminal domain. Superimposition of two domains shows that both the ligands exist exactly at the same position. These ligands may have a probable role in structural integrity and formation of swiss-roll topology. This protein might be acting as a depository of pyroglutamate and acetate, which are required in different metabolic pathways or could be a source of carbon and nitrogen in the germination process. Along with above Mg is also present in the structure. Surface electrostatic potential map of the protein showed an uneven distribution of charge on the protein surface. Although there was a little difference, the basic structure was close to those of 7S Adzuki bean, canavalin, AraH1 and phaseolin. All these structures have very low sequence identity, suggesting that multiple and varied sequences can yield similar three-dimensional structure and explaining evolutionary divergence.
Another protein, SM80.2, was also purified from the defatted seed powder by 80% ammonium sulphate fractionation. The purified protein corresponds to a molecular weight of 11.7 kDa as analyzed by mass determination using mass spectrometry. N-terminal sequencing was also done for the purified protein which identified 20 residues of the polypeptide. The purified protein shows homology with other known 2S albumin family proteins. The protein was crystallized to obtain hexagonal shaped crystals which belonged to space group P6, with unit- cell parameters a = b = 87.48, c = 49.67 Å. As no homologous crystal structures of closely related proteins were available, ab initio phasing was pursued utilizing eight inherited sulphur atoms (cysteine). Experimental anomalous intensities were used by the PHENIX package to obtain initial phase information. Data were collected on single crystal, first to 1.87 Å, which were used for high resolution structure refinement, and then to 2.5 Å, at synchrotron source (BM14, ESRF, Grenoble) which were used for SAD phasing. Program SOLVE successfully
133 identified in total seventeen sulphur sites from the Bijvoet pair differences. A preliminary model was built automatically by Autosol. A total of 150 resides were built and 8 chains were placed with Rwork and Rfree values of 38.32 and 41.81 respectively.
Future Plans
Bioinformatics and crystallographic analyses of antigen-antibody recognition as well as broader aspects of host-pathogen interactions will be continued with the ultimate goal to correlate the structural principles with physiological implications. Structural proteomics of plant seed allergens and pollen allergens from Oryza sativa will be continued towards crystallographic analysis and structure-function correlation.
Publications Original peer-reviewed articles
1. Tapryal S, Gaur V, Kaur KJ, Salunke DM* (2013) Structural evaluation of a mimicry recognizing paratope: plasticity in antigen–antibody interactions manifests in molecular mimicry. J Immunol 191: 456-463.
2. Sharma R, Lomash S, Salunke DM* (2013) Putative bioactive motif of tritrpticin revealed by an antibody with biological receptor like properties. PloS One 8: e75582.
3. Gill J, Jayswal P, Salunke DM* (2014) Antigen exposure leads to rigidification of germline antibody combining site. J Bioinfor Comput Biol (in press).
Review/Proceeding 1. Salunke DM*, Gill J, Dwevedi A (2013) Comparative structural proteomics of allergenic proteins from plant pollen. J Ind Inst Sci 94: 119-126.
*Corresponding author
134 Ribonucleases and heat shock proteins: Involvement in host defense
Principal Investigator Janendra K. Batra
Research Associates Manish Gupta Deepak Pandey
Ph.D. Students Ayush Attrey Priyanka Parijat Virendra K Patel Alla Singh Prajna Tripathi Santosh K Yadav
Theme of research
The work is focused on the following two major themes.
1. Investigation of the role of human ribonucleases, particularly eosinophil ribonucleases, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in host defense. Also, human ribonucleases, and natural protein toxins targeting RNA or ribosomes are being analyzed for structure-function relationships to understand their molecular mechanism of action, and to explore them to design knowledge-based recombinant toxins.
2. Investigation of crucial housekeeping proteins of M. tuberculosis for their role in survival and virulence of the pathogen. We are studying the functioning of caseinolytic protease (Clp) machinery, and RNase P mediated tRNA maturation in M. tuberculosis. Clp proteases regulate the expression of virulence genes, and also help bacterial pathogens in countering stress in the host. RNase P is a key housekeeping enzyme involved in tRNA maturation, and is structurally completely different in bacteria and human. Both, Clp proteins and RNase P could be promising drug targets in M. tuberculosis.
Objectives
A. Investigation of molecular mechanism of biological actions of human ribonucleases and their role in host defense. Our study is focused on understanding the mechanism of action of ECP and EDN, identification of their intracellular targets, and their modulation during eosinophil differentiation and maturation.
B. Construction and evaluation of recombinant toxins as potential therapeutics. The aim is to rationally design, engineer and characterize specific and potent recombinant toxins for targeted therapy employing human RNases, ribosome-inactivating protein, saporin and ribonucleolytic toxins, restrictocin.
135
C. Investigation of the involvement of Clp proteases in pathogenic mechanism of M. tuberculosis. We are addressing the molecular basis of the interplay between substrate, adaptor and various members of the Clp proteolytic machinery in M. tuberculosis to explore their involvement in survival and pathogenesis.
D. Structure-function analysis of ribonuclease P of M. tuberculosis. The objective of our study is to understand the mechanism of function of the mycobacterial RNase P.
Work reported in 2012-2013
Investigation of the involvement of Clp proteases in pathogenic mechanism of M. tuberculosis
Stress regulation and persistence mechanisms in Mycobacteria There are two heat shock protein repressors, HspR and HrcA annotated in the genome of M. tuberculosis. HspR and HrcA of M. smegmatis and M. tuberculosis were expressed in E. coli and recombinant proteins purified from inclusion bodies. The binding of mycobacterial HspR was investigated with three putative HAIR motifs upstream of dnaK, and one upstream of clpB gene. The study has identified regions in dnaK and clpB genes that bind HspR.
Structure-function analysis of Clp proteins of M. tuberculosis M. tuberculosis ClpX contains a zinc binding domain at the N-terminus and a single ATPase (AAA+) domain divided into a large ATPase domain, containing the Walker motif, and a small C-terminal ATPase domain. The recombinant M. tuberculosis ClpX protein showed an inherent ATPase and chaperonic activities. M. tuberculosis ClpX was found to function independently in ATP dependent manner to unfold the model substrate protein, GFP-ssrA, and trap the unfolded GFP-ssrA to keep it in an unfolded state. Further, using six internal deletion mutants within the small AAA+ domain of ClpX, we have identified two motifs, respectively spanning residues 316-336 and 356-376 to be required for oligomerization and in turn the chaperonic activity of M. tuberculosis ClpX. The mutants, lacking either of these motifs were defective in oligomerization, and lost the unfoldase and trapping activities.
The ClpX hexamer has a translocation channel, and three different pore loops called GYVG, pore 2, and RKH which play crucial role in binding the SsrA tag in its substrates. To establish the role of these loops in M. tuberculosis ClpX in SsrA-tag recognition, in each of these loops, a conserved amino acid residue was mutated to generate three ClpX mutants. The conserved tyrosine in the GYVG loop was found to be critical for the substrate interaction ability of ClpX.
Structure-function analysis of ribonuclease P of M. tuberculosis
To investigate the role of M. tuberculosis RNase P in vivo, we are making conditional knockouts of rnpA gene, encoding the RNase P protein in M. tuberculosis, H37Rv. As the
136 rnpA gene appears to an essential gene, we decided to integrate a copy of rnpA gene under inducible tetracycline promoter in the genome, before attempting to disrupt the endogenous copy. Through pTC-mcs vector, containing teracycline promoter and integrase gene from mycobacteriophage L5, the genomic integration of rnpA in sense orientation was achieved. In addition, one strain of M. tuberculosis that contains an integrated copy of rnpA gene in the reverse, antisense orientation was constructed. It is aimed to silence the endogenous rnpA locus by introducing stop codon through point mutations, using a recombineering plasmid pJV62.
Progress of work during the current reporting year (2013-2014)
Construction and evaluation of recombinant toxins as potential therapeutics
Anti-HIV activity of ribosome inactivating protein, saporin and ribotoxin, restrictocin The anti-HIV activity of saporin and restrictocin was investigated in HIV-1 infected TZM-bl cell line, which expresses CD4, CXCR4 and CCR5 and contains Tat-inducible Luc and β- Gal reporter genes, and CEM-GFP cell line, which contains Tat-inducible EGFP reporter gene.
Saporin showed a dose dependent inhibition of HIV propagation in both model cell lines. Further, we used three mutants of saporin, namely Y16A, W208A and Y72A, generated earlier in our laboratory, which respectively lack either N-glycosidase or DNA fragmentation or both activities to investigate if any of these two activities is important for the anti-HIV activity of saporin. Out of the three saporin mutants, only Y16A showed anti- HIV effect, which too was relatively less as compared to that of the wild type saporin. The other mutants did not show any anti-HIV activity even at higher concentrations. In all cases, the viability of cells was not affected at the concentrations of proteins where anti-HIV activity was observed. Further, the p24 levels were found to be reduced in a dose dependent manner in the supernatants of cells treated with saporin and Y16A mutant. The results indicate that for the anti-HIV activity of saporin its N-glycosidase and DNA fragmentation activities are important.
Restrictocin also showed a dose dependent anti-HIV effect in both the cell lines. Further, using three mutants of restrictocin, namely Y47A, H49A and H136A we investigated if the specific RNase activity of restrictocin was involved in its anti-HIV activity. The mutants Y47A and H49A lack the specific ribonuclease activity of restrictocin but they contain high nonspecific ribonuclease activity, whereas the H136A mutant is ribonucleolytically inactive. None of the mutants of restrictocin showed any anti-HIV activity in both the cell systems, suggesting that indeed the specific RNase activity of restrictocin is important for its anti- HIV activity. We also studied the effect of human pancreatic ribonuclease (HPR) and RNase A on HIV propagation to investigate if any potent RNase would show anti-HIV effect. Both, HPR and RNase A did not manifest any anti-HIV activity.
137 Investigation of the mechanism of apoptosis induction by saporin Earlier, we have shown that saporin induces apoptosis in mammalian cells through the intrinsic pathway. The current study was focussed onto identifying the triggers of apoptosis induced by saporin. Saporin induced time dependent PARP cleavage in Hela cells. Saporin treated Hela cells showed a dose and time dependent loss of mitochondrial membrane potential accompanied by a dose and time depended reactive oxygen species generation. We found SAP/JNK, ERK and p38 to be phosphorylated in Hela cells treated with saporin as compared to untreated control cells. However, the time kinetics of phosphorylation of the three kinases in saporin treated cells was different. There was a time dependent increase in the phosphorylation of SAP/JNK followed by a reduction beyond a time point. P38 also showed an increase in phosphorylation in a time dependent manner, however instead of a decrease there was a plateau at the later time points. ERK showed a similar increased level of phosphorylation at all time points. The levels of p21WAF/CIP1, a cell cycle protein involved in cell growth inhibition and ER stress regulating protein, Bip decreased with time in saporin treated Hela cells.
Investigation of the involvement of Clp proteases in pathogenic mechanism of M. tuberculosis
Stress regulation and persistence mechanisms in Mycobacteria In M. tuberculosis genome, the upstream region of groES and hrcA genes contains the probable CIRCE DNA binding site for HrcA. A radiolabelled 200bp fragment of this region was used to check the DNA binding ability of HrcA. With M. tuberculosis and M. smegmatis cell extracts, a dose dependent mobility shift of the 200bp DNA was observed. However, the recombinant HrcA of M. smegmatis and M. tuberculosis, purified by denaturation and renaturation from E. coli inclusion bodies, was unable to bind to this DNA. E. coli extract, which does not contain the HrcA protein, did not bring about any shift in the mobility of CIRCE DNA. To test if other cellular components are required for HrcA to bind to the CIRCE DNA element, the binding activity of recombinant HrcA was analysed in the presence of M. tuberculosis and M. smegmatis cell extracts. We observed a dose dependent increase in recombinant HrcA binding to the target DNA in the presence of mycobacterial cell extracts. Further, we also used crude cell extracts from a M. tuberculosis CDC1551 strain which lacks the hrcA gene. While the cell extract of hrcA knockout strain alone did not show any shift, upon addition of recombinant protein, there was appearance of a DNA- protein complex, which was dose dependent. These observations indicate that HrcA protein requires other factors from the mycobacterial cell extracts to be able to bind to CIRCE DNA. Further studies are going on to identify the interacting partner(s) of HrcA, which may be required for its DNA binding and in turn the transcriptional repression activity.
The DNA binding properties of HspR were analysed using a 50 bp DNA fragment from the HAIR motif, upstream of dnaK gene. A mutant of the DNA fragment, and a non specific DNA of 50 bp were used as controls. Both, HspR of M. smegmatis and M. tuberculosis bound to the HAIR DNA. HspR also showed binding to the mutant as well as the non- specific control DNA targets. However, HspR was found to have higher affinity towards HAIR element. Apparently, the specificity of HspR binding to HAIR element is rendered by other molecule(s) that remain to be identified. Further, the oligomeric status of recombinant
138 HspR from M. smegmatis and M. tuberculosis, purified by denaturation and renaturation from E. coli inclusion bodies, was analysed. The protein was found to exist in a dimeric form, which was not affected upon treatment with 1M KCl.
We are phenotypically characterizing the effect of various stress conditions on two knockout strains of hrcA and clpB genes of M. tuberculosis CDC1551. The wild type and knockout strains, without antibiotics had similar growth pattern, however in the presence of antibiotics the knockouts had reduced growth compared to the wild type. In response to a short or a continuous heat stress, as compared to the wild type CD1551 strain, the growth of the clpB knockout was significantly reduced, however the growth of the hrcA knockout was not affected. The preliminary observations indicate that both hrcA and clpB are required by the pathogen for its survival under adverse conditions.
Structure-function analysis of Clp proteins of M. tuberculosis The M. tuberculosis genome has revealed the presence of five members of the Clp family namely, ClpP1, ClpP2, ClpC1, ClpX and ClpC2, annotated as Rv2461c, Rv2460c, Rv3596c, Rv2457c and Rv2667 respectively. Though, the function of ClpC2 (Rv2667) is unknown, it appears to be a possible ATP-binding subunit of ATP-dependent protease. To investigate the function of mycobacterial ClpC2, we cloned and expressed it in E. coli, and purified the protein to homogeneity. ClpC2, unlike ClpC1, didn’t show any ATPase activity. Further characterization of the protein is underway.
Structure-function analysis of ribonuclease P of M. tuberculosis
On the basis of multiple sequence alignment, Phe23, Val27, Ala70, Arg72, Ala77, Arg93 and Asp124 were identified to be unique in the protein component M. tuberculosis RNase P. Eight variants of the M. tuberculosis RNase P protein component were generated that contained the unique residues substituted to those conserved in E. coli and B. subtilis. The CD analysis of these variants showed them to have a secondary structure similar to that of the wild-type mycobacterial RNase P protein. On the basis of the enzymatic activities of holoenzymes reconstituted with the variants, they could be grouped in three categories. One group that includes F23A, A70K and R72A variants showed significantly reduced activity; the second group that includes A77F and D124S had partial activity; and the third group comprising of V27F, R72L and R93A mutants showed non-specific activity on pre-tRNAAla. These observations suggest that the selected residues play different roles in different protein components with respect to their interaction with the respective RNA component.
Future plans 1. The assembly mechanism of Clp complex of M. tuberculosis will be investigated using recombinant ClpP1, Clp2, ClpX and ClpC1 and their variants. 2. The regulation of stress response in M. tuberculosis will be further investigated in vitro and in vivo. 3. The role of mycobacterial RNase P in pathogenesis of M. tuberculosis will be investigated by inhibiting its expression in vivo. Structure-function analysis of RNase P will be continued.
139 4. The anti-HIV activity of EDN, ECP and restrictocin will be further studied. 5. Intracellular targets of eosinophil proteins will be validated. The role of identified targets will be investigated in the activity of eosinophil proteins.
Action taken on the RAP/SAC 2013 recommendations
Clarifications sought, during the laboratory visit were provided to the satisfaction of the RAP/SAC members.
Publications Original peer-reviewed article
1. Shah U, Haquea MA, Zaidia S, Hassan MI, Islam A, Batra JK, Singh TP, Ahmad F* (2013) Effect of sequential deletion of extra N-terminal residues on the structure and stability of yeast iso-1-cytochrome-c. J Biomol Struct Dyn (in press).
Review/Proceeding
1. Chopra A, Batra JK* (2013) Antimicrobial activity of human eosinophil granule proteins. Meth Mol Biol (in press).
*Corresponding author
140 Role of carbohydrates in modulating the structure and function of glycopeptides
Principal Investigator Kanwal J. Kaur
Ph.D. students Deepti Sripad Lele PRV Shabareesh Rohini Dwivedi
Project Fellow Supriya Vishwakarma
Collaborator Dinakar M. Salunke
Theme of research
The project is aimed for understanding the role of carbohydrate domains in modulating the structure and function of glycopeptides by involving different model systems such as antimicrobial and thrombin-inhibitory glycopeptides.
Objectives
1. Synthesis and structural characterization of glycosylated amino acids. 2. Structure-function analysis of the synthetic glycoconjugates.
Work reported in 2012-2013
Antimicrobial peptides
Continuing the studies for understanding the role of glycosylation in the antibacterial activity, an example of disaccharide (Galβ(1→3)GalNAcα1-) containing antibacterial peptide, drosocin, was undertaken. The synthesis of disaccharide building block, Nα-Fmoc- Thr(Ac4-β-D-Gal-(1Æ3)-Ac2-α-D-GalNAc)-OH, was standardized by the methodology which involved sixteen steps from free galactose. The disaccharide containing drosocin (di- drosocin) was synthesized and then assayed for its comparative spectrum of antimicrobial activity against different bacterial strains. It was observed that though the di-drosocin exhibited higher level of antibacterial activity than non-glysoylated drosocin but it showed lower level of activity than mono-glycosylated drosocin against all the tested bacterial strains. The results indicated that the conformation of the peptide is affected by sugars and different sugars alter the conformation of same peptide differently.
141 Bacterial DnaK has been reported as one of the possible targets for the proline rich class of antibacterial peptides. In order to understand bioactive conformation of Drosocin, we need to have its structure in presence of its plausible receptor. Hence we cloned and expressed the substrate binding domain of E.coli DnaK. Its binding with mono-glycosylated as well as non- glycosylated analogues of Drosocin was confirmed using SPR. The co-crystallization trials were done with DnaK and Drosocin analogues.
Thrombin-inhibitory glycopeptides
With an aim to provide a structure based understanding of functionality in differentially glycosylated peptides, we have also undertaken model system of thrombin inhibitory glycopeptides because the receptor for this class of peptides, thrombin, is structurally well characterized. Expanding our repertoire of the differentially glycosylated variegin analogues, α-GalNAc and α-Mannosyl bearing variegin analogues were synthesized as well as Hirudin variants, α-GalNAc-Hirudin P6, non-glycosylated Hirudin P6 and Hirudin P18 were synthesized. Functional characterization of the non-glycosylated and mannosylated variegins was carried out for their abilities to inhibit thrombin amidolytic activity by chromogenic assay. Both the analogues of variegin showed the comparable IC50 values.
Progress of work during the current reporting year (2013-2014)
Antimicrobial peptides
Earlier, we have reported the effect of different sugars and their linkages on the activity of two Proline rich antibacterial glycopeptides, formaecin and drosocin. In glycoproteins, usually Thr and Ser amino acids are O-glycosylated on their side chain hydroxyl groups. It has been reported that the conformational impact of α-GalNAc attached to Thr residue differs significantly from that attached to Ser residue. To obtain insights into the effect of glycosylated amino acid variation on the structural and functional properties of drosocin, an analogue was synthesized by substituting its glycosylated-Thr at 11th position with glycosylated-Ser and named as S11-α-D-GalNAc-Drosocin. For synthesizing this α glycosylated peptide, first critical building block, N -Fmoc-Ser (Ac3-α-D-GalNAc)-OH was synthesized and then used for peptide synthesis.
We have shown earlier that drosocin possesses higher antibacterial activity than its non- glycosylated form. To compare the antibacterial activity of S11-α-D-GalNAc-Drosocin with its non-glycosylated variant, the S11-Drosocin was also synthesized. All the peptides were synthesized by solid phase synthesis following Fmoc chemistry and finally purified peptides were characterized by mass spectrometry.
In order to analyze the antibacterial effect of Thr substitution with Ser in Drosocin analogues, MIC of each peptide was determined against Gram negative bacterial strains of E. coli ATCC 25922, E. coli ATCC 11775, E. coli ATCC 35218, Salmonella typhimurium ATCC 14028
142 and S. typhi Vi+. Native mono-glycosylated Drosocin (T11-α-D-GalNAc-Drosocin) which contained glycosylation at Thr, displayed higher level of antimicrobial activity compared to other peptide analogues. Non-glycosylated Drosocin showed two fold increase in MIC against all the tested E. coli strains compared to that of Drosocin, whereas around seven to ten fold higher MIC than native glycosylated Drosocin was observed against Salmonella strains. These observations also confirmed the importance of sugar in antimicrobial activity of Drosocin. Substituting Thr with Ser in non-glycosylated Drosocin did not affect the MIC significantly, indicating that Ser or Thr alone did not determine differential antimicrobial behaviour of Drosocin peptides. The comparison of antibacterial activity of S11-α-D- GalNAc-Drosocin and Drosocin (T11-α-D-GalNAc-Drosocin) demonstrated that the MIC for S11-α-D-GalNAc-Drosocin was around five fold higher than T11-α-D-GalNAc-Drosocin against E. coli ATCC 25922 and ATCC 11755, whereas around two times higher against E. coli ATCC 35218 and twenty fold higher against tested Salmonella strains. The only difference between T11-α-D-GalNAc-Drosocin and S11-α-D-GalNAc-Drosocin was presence of glycosylated-Thr in former and glycosylated-Ser in later. These findings suggested that glycosylation of Thr was a prerequisite for displaying full antimicrobial activity of Drosocin. With just a difference of γ-methyl group, S11-α-D-GalNAc-Drosocin could not display lethal action comparable to that of T11-α-D-GalNAc-Drosocin.
Interestingly, S11-α-D-GalNAc-Drosocin showed around three times higher MIC than its non-glycosylated counterpart, S11-Drosocin, against bacterial strains of E. coli and Salmonella. This result was intriguing, suggesting that the addition of N-acetyl galactosamine was not the only reason for higher antimicrobial activity of T11-α-D-GalNAc-Drosocin. These observations further confirmed that there was a difference between the conformational impact of glycosylated-Thr and glycosylated-Ser on the peptide backbone.
To analyze the structural effects of substitution of glycosylated-Thr, the comparative conformational properties of Drosocin and its analogues were analyzed by CD spectroscopy in PB (10mM, pH7.4), 90% TFE, and 10mM SDS. All the peptides exhibited absence of any regular secondary structure in PB with a negative band at around 200nm. All the analogues showed decrease in mean residue ellipticity compared to Drosocin indicating destabilization of structure. In 90% TFE, the overall spectral profiles for all the analogues of Drosocin were generally similar whereas the absolute values of the molar ellipticities varied slightly among the analogues. The structure promoting effect of TFE was significantly less marked on the peptides. In presence of the membrane mimicking environment like SDS, all the peptides showed similar CD spectra which were characterized by two negative bands at around 200nm and 204nm somewhat red shifted but with slight opening as compared to that observed in TFE. In TFE and SDS environment, the peptides became more structured as shown by the deviation in the curves to a more positive ellipticity. The CD spectra of Drosocin and its analogues did not show any specific difference among themselves. Thus, the conformational studies using CD could not resolve the local conformational changes occurring in the peptide backbone due to modifications in Drosocin.
143 Thrombin-inhibitory glycopeptides
With an aim to study the impact of glycosylation on the inhibitory potential of active site directed, short peptidyl inhibitors of thrombin, two non-glycosylated tetra peptides were synthesized. The synthesized peptides were purified to homogeneity using HPLC and characterized by MALDI-TOF. Functional characterization of the above synthesized peptides along with few previously synthesized analogues was carried out by chromogenic assays and competitive fibrinogen assays. The results indicated that D-ChaPβhomoRG is more efficient than fPβhomoRG in inhibiting thrombin’s activity.
The inhibitory potential of Hirudin P6 C-terminal 23 residue peptide and its cognate glycosylated analogue (α-GalNAc HP6) was estimated by fibrinogen competitive assays. For this, 3nM of human thrombin was pre-incubated with different concentrations of peptides for 15mins at room temperature and the rate of fibrin polymerization was monitored by adding 3mg/ml of human fibrinogen at 37°C for 10mins at a wavelength of 405nm. These experiments were performed in triplicates and dose response curves were plotted to obtain the IC50 values. The results indicated the higher inhibitory activity of glycosylated HP6 (α- GalNAc HP6) than its non-glycosylated form and suggesting the effect of sugar in modifying the functional activity of thrombin-inhibitory peptide.
Future Plans The detailed comparative structure-function studies of glycosylated and non-glycosylated antibacterial peptides and thrombin-inhibitory peptides will be continued.
Action taken on the RAP/SAC 2013 recommendations
No specific recommendations were made.
Publications Original peer-reviewed articles
1. Lele DS, Talat S, Kaur KJ* (2013) The presence of arginine in the Pro-Arg-Pro motif augments the lethality of proline rich antimicrobial peptides of insect source. Int J Pept Res Ther 19: 323-330.
2. Tapryal S, Gaur V, Kaur KJ, Salunke DM* (2013) Structural evaluation of a mimicry- recognizing paratope: plasticity in antigen-antibody interactions manifests in molecular mimicry. J Immunol 191: 456-463.
*Corresponding author
144 Structural studies on proteins, dynamics and ligand interactions using NMR
Principal Investigator Monica Sundd
Ph.D. Students Ambrish Kumar Rohit Singh Dangi Usha Singh Usha Yadav
Research Fellow Pinakin Makhwana
Theme of research
The theme of our research is to understand the structure, backbone dynamics and interactions of proteins using NMR, and other biophysical techniques in order to understand their function. We are working on proteins involved in fatty acid metabolism in Leishmania viz. the acyl carrier protein, acyl CoA binding proteins, enzymes of type II fatty acid biosynthesis pathway etc.
Objectives
The primary objective of the project involves cloning, expression, purification and structural characterization of various proteins using NMR and other biophysical techniques to understand their interaction with naturally occurring partners with a goal to better understand their biological function. We are presently pursuing two projects in this regard that are interlinked:
1. Understanding the sructure and function of the type II fatty acid biosynthesis pathway of Leishmania spp. 2. Structural characterization of the proteins involved in fatty acid metabolism in Leishmania spp.
Work reported in 2012-2013
1. Understanding the sructure and function of the type II fatty acid biosynthesis pathway of Leishmania spp.
In the year 2012-2013, we completed the structure solution of Leishmania major holo-acyl carrier protein using NMR and also characterized its acyl-ACP intermediates. Our studies showed that the holo-ACP of Leishmania is a four helix bundle protein, with a hydrophobic cavity that protects the acyl chain. Using NMR, we also characterized the acyl ACP intermediates of Leishmania formed during fatty acid biosynthesis and showed that the ACP
145 molecule can protect upto C8-ACP. The acyl-ACP intermediates longer than C8- were highly unstable at room temperature in comparison to P. falciparum acyl-intermediates. We also carried out some in vivo studies on this pathway by expressing the associated proteins in Leishmania donovani and our data suggested that the ACP along with other enzymes like ENR, Phosphopantetheine transferase are localized in the mitochondria.
2. Structural characterization of the proteins involved in fatty acid metabolism in Leishmania
Last year, we had just initiated this work that involved cloning of the three ACBPs of L. major viz. ACBP89, ACBP96 and ACBP103.
Progress of work during the current reporting year (2013-2014)
1. Understanding the sructure and function of the type II fatty acid biosynthesis pathway of Leishmania spp.
After analyzing the NMR data on the acyl-intermediates of Leishmania ACP, we arrived at the conclusion that Leishmania type II pathway synthesizes only short acyl chains. Side by side, we determined the molecular mass of the acyl intermediates using MADLI-tof mass spectrometry. Mass spectrometry data supported our NMR findings that acyl-intermediates longer than C10-ACP were highly unstable. In case of C12- and C14-ACP, during enzymatic conversion of apo- to the acyl-intermediates, the acyl chain gets hydrolysed and hence displays the molecular mass of holo-ACP. Even C10-ACP displayed only 70% peaks corresponding to the intermediate form in our NMR 1H15N HSQC experiment and 30% peaks corresponding to holo-ACP supporting the observation that latter is extremely unstable.
In order to understand the molecular basis of the unusual specificity of Leishmania ACP, residues that are remarkably different in the latter compared to other type II ACPs were identified and mutated to the residues present in E. coli ACP. These involved mutations Leu35Ala, Asn36Asp, Phe45Met, His58Asp etc. and the samples were analyzed using NMR. Of these mutations, L35A failed to fold properly, suggesting an important role of Leu 35 in Leishmania ACP. Notably, this residue is conserved in all type I ACPs that do not form stable acyl-intermediates in solution. Rest of the mutants displayed a very similar acyl chain specificity as the wild type protein. Apart from these residues, we observed that seven additional hydrophobic residues are present in Leishmania ACP compared to other type II ACPs and most of them are localized in the hydrophobic core. Combining our NMR data, mass spectrometry data and mutagenesis studies, we posit that the presence of the hydrophobic residues makes the cavity of the ACP molecule rigid, thus preventing it from expansion during acyl chain elongation. It seems, Leishmania ACP can form acyl chains only upto the length it can accommodate in its core without expansion. C8-ACP is a precursor of lipoic acid synthesis, required for the functioning of several enzyme complexes. We speculate that this pathway has been designed by nature to fulfil the lipoic acid requirement in the mitochondria as it cannot synthesize long acyl chains.
146 2. Structural characterization of the proteins involved in fatty acid metabolism in Leishmania
In Leishmania, the amastigote stage (blood stage) depends largely on the beta oxidation of fatty acids while the promastigote stage (insect gut stage) thrives on glucose. A major fraction of this lipid requirement in the amastigote stage is supplied by the host. Hence, there must be proteins present in Leishmania that help in the dissemination of the acyl Co-As, generated in this process. In Leishmania, we have identified 3 ACBPs (putative acyl CoA binding proteins) that possibly perform this function. We have cloned, expressed and purified the three ACBPs of L. major in E. coli. The three proteins have been expressed in minimal media and all three proteins show good dispersion in an NMR spectrum. The chemical shifts of these proteins have been partly assigned. We also analyzed the binding affinity of these ACBPs towards standard acyl-CoAs like C4-, C8-, C10-, C12-, C14- C16-, C18- and C20-CoAs using ITC. The three ACBPs show very different affinities underscoring their importance in the survival of Leishmania.
Future plans
We plan to express the enzymes of the type II pathway in L. donovani, viz. Enoyl-ACP reductase, Phosphopantetheine transferase etc. in order to characterize them and study their interaction with the Leishmania ACP molecule. We are in the process of identifying the binding partners of Leishmania ACP as well as the ACBPs to better understand their mechanism of function. We also plan to structurally characterize the ACBPs (acyl CoA binding proteins) of Leishmania, and identify the residues that interact with the acyl CoAs.
Action taken on the RAP/SAC 2013 recommendation
No specific recommendation was made.
147 To develop strategies for making sensors and actuators for biological processes
Investigator Pramod K. Upadhyay
Ph.D. students Jashdeep Bhattacharjee Srikant Ayer Barun Das Alaknanda Mishra Preeti Sahay
Collaborators Sangeeta Bhaskar, NII Amulya K. Panda, NII Asok Mukhopadhyay, NII P. Nagarajan, NII P. Khanduri, St. Stephen’s Hospital, Delhi J.M. Puliyel, St. Stephen’s Hospital, Delhi Rajnish Juneja, AIIMS, New Delhi S. Ramakrishnan, AIIMS, New Delhi R. Lodha, AIIMS, New Delhi Sara Varughese, Christian Blind Mission, Bengaluru Aseem Bhatnagar, INMAS, Delhi Subash Khushu, INMAS, Delhi
Theme of research
To develop systems for monitoring biological processes.
Objectives
1. To develop tools for needle free immunization. 2. To study the biological processes like differentiation, hybridization etc. and to develop devices and sensors based on such studies.
Work reported in 2012-2013
A. Ex-vivo 3D liver culture
The successful generation of hepatocyte like cells in vitro will have clinical applications as it may give a cell based therapeutic option for patients who are suffering from liver cirrhosis. Hematopoietic stem cells (HSCs) are known to differentiate to hepatocytes in vitro but it is difficult to get significant number of HSCs from adults. Earlier we have shown how to
148 reprogram peripheral blood mononuclear cells (PBMCs) to ‘hematopoietic stem like cells’. The hepatocytes like cells from the monocytes of HBV infected (HBsAg positive) patient were generated. After 6 days of reprogramming the monocytes and 14 days of differentiation, the cells were examined for the presence of cytosolic albumin and Connexin 32. To characterize the differentiated cells at transcript level relative expression of transcripts in differentiated hepatocytes like cells derived from the monocyte of HBsAg positive blood sample were determined with respect to HepG2 (positive control) and hepatocytes like cells derived from the monocyte of healthy blood sample. It was found that hepatocyte like cells have expression of Albumin and HNF-4α.
B. Trans-differentiation of PBMCs to endothelial-like cells
Endothelial cells occupy the core position in the successful treatment of vascular diseases. PBMCs can be trans-differentiated to endothelial like cell by culturing them in an appropriate angiogenic medium. A composite culture of PBMCs obtained by means of a density gradient centrifugation was cultured in EGM-2 medium, supplemented with a cocktail of angiogenic growth factors including VEGF, EGF, FGF and IGF and fortified with serum. After 30 days in culture, the PBMCs differentiated into endothelial-like cells as shown by the expression of various endothelial markers verified by immunostaining, western bloting and RNA expression profiling using qPCR.
Progress of work during the current reporting year (2013-2014)
A. Differentiation of PBMCs to endothelial-like cells
PBMCs can be differentiated to endothelial like cell by culturing them in an appropriate angiogenic medium. PBMCs being abundant in number and easily harvestable, they can be a promising source of autologous endothelial cells for use in vascular treatment. Different approaches were investigated for differentiating human PBMCs into endothelial cells.
In the first approach, endothelial progenitor cells (EPCs) that are found in very minute amounts (around 0.04%) in the peripheral blood were differentiated into endothelial cells. EPCs are a population of highly proliferative, bone marrow derived progenitor cells found in the peripheral circulation. They were isolated by magnetically labeled antibody against CD133. The CD133+ cells were then cultured in IMDM medium with 10% serum in a fibronectin coated dish at a density of 3 x 104 cells/cm2. Characterization of the differentiated cells were done by incorporating DiI labeled acetylated low density lipoprotein (DiI-acLDL) in the culture medium and further incubating for 4 h at 37oC. The nucleus was counterstained by DAPI. Endothelial cells have the characteristic feature of uptaking acLDL since they are unique in having surface receptors for it. Figure 1 shows the uptake of DiI-acLDL by differentiated endothelial cells.
149
Figure 1: Uptake of DiI-acLDL by differentiated endothelial cells.
In the second approach, the PBMCs were trans-differentiated into endothelial cells by culturing them in EGM-2 medium supplemented with angiogenic cytokines like VEGF, FGF, IGF in addition to hydrocortisone and heparin. The cells were plated at a density of 1 x 106 cells/cm2 in fibronectin coated culture dish for a period of 30 days at 37oC. Epidermal growth factor, a potent mitogen was incorporated into the medium to induce proliferation of the differentiated cells. The characterization of the cells was done by RTqPCR, western blotting as well as immunocytochemistry. The RTqPCR analysis showed that after 30 days of culture there was a decrease in the expression of CD14 which is a monocytic marker by the differentiated endothelial cells, while there was an increase in the expression of various endothelial markers like CD31, VE-Cadherin, Von Willebrand factor (vWF), VEGFR1 and VEGFR2. The Ki67, a marker for proliferation, showed a 4 fold increase as compared to the Day 1 cells, indicating that the differentiated endothelial cells also show proliferation. The western blot of differentiated endothelial cells also confirmed the expression of vWF and VE cadherin which were absent in day 1 cells. The immunocytochemistry pictures of differentiated endothelial cells expressing vWF and VE cadherin are shown in Figure 2.
Figure 2: Immunocytochemistry of differentiated cells for vWF and VE-cadherin (200X).
In the third approach, a two steps procedure was followed for the differentiation of monocytes into endothelial cells. In the first step, the monocytes were de-differentiated into stem cell like cells called reprogrammed monocytes (RM). The de-differentiated medium consisted of IMDM incorporated with growth factors like IL3 and MCSF with serum at
150 0.5%. The cells were seeded at a density of 1 x 106 cells/ cm2 in a fibronectin coated culture dish and the culture was continued for 6 days to obtain reprogrammed monocytes. In the next step, the reprogrammed monocytes were again re-differentiated into endothelial cells by culturing them in medium containing 10% serum and 100 μg/ml endothelial cell growth supplement (ECGS) for 15 days. The endothelial-like cells obtained at the end of the culture term were characterized by RTqPCR and western blotting. In the RTqPCR analysis similar to trans-differentiation results were obtained; there was an increase in the expression of various endothelial markers like CD31, VE-Cadherin, von Willebrand factor (vWF), VEGFR1 and VEGFR2. However in the re-differentiation approach, the Ki67, a marker for proliferation, shows a 10 fold increase as compared to the Day 1 cells, indicating that the re-differentiated endothelial cells are highly proliferative. These observations were confirmed by the western blot. Decellularization of a rat blood vessel A blood vessel is composed of a collagen matrix on which are overlaid the vascular cells i.e. endothelial cells on the luminal side and smooth muscle cells on the external surface. An allogenic or xenogenic transplantation of this vessel can lead to acute rejection of the graft. Decellularization removes the cellular component of the graft leaving behind a collagen scaffold. The structure of collagen is conserved through species making this acellular collagen scaffold non-immunogenic. Decellularization was carried out by running 1% triton-x 100 in MQ water through the vessel using a perfusion pump setup. The time period of decellularization was determined by a kinetic study in which the graft was decellularized for different time periods and stained by Hematoxylin and Eosin to check the efficiency of decellularization. The process of decellularization was completed in 24 h. It was confirmed by the complete absence of nuclear content (Hematoxylin stain). The decellularized rat blood vessel was checked for its potential to elicit an immune response. A 1 cm length of the rat blood vessel, either decellularized or non-decellularized with intact cellular component was implanted subcutaneously into 2 groups (n = 3) of Balb C mice. The mice implanted with decellularized vessel does not show the presence of IFNγ (an inflammatory cytokine) in the serum, whereas those implanted with non-decellularized vessel shows high serum IFNγ.
B. Differentiation of PBMCs to hepatocyte-like cells
The reprogramming of PBMCs was further investigated. It was observed that the concentration of serum played a critical role in the degree of ‘acquired plasticity’ in the reprogrammed Monocytes (RM). Non activated monocytes were isolated from the peripheral blood of healthy volunteer and cultured with four different types of serums namely, autologous human serum (AHS), human cord serum (HCS), embryonic stem cell grade fetal bovine serum (ESC) and fetal bovine serum (FBS). After six days of culture, the cells were harvested and analysed by flowcytometry for the abundance of CD34 and CD117 (hematopoietic stem cell markers) on the cells having phenotype CD45+CD14+CD16+. The sample size was n = 5. The extent of ‘acquired plasticity’ in RM, as measured by the
151 percentage abundance of CD34 and CD117, was highest by HCS followed by ESC, AHS and FBS. Similar observations were made in the reprograming of monocytes isolated from HBsAg (positive) blood in which n = 9.
Engrafting RM and hepatocyte like cells
Partial hepatectomy of left liver lobe of SCID mice were performed to generate the liver injury model in immune-compromised mice. In order to check the engraftment of administered reprogrammed or differentiated human cells in the regenerated portion of mouse injured liver, cells were transplanted intraspleenically immediately after the partial hepatectomy. 24 days after partial hepatectomy and cell transplantation the mouse was euthanized and the regenerated liver was isolated. The presence of human specific Glyceraldehyde-3-phosphate dehydogenase (hu-GAPDH) was observed in RT-PCR of different liver lobes and spleen. Detailed investigations are underway.
Future plans
1. To establish the 3D culture of hepatocytes like cells in a recellurised liver and compare its biochemical activity with that of intact liver. 2. To construct small diameter vascular grafts by using decellurised vessels and differentiated PBMCs. 3. To investigate aerogenic route immunization technology on non-human primates.
Action taken on the RAP/SAC 2013 recommendation
No specific recommendation was made.
Publications Original peer-reviewed articles
#1. Roy A, Singh M, Upadhyay P*, Bhaskar S* (2013) Nanoparticle mediated co-delivery of Paclitaxel and a TLR-4 agonist leads to tumor regression and enhanced immune response in the tumor microenvironment of mice. Int J Pharm 445: 171-180.
2. Arindkar S, Bhattacharjee J, Kumar JM, Das B, Upadhyay P, Asif S, Juyal RC, Majumdar SS, Nagarajan P* (2013) Antigen peptide transporter 1 is involved in the development of fructose-induced hepatic steatosis in mice. J Gastroenterol Hepatol 28: 1403-1399.
3. Bhattacharjee J, Kumar JM, Arindkar S, Das B, Pramod U, Juyal RC, Majumdar SS, Nagarajan P* (2014) Role of immunodeficient animal models in the development of fructose induced NAFLD. J Nutr Biochem 25: 219-226.
152 Reviews/Chapters/Etc.
1. Upadhyay P* (2013) Tuberculosis vaccine trials. The Lancet 381: 2253-2254.
2. Upadhyay P* (2013) New therapeutic approaches for airway hyperimmune response are required. Indian Pediatr 50: 1087.
*Corresponding author(s) #In press last year, since published
153 Protease-catalyzed splicing of peptide bond
Principal Investigator Rajendra P. Roy
Ph.D. Students Tora Biswas Shikha Singh S Vijay Kumar Prity Yadav Avinash Kumar
Collaborators D. Sehgal, NII S Ramakumar, IISc, Bangalore D Chowdhury, JNU, New Delhi
Theme of research
We study the principles underlying peptide ligation reactions catalyzed by proteases and transpeptidases with a view to apply them to the semisynthesis of proteins, assembly of well defined bioconjugates and protein dendrimers that may be useful in a variety of biotechnological applications. Transpeptidase sortase that catalyzes covalent anchoring of surface proteins to the cell wall in gram-positive bacteria has turned out to be a wonderful enzyme in this endeavor. The propensity of sortase to ligate LPXTG proteins to an aminoglycine-derivatized moiety offers unprecedented opportunities in synthetic protein chemistry.
Objectives
1. Sortase-mediated protein labeling and conjugation 2. Studies on structure, dynamics and function of sortases.
Work reported in 2012-2013
Objective 1
We had developed a bioorthogonal Sortase-Click reaction suite for defined assembly of multivalent proteins by coupling sortase-mediated ligation and azide-alkyne Huisgen cycloaddtion reactions. We had utilized lysine-based multiple antigenic peptide (MAP) and β-cyclodextrin (β-CD) scaffolds for this purpose.
154 Objective 2
Delineation of catalytic residues of pSrtA (archetypal sortase A from S. pneumoniae) through a combination of mutagenesis and kinetic experiments was reported. Preliminary results about crystal structure of pSrtA were presented. The mechanism of substrate recognition by housekeeping sortase of S. aureus (SrtA) and its linkage to enzyme dynamics was described using experiments and simulations.
Progress of work during the current reporting year (2013-2014)
Objective 1
During the period of present review, several dendritic scaffolds representing a variety of shapes and pattern were synthesized and characterized. These linear, branched or cyclic scaffolds were designed to display orthogonal groups compatible with labeled proteins. For this, ubiquitin and a immunogenic domain (D3) of RrgB, a major pilin protein of S. pneumoniae were expressed with a sortase-recognition sequence. The respective purified proteins were labeled with clickable groups and conjugated to the desired multivalent complementary dendritic scaffolds. Mass spectrometric characterization in each case defined the homogeneity of the protein dendrimers. Together, these results establish the generality of Sortase-Click reaction as a method of choice for well defined assembly of multivalent proteins. The current efforts are geared toward adapting the process to a copper-free click chemistry module and innovating ways to introduce two or more proteins on a single scaffold.
Contemporaneously, we have also been seeking strategies for semisynthesis of Ubiquitin or SUMO conjugates that can serve as substrate for deubiquitinating or desumoylating enzymes. The synthesis of isopeptide-liked chemically defined protein/peptide substrates represents a challenge and a chemo-enzymatic approach can be very useful in this regard.
Objective 2
In the past year, studies on structural aspects of substrate recognition by sortase A (SrtA) were continued. The LPXTG recognition motif located near the C-terminus of bacterial protein is always followed by a stretch of residues. Therefore it was pertinent to see if an isolated LPXTG pentapeptide would serve as an effective SrtA substrate. We first examined the propensity of three LPXTG pentapeptide sequences containing Glu, Ala or Asn respectively at the X position to undergo SrtA-mediated transpeptidation reaction. Interestingly, the native LPXTG pentapeptide sequences (X= A, E or N) could be converted into a productive substrate by capping their ends with acetyl and amide groups respectively. Consistent with this, presence of an Ala residue (ALPNTGA) at the termini elicited the same effect and resulted in similar or slightly better yield of the product. Together the above results show that the charge neutrality achieved at the termini of the LPXTG motif either by acetylation/amidation or due to the presence of an additional residue exerts similar effect on
155 the transpeptidation reaction. This could also be interpreted to mean that the amide or peptide linkage (-CONH-) flanking the LPXTG sequence may have some role to play in the recognition of a catalytically competent substrate. In order to see that this was indeed true, we replaced the acetyl group by a methyl and subjected the N-methylated and C-amidated LPNTG peptide to transpeptidation reaction. Interestingly, this peptide behaved much like peptides with free termini and did not form any product thereby suggesting a crucial role of the peptide bond preceding the Leu residue in the process of productive substrate recognition by SrtA.
To further elucidate the chemical features of a minimalist bona fide productive SrtA substrate, we explored the propensity of threonine amide as substrate0. We reasoned that intrinsic amidase activity of SrtA may facilitate acylation of an amide substrate to form an acyl-enzyme intermediate. Accordingly, a tetrapeptide Ac-LPNT-NH2 was synthesized and subjected to transpeptidation reaction. Under standard conditions, SrtA catalyzed the transpeptidation reaction albeit at a slower rate but produced higher equilibrium yield relative to counterpart LPNTG pentapeptide. The reason for the higher conversion in the case of Ac- LPNT-NH2 is perhaps linked to the reversibility of the transpeptidation reaction which is somewhat compromised because ammonia byproduct may not be as good a substrate for reverse reaction as aminoglycine. SrtA-mediated transpeptidation reaction with an another version of the above peptide (Ac-LPNT-NH-CH3) carrying a methyl substitution in the amide (threonine methylamide) occurred with a relatively slower rate but produced equilibrium yield that compared well with Ac-LPNTG-NH2. Taken together, our results establish acetylated and amidated LPNT tetrapeptide as the minimalist recognition motif of SrtA and show that the LPETG peptide seen in the crystal structure of SrtA-substrate complex is not bound to the enzyme in a catalytically competent state.
Future plans
Investigations involving basic and applied aspects of sortase enzymes will continue.
Action taken on the RAP/SAC 2013 recommendation
No specific recommendations were made.
156 Therapeutic interventions in chronic diseases
Principal Investigator Sarika Gupta Research Associates Archana Saini Sakshi Arora Tandrika Chattopadhyay
Ph.D. Students Rajiv Ranjan Singh Nuzhat Ahsan Kapil Manglani Madhuraka Pal Ibrar Ahmed Siddique
Research fellow Mayuri Khandelwal
Collaborators A. Surolia, IISc, Bangalore Biswajit Kundu, IIT Delhi
Theme of research
My group is a multi-disciplinary group adapting an integrated approach in drug discovery that combines medicinal chemistry, basic biology and biochemistry principles for efficient drug design process. Interests of the group lie in identifying underlying principles in a disease pathogenesis to discover new targets, designing molecular intervention strategies and confirming the biological/therapeutic activities of the designed compounds. The small molecule regulators contribute to both drug development and understanding biological systems in human body.
Objectives
1. To find out whether membrane cholesterol and AMPK / ACC pathway play any role in simvastatin induced neuritogenesis (SIN). We choose simvastatin (SIM) because of its well known role as a therapeutic agent in various neurological diseases and inducer of neuritogenesis. SH-SY5Y cells were used as target cells because of their ability to develop well differentiated neurites. 2. Studies have demonstrated that a moderate intake of amino acids is associated with development of bone health. Methionine, a sulphur-containing essential amino acid, has been largely implicated for improving cartilage formation, however its physiological significance on bone integrity and functionality have not been elucidated. We investigated whether methionine can prevent osteoporotic bone loss.
157 Work reported in 2012-2013
Homocysteine, the by-product of methionine metabolism aggravated bone remodelling through a set of oxidative events that impaired normal synthesis pattern of OPG and RANK ligand in the osteoblast. We discovered that homocysteine acted via activation of protein phosphatase 2A (PP2A), a negative regulator of insulin signaling that inhibited normal expression of redox regulator forkhead O1 (FOXO1) which was integral for OPG synthesis in the osteoblast. An increased RANK ligand synthesis was found to the consequence of activated c-Jun/JNK MAP kinase signaling in the osteoblast. These untoward effects of homocysteine were alleviated in the presence of a modified sulphur containing amino acid, N-acetyl cysteine.
Progress of work during the current reporting year (2013-2014)
1. Simvastatin induced neurite outgrowth unveils role of cell surface cholesterol and acetyl CoA carboxylase in SH-SY5Y cells
Statins are known to modulate cell surface cholesterol (CSC) and AMP-activated protein kinase (AMPK) in non-neural cells; however no study demonstrates whether CSC and AMPK may regulate simvastatin induced neuritogenesis (SIN). We found that simvastatin (SIM) maintains CSC as shown by Fillipin III staining, Flotillin-2 protein expression / localization and phosphorylation of various receptor tyrosine kinases (RTKs) in the plasma membrane. Modulation of CSC revealed that SIN is critically dependent on this CSC. Simultaneously, phospho array for mitogen activated protein kinases (MAPKs) revealed PI3K / Akt as intracellular pathway which modulates lipid pathway by inhibiting AMPK activation. Though, SIM led to a transient increase in AMPK phosphorylation followed by a sudden decline; the effect was independent of PI3K. Strikingly, AMPK phosphorylation was regulated by protein phosphatase 2A (PP2A) activity which was enhanced upon SIM treatment as evidenced by increase in threonine phosphorylation. Moreover, it was observed that addition of AMP analogue and PP2A inhibitor inhibited SIN. Bio-composition of neurites shows that lipids form a major part of neurites and AMPK is known to regulate lipid metabolism majorly through acetyl CoA carboxylase (ACC). AMPK activity is negative regulator of ACC activity and we found that phosphorylation of ACC started to decrease after 6 hrs which becomes more pronounced at 12 h. Addition of ACC inhibitor showed that SIN is dependent on ACC activity. Simultaneously, addition of Fatty acid synthase (FAS) inhibitor confirmed that endogenous lipid pathway is important for SIN. We further investigated SREBP-1 pathway activation which controls ACC and FAS at transcriptional level. However, SIM did not affect SREBP-1 processing and transcription of its target genes likes ACC1 and FAS. This study highlights a distinct role of CSC and ACC in SIN which might have implication in process of neuronal differentiation induced by other agents. In conclusion, this study unravels a co-ordinated action of CSC and ACC in SIN. We highlight two major events involved during SIN: 1) retention of CSC which acts as stabilizer to orchestrate signaling events necessary to promote neuritogenesis; 2) initiation of fatty acid biosynthesis by ACC activation through PP2A phosphatase dependent de-phosphorylation of AMPK (Figure 1 & 2).
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Figure 1: Dephosphorylation of AMPK by PP2A phosphatase promotes neuritogenic effect of SIM in SH- SY5Y cells. Immunoblot in A, B, C and D showing modulation of protein expression of AMPK, p-AMPK (Thr-172), Akt, pAkt (ser-473), pAkt (Thr-308), PP2Ac and PP2Ac (p-Tyr) after SIM treatment for 12 hrs. A) protein expression of AMPK and p-AMPK (Thr-172) at 2 hrs, 6 hrs and 12 hrs after SIM treatment. B) AMPK and p-AMPK (Thr-172) levels at 6 hrs in presence of SIM and PI3K inhibitors like wortmannin and LY294002, 10 μM each. C) Akt, pAkt (ser-473) and pAkt (Thr-308) levels at 2 hrs, 6 hrs and 12 hrs after SIM addition. p < 0.05* pAkt (Thr-308). D) Time dependent decrease in tyrosine phosphorylation of PP2Ac after SIM treatment till 12 hrs. Total PP2Ac protein levels remained same during SIM treatment. Simultaneously, inhibition of PP2A activity by Fostriecin showed increase in pAMPK (Thr-172) levels in presence or absence of SIM at 6 hrs E) Morphology of SH-SY5Y cells at 10 X magnification under light microscope showing significant (p < 0.001) decline in neuritogenic effect of SIM in presence of Fostriecin and an AMP analogue i.e. AICAR. Error bar graph shows the difference in neurite length between SIM, SIM + Fostriecin and SIM + AICAR treated cells for 12 hrs (mean ± SEM; N = 50 cells per condition from 3 separate cultures). Scale Bar = 100 μm.
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Figure 2: Dephosphorylation of AMPK substrate i.e. acetyl CoA carboxylase (ACC) initiates neuritogenic effect of SIM. A) Immunoblot showing SIM induced change in phosphorylation of ACC over a period of 2 hrs, 6 hrs and 12 hrs. SIM led to a significant (p < 0.05*) increase in pACC (Ser-79) levels at 2 hrs which was followed by a gradual decrease. Total ACC levels showed no change during SIM treatment. Error bar graph shows fold change (normalized to control) in pACC (Ser-79) levels at different time points (mean ± SEM; n = 4). B) Immunoblot showing modulation of ACC phosphorylation by SIM follows AMPK pathway. As evident, addition of AICAR (an AMPK activator) increased the phosphorylation of ACC either alone or in presence of SIM till 12 hrs. C) Morphology of SH-SY5Y cells under light microscope showing significant (p < 0.001) inhibition of SIM induced neuritogenesis in presence of acetyl CoA carboxylase (ACC) inhibitor i.e. 10 μM TOFA and fatty acid synthase (FAS) inhibitor i.e. 10 μM Cerulenin (Cerul.). Error bar graph shows the comparative difference in neurite lengths SIM, TOFA, SIM + TOFA, Cerulenin and SIM + Cerulenin treated cells for a period of 12 hrs (mean ± SEM; N = 50 cells per condition from 3 separate cultures). Morphology of PC12 cells under light microscope in presence of NGF or NGF + TOFA for a period of 3 days. Scale Bar = 100 μm.
2. Methionine down-regulates TLR4/MyD88/NF-κB signalling in osteoclast precursors to reduce bone loss during osteoporosis
Post menopausal osteoporosis is a common form of osteoporosis in women characterized by high bone turnover and negative remodelling balance. The incidence of post-menopausal osteoporosis is 1/10th of women aged 60, 1/5th of women aged 70 and 2/5th of women aged 80. Osteoporotic medications like anti-resorptive drugs (that slow down bone resorption) and anabolic drugs (that increase the rate of bone formation) have not been able to alleviate osteoporosis mainly because of different responses by patients to the same medicine.
In the present study, methionine, a nutritionally essential amino acid has been employed as a tool to evaluate whether an anti-osteoporotic pharmacological agent can perform similarly at varying lengths of time. Administration of an essential amino acid methionine to a rat osteoporotic system prevented pathophysiological bone resorption (Figure 3). Methionine
160 inhibited trans-differentiation of blood mononuclear cells to functional osteoclasts and this was due to its ability to down-regulate TLR-4/MyD88/NF-ĸB cascade in developing osteoclasts, a signaling mechanism that was originally thought to be involved only in pathogen recognition (Figure 4). A combination therapy of Alendronate (bisphosphonate) + methionine significantly improved bone physiology of osteoporotic rats (even at a significantly lower dose of Alendronate) highlighting methionine as a pharmacological drug for anti-resorptive therapy. Intriguingly, we discovered that after a deflection point (higher doses and prolonged therapy), methionine turned from a pharmacological agent to a hyperhomocysteinemia inducing substance producing many pathophysiological changes similar to that of cancellous osteopenia, the beginning stage of osteoporosis.
Figure 3: Effect of administration of methionine on bone physiology. A. & B. Changes in the level of CTX and TRAP5b in serum samples of normal, ovariectomized and treatment groups during the experimental period. Methionine at doses 100, 250 or 500 mg/kg body weight was administered to rats in drinking water for 10 weeks. Alendronate (200 µg/kg body weight) was administered orally to ovariectomized rats at an interval of 5 days for one month. For combination therapy, rats were administered alendronate (100 µg/kg body weight) and methionine (250 mg/kg body weight). Both the treatments were performed 6 hours apart. CTX and TRAP5b were measured by immunoassay. Values are mean ± SEM; n=4. *P<0.05 compared to normal rat. **P<0.05 compared to ovariectomized rat. ***P<0.05 compared to alendronate (200 µg/kg body weight) administered OVX rats. C. Panel 1: Radiographs of femur were acquired on Kodak FX Pro in the X-ray mode. Each animal
161 received an approximate radiation dose of 1.4 Rad during imaging. Panel 2: Images of femoral bone head of normal and experimental rats acquired using Image J after H&E staining. Red arrows indicate changes in growth plate. Panel 3: H&E staining of femur showing changes in trabecular structure between normal and experimental rats. D. M-CSF and soluble RANK ligand levels in serum by ELISA. Values are mean ± SEM; n=4. *P<0.05 compared to normal rat. **P<0.05 compared to ovariectomized rat.
Figure 4: Effect of methionine administration on osteoclastogenesis in vivo. Methionine at a dose of 250 mg/kg body weight was administered in drinking water for 10 weeks. Alendronate (200 µg/kg body weight) was administered orally to ovariectomized rats at an interval of 5 days for one month. For combination therapy, rats were administered alendronate (100 µg/kg body weight) and methionine (250 mg/kg body weight). Both the treatments were performed 6 hours apart. Blood mononuclear cells isolated from these rats were cultured in the presence of M-CSF (25 μg/L, days 1-11) and RANKL (20 μg/L, days 6-20) ex vivo. A. Representative histogram showing changes in TRAP5b secretion in response to methionine and alendronate administration. Values are mean ± SEM; n=4. *P<0.05 compared to normal rat. **P<0.05 compared to ovariectomized rat. B. Changes in pro-inflammatory cytokine production in blood derived osteoclast cultures ex vivo. Pro- inflammatory cytokines were assayed by multiplex kits from Millipore on Bioplex 200TM (Bio-Rad, Hercules, CA, USA). Values are mean ± SEM; n=4. *P<0.05 compared to normal rat. #P<0.05 compared to ovariectomized rat. C. & D. Changes in the expression status of TLR-4, MyD88 and NF-κB in developing osteoclasts following ovariectomy and methionine or alendronate treatment by Western blot. *P<0.05 compared
162 to basal. Values are mean ± SEM; n=4. #P<0.05 compared to ovariectomized rat. ƚ P<0.05 compared to alendronate administered rat.
Future plans
1. Screening of small molecular inhibitors of amyloid beta, alpha synuclein and transthyretin aggregation and their preventive and therapeutic efficacy in animal models. 2. Understanding the mechanism of cytotoxicity of transthyretin oligomers. 3. Role of Bone Morphogenetic Proteins and testosterone in Glucose Homeostasis and Diabetes. 4. To study the interaction of ERAP with -synuclein and role in Parkinson’s disease. 5. Investigating the role of gelsolin as a common cellular player for modulating amyloid load and neurodegeneration.
Action taken on the RAP/SAC 2013 recommendations
All suggestions by the RAPSAC members were addressed and incorporated in the research program.
Publications Original peer-reviewed articles
#1. Vijayan V, Khandelwal M, Manglani K, Ranjan Singh R, Gupta S*, Surolia A* (2013) Homocysteine alters osteoprotegerin/RANKL System in the osteoblast to promote bone loss: Pivotal role of redox regulator forkhead O1. Free Radic Biol Med 61C: 72-84.
2. Raina V, Gupta S*, Yadav S, Surolia A* (2013) Simvastatin induced neurite outgrowth unveils role of cell surface cholesterol and acetyl CoA carboxylase in SH-SY5Y cells. PLoS One 8: e74547.
3. Vijayan V, Khandelwal M, Manglani K, Gupta S*, Surolia A* (2014) Methionine down- regulates TLR4/MyD88/NF-κB signalling in osteoclast precursors to reduce bone loss during osteoporosis. Br J Pharmacol 171: 107-121.
4. Pathak, CM, Singh RR, Yadav S, Kapoor N, Raina V, Gupta S*, Surolia A* (2014) Evaluation of Benzothiophene carboxamides as analgesics and anti-inflammatory agents. IUBMB Life (in press).
163 Patents Granted
1. Avadhesha Surolia, Sarika Gupta, Mahendra Pal Singh, Tandrika Chattopadhyay. Composition useful for treatment of diabetes & disorder. EP2133091, Granted on 19.04.2013.
2. Avadhesha Surolia, Sarika Gupta, Mahendra Pal Singh, Tandrika Chattopadhyay. Composition useful for treatment of diabetes & disorder. USA 12/419,669, Granted on 23.04.2013.
*Corresponding authors
#In press last year, since published
164 Chemical glycobiology: Glycoform modulation, carbohydrate-based drug design, and glycomics
Principal Investigator Srinivasa-Gopalan Sampathkumar
Ph.D. Students Asif Shajahan Syed Meheboob Ahmed Surbhi Goswami Vandita Dwivedi
Collaborators C. V. Ramana, NCL, Pune B. L. V. Prasad, NCL, Pune
Theme of research
Our laboratory of chemical glycobiology strives to develop carbohydrate-based small molecules as probes, tools, and inhibitors to shed light on the importance of glycosylation in biological processes. Understanding the functional roles of glycocalyx – which covers the mammalian cells consisting of glycoproteins, glycolipids, glycosaminoglycans, and glycosylphosphatidyl inositol anchors – has been challenging due to inherent complexity and non-template driven biosynthesis. We design and synthesize novel molecules that intercept glycan biosynthesis and enable bio-orthogonal functionalization. We investigate effects of these molecules in vitro in mammalian cells, characterize changes to glycosylation using biochemical, cell biological, and glycoproteomic methodologies, and in vivo in living animals.
Objectives
Our main objective is to harness the power of carbohydrate-based synthetic small molecules to unravel the importance of glycosylation in biological systems, as follows: 1. Development of non-natural monosaccharide analogues for interception and metabolic glycan engineering (MGE), wherein the promiscuity of enzymes of glycan biosynthesis is exploited for processing of non-natural analogues. Small molecules that alter glycosylation of cell surface antigens could potentially provide valuable inhibitors of glycan biosynthetic pathways and disease modulators for auto-immune diseases. 2. Development of novel carbohydrate-neuroactive (CH-NA) hybrid molecules in order to achieve MGE of the central nervous system (CNS) across the blood-brain barrier (BBB). Although MGE has been successfully shown in peripheral organs living animals, no expression of non-natural glycans were found in organs of the CNS. Our approach which enables MGE in brain provides a vital access key to study importance of glycosylation in CNS development, diseases, and disorders.
165 3. Development of glycopeptidomimetics (GPM) – based small molecules carrying a zinc binding group (ZBG) on the carbohydrate moiety as novel inhibitors of matrix metalloproteinases (MMP), which are known to trigger cancer metastasis. 4. Development of inhibitors of human neuraminidase (sialidase) – NEU3 which is expressed on the cell surface and might regulate ganglioside mediated processes.
Work reported in 2012-2013
A. Modulation of glycoforms of cell surface antigens using non-natural monosaccharide analogues via metabolic glycan engineering (MGE)
Towards our development of MGE of mucin-type O-glycosylation pathway, synthesis and characterization (NMR – 1H and 13C, HPLC, Mass spectrometry) of a panel of peracetylated N-acetyl-D-galactosamine (GalNAc) analogues were reported. N-acyl variants containing N- acetyl, N-acetoxyacetyl, N-acetylthioacetyl (Ac5GalNTGc, 1), N-propanoyl, N-azidoacetyl, N-methylthioacetyl, N-3-acetylthiopropanoyl, N-4-acetylthiobutanoyl derivatives were synthesized. Additionally, synthesis of peracetyl N-thioglycolyl-D-glucosamine - as a control for epimerization by GALE (UDP-GlcNAc C-4 epimerase) and peracetylated benzyl-α- GalNAc – as a substrate-decoy based inhibitor were reported. Results of in vitro studies in Jurkat (human T-lymphoma) cells showed that Ac5GalNTGc (1), but not the control analogues, was able to selectively induce hypo-sialylation and hypo-glycosylation of CD43 in a thiol-dependent manner. Metabolic expression of N-thioglycolyl-D-galactosamine (GalNTGc) on CD43 was confirmed by thiol-selective (Michael addition reaction) biotinylation, using biotin-linker-maleimide, of CD43-myc/FLAG from stably-transfected Jurkat cells. Metabolic incorporation of GalNTGc in CD43 was governed by the UDP- GalNAc: polypeptide transferases (ppGalNAc-T, E. C. 2.4.1.41) of which 14 isoforms are known in humans. Comparative qPCR profiling of expression of ppGalNAc-T isoforms in Jurkat and K562 (human chronic myeloid leukemia) cells revealed unique and differential expression patterns.
B. Development of carbohydrate – neuroactive (CH-NA) hybrid molecules for metabolic glycan engineering (MGE) across the blood-brain barrier (BBB)
Studies by Reutter and Bertozzi have shown that administration of Ac4ManNProp and Ac4ManNAz (intraperitoneally) in mice resulted in expression of SiaProp and SiaAz, respectively, in peripheral organs (such as spleen, heart, and liver). However, very little expression was detected in organs of the CNS. Lack of expression of SiaProp / SiaAz in the brain could be attributed to poor transport of non-natural analogues across the BBB. To overcome this we reported the synthesis and characterization of a panel of carbohydrate- neuroactive (CH-NA) hybrid molecules. CH-NA hybrids were found to be metabolized by Jurkat and SH-SY5Y cells with equal efficiency compared to the parent non-hybrid compound, Ac4ManNAz, as determined by flow cytometric estimation of SiaAz expression on cell surface. Cell surface azides were estimated via the copper (I) catalyzed azide-alkyne click (Cu-AAC) reaction using a biotin-linker-alkyne derivative followed by FITC-avidin staining.
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C. Design, synthesis, and development of carbohydrate-based small molecules for inhibition of matrix metalloproteinases (MMP)
Towards development of glycopeptidomimetics (GPM) as potential MMP inhibitors, we have designed several chemical compounds. Synthesis of protected carbohydrate derivatives carrying ZBG at C-6 position and their preliminary toxicity studies in cancer cell lines were reported.
Progress of work during the current reporting year (2013-2014)
A. Inhibition of mucin-type O-glycosylation of cell surface molecules using non-natural monosaccharide analogs via metabolic glycan engineering (MGE)
To develop carbohydrate-based small molecule inhibitors of biosynthesis of mucin-type O- glycans by application of the metabolic glycan engineering (MGE) methodology. Such small molecules would act as a first step in manipulating mucin-type O-glycans and may serve as valuable tools in probing the dynamic structure-function relationships of mucins in biological processes. Most of CD antigens carry both N-linked and O-linked glycans which are built post-translationally by an army of glycosyl transferases in endoplasmic reticulum (ER) and Golgi. Epitopes of biomedical significance such as blood groups (H, A, and B) and sialyl- Lewis X/Y/A are expressed on O-glycans and are known to play key roles in immune recognition, homing, and extravasation.
Disrupting the sugar-coat: Thiol-dependent inhibition of mucin-type O-glycans by non- natural GalNAc analogs Biosynthesis of mucin-type O-glycans is initiated by the addition of N-acetyl-D- galactosamine (GalNAc) to Ser/Thr (S/T) on the polypeptide backbone. It is known that mucin-type O-glycans could be engineered to express non-natural functional groups by exploiting the promiscuity of the GalNAc salvage pathway. Our results showed that peracetyl N-thioglycolyl-D-galactosamine (Ac5GalNTGc, 1) inhibited elaboration of O-glycans specifically in a thiol-dependent manner. Upon treatment with 1 (100 μM, 48 h), the binding of Maackia amurensis lectin (MAL-II) (which binds to NeuAcα(2→3)Gal-) and peanut agglutinin (PNA) (which binds to Galβ(1→3)GalNAc-α-S/T) were abrogated in Jurkat (human T-cell leukemia) cells. Treatment with vehicle (dimethyl sulfoxide), the corresponding N-glycolyl (Ac5GalNGc, 2) or N-acetyl (Ac4GalNAc, 3) derivative did not affect binding of MAL-II and PNA. GalNAc analogs 1, 2, and 3 did not affect cell viability when incubated at 200 μM for 48 h thus ruling out that inhibition of O-glycans is not a consequence of compound-related toxicity. MALDI-TOF/TOF mass spectrometry and Glycoworkbench analysis of permethylated O-glycans from Jurkat cells provided evidence for the presence of substantial levels of T- (Galβ(1→3)GalNAcα-S/T), sialyl-T, di-sialyl-T antigen structures. Mass spectrometry revealed reduction in the levels of T- and sialyl-T and absence of disialyl-T structures upon treatment with 1.
167 Both sulfhydryl and galactosamine moieties are critical for inhibition The chain length variants N-(3-acetylthiopropanoyl) (Ac5GalNTPr, 4) and N-(4- acetylthiobutanoyl) (Ac5GalNTBut, 5) did not affect CD43 glycoforms indicating steric limitations for processing through GalNAc salvage pathway. Importance of sulfhydryl group released from 1 for inhibition was highlighted by the inability of N-methylthioacetyl (Ac4GalNMeTA, 6) analogue to affect CD43 glycoforms. Specificity of 1 for GalNAc salvage pathway was illustrated by studies using peracetyl N-thioglycolyl-D-glucosamine (Ac5GlcNTGc, 7) – a C-4 epimer of 1 – which did not alter CD43 glycoforms. Additionally incubation of Jurkat cells with either free α-O-benzyl 2-acetamido-2-deoxy-D- galactopyranoside (α-Bn-GalNAc, 8) or its peracetyl derivative (Ac3-α-Bn-GalNAc, 9) did not mimic effects observed with 1 suggesting that 1 does not act via a substrate-decoy mechanism.
Antigen-selective and cell-type selective inhibition of mucin-type O-glycans by 1 The effects of metabolic processing of 1 are likely to be governed by (i) utilization of UDP- GalNTGc in place of UDP-GalNAc (‘natural’ donor) by ppGalNAcTs – of which fourteen isoforms are known in humans – and (ii) nature of the polypeptide acceptors. A comparative study revealed relatively little changes to CD45 glycoforms in contract to CD43. Similarly, CD162/PSGL-1 was also found to be altered only moderately. These results suggest that 1 exhibits ppGalNAcT isoform-selective and antigen-selective effects for inhibition of mucin- type O-glycans. Additionally, a study of effect of 1 on CD43 glycoforms in a panel of human hematopoietic cell lines revealed differential effects.
B. Development of carbohydrate – neuroactive (CH-NA) hybrid molecules for metabolic glycan engineering (MGE) across the blood-brain barrier (BBB)
To design and develop CH-NA hybrid molecules for achieving MGE across the blood-brain barrier (BBB). The ability to modulate glycan structures in brain across BBB would provide key access to study the importance of glycoconjugates in development and disorders of the central nervous system (CNS) in living animals. Sialoglycoconjugates, such as polysialic acid – neural cell adhesion molecule (PSA-NCAM) are abundantly present in CNS and are critically important for development, learning, memory, and nerve regeneration. Ability to manipulate sialoglycoconjugates in living animals might shed light on the changes to glycosylation under normal and disease conditions.
Expression of NeuAz in glycoproteins of brain in mice Mice (C57BL/6j and Balb/c) were treated with non-hybrid Ac4ManNAz via multiple routes of administration – (i) intraperitoneally (i.p.), (ii) intravenously (i.v.), (iii) i.v. along with mannitol (i.p.) – euthanized and tissues were harvested. Lysates of tissues were subjected to copper (I) – assisted alkyne-azide click (Cu-AAC) reaction using a biotin-linker-alkyne reagent and studied by far-western blotting. Expression of NeuAz was found in heart but not in brain. However, intracranial administration of Ac4ManNAz resulted in robust NeuAz expression in brain tissue thus confirming limited access to CNS via i.v. route. Strikingly, administration of CH-NA hybrids via i.v. route resulted in NeuAz expression in both heart and brain thus providing a ‘proof-of-concept’ validation of our strategy of piggy-backing monosaccharide analogs on neuroactive molecules.
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Down regulation of PSA on NCAM in brain in mice Having achieved successful engineering of sialic acid biosynthetic pathway across BBB, CH- NA hybrids were synthesized carrying N-butanoyl-D-mannosamine (ManNBut) moiety. ManNBut has been shown to intercept assembly of long polysialic acid chains in vitro. ManNBut-NA hybrids when injected i.v. in mice showed ~ 50% reduction in PSA levels on NCAM compared to mice treated with Ac4ManNBut (non-hybrid) thus confirming the unique ability of CH-NA hybrids to cross BBB.
C. Design, synthesis, and development of carbohydrate-based small molecules for inhibition of matrix metalloproteinases (MMP)
To design and develop carbohydrate-based small molecules as potential inhibitors of MMPs and as anti-metastatic agents. MMPs, on the one hand, are critically important in normal development and tissue remodeling. On the other hand, MMPs play a detrimental role by aiding cancer cells to break-away from primary site and enhance metastasis. Development of MMP inhibitors (MMPi) as anti-metastatic agents has been a focus of intense research for more than three decades.
Design and synthesis of and glycopeptidomimetics (GPM) - based MMPi We have designed and synthesized a panel of monosaccharide derivatives carrying a zinc binding group (ZBG) at the C-6 position. Starting from multiple monosaccharides (Glc/Gal/Man) several derivatives carrying a hydroxamic acid (-NHOH) were synthesized in a multi-step synthesis using selective protection and deprotection strategies. All small molecules were characterized thoroughly by NMR (1H and 13C) and mass spectrometry. Additionally we have synthesized selectively protected monosaccharide donor derivatives suitable for glycosidation to a Ser/Thr containing peptide derived acceptor using Lemieux, Schmidt, and Königs-Knorr reactions under moisture-free conditions.
D. Development of nanotools for glycobiology: Design and development of inhibitors for human sialidases
To develop small molecules for inhibition of human sialidases, particularly NEU3. Mammalian glycocalyx is modified in a dynamic manner during development and homeostasis. Particularly, sialic acid present at the termini of glycoproteins and glycolipids on cell surface are removed by human sialidase – of which four isoforms NEU 1 – 4 are known – in response to environment.
Expression of NEU 1 – 4 in mammalian cells Mammalian cells such as SH-SY5Y (human neuroblastoma), CaCO-2 (human colon adenocarcinoma) were screened for the endogenous expression of NEU 1 – 4 by RT-PCR. NEU3 was chosen for our studies as it is known to be present on plasma membrane and to hydrolyze ganglioside GD3 to GM3. Expression of stable Cos-1 (African green monkey kidney) cells expressing NEU3-FLAG (a kind gift from Dr. Takeo Miyagi, Japan) have been established and verified by western blotting and microscopy.
169 Future plans
Our current plans and future directions include consolidating our in vitro data and pursuing both analytical characterization and studied in vivo in animals. 1. Considering that glycans aid cell migration, cell-type selective modulation of glycoforms by carbohydrate-based small molecules could potentially open up new avenues for attenuation of auto-immune diseases. Studies on effect of GalNAc analogs on glycoform modulation in hematopoietic mouse cells and in vivo in mice are currently underway. 2. Studies on the isolation of membrane proteins and Cu-AAC based enrichment of NeuAz- carrying glycoproteins from brain tissue and their proteomics based identification using nano-LC-HR-MS are currently underway. 3. Evaluation of small molecules for MMP inhibition, including thermolysin and MMPs, using colorimetry and fluorimetry are currently underway. Evaluation of compound effects on toxicity and in vitro metastasis models have been initiated. 4. Small molecules designed keeping the di-sialyl motif of GD3 would be evaluated for inhibition of immunoprecipitated NEU3.
Action taken on the RAP/SAC 2013 recommendations
Suggestions provided by members of RAP/SAC have been duly incorporated.
Publication Original peer-reviewed article
1. Agarwal K, Kaul, R, Garg M, Shajahan A, Jha SK, Sampathkumar SG* (2013) Inhibition of mucin-type O-glycosylation through metabolic processing and incorporation of N- thioglycolyl-D-galactosamine peracetate (Ac5GalNTGc). J Am Chem Soc 135: 14189- 14197.
*Corresponding author
170 Biophysical and biochemical characterization of Leishmania phosphoglycerate kinase: an enzyme in the glycolytic pathway of parasitic protozoa
Principal Investigator Vidya Raghunathan
Collaborator S. Raghothama, NMR Centre, IISC, Bangalore
Theme of Research
It is known that Leishmania sp. unlike mammalian counterparts uses multiple isoforms for many enzymes of the energy pathway, one of which is phosphoglycerate kinse or PGK. Leishmania PGK isoforms has some distinct structural features, as PGKB and PGKC differ in a handful of internal residues and in the presence of a long extension at the C-terminus of PGKC. Drug development efforts can be targeted, either at the glycosome itself or at the enzymes present within them for which, targeting unique structural features is critical.
We are interested to use nucleur magnetic resonance spectroscopy to study the enzymology as well as structure of PGK isoforms. We also want map the metabolic profile of Leishmania spp cultures and correlate this to the enzymological studies with purified proteins.
Objectives
1. Expression, purification and determination of specific activities of PGKB-Lmex and PGKC-Lmex and steady state kinetics study. 2. Comparison between PGKB-Lmex and PGKC-Lmex of, pH optimum of activity and enzyme inhibition by salt and suramin. 3. 31P NMR studies using substrate / enzyme (PGKB-Lmex or PGKC-Lmex) mixtures, with, either no metal, MgCl2, CaCl2, MnCl2 or CoCl2 to determine the change in the dissociation constant of substrate with metal ions. Comparison with data from similar experiments in literature with yeast PGK using Mg-ADP and Mg-ATP. 4. Peptide based studies of glycosomal membrane association of PGKC-Lmex. The peptides used in these studies will be evaluated as useful models to understand the structural basis of the biochemical differences between PGKC-Lmex and PGKB-Lmex. 5. Conformational studies by NMR using site non-radioactive isotope labeling 6. Using promastigote and amasitoge cultures of Leishmania spp for metabolome mapping. The concentration of specific metabolites in the cell at a particular time can be monitored at the micromillimolar level by 31P, 1H and 13C NMR spectroscopy. The metabolites that can be detected are alanine, lactate, acetate, pyruvate, succinate, glycerol, urea, CO2, oxalate, valine, glutamine and arginine.
171 Work reported in 2012-2013
Therefore, looking at the previous reports we took the glycolysis reaction for testing the activities of PGKB and PGKC from Leishmania mexicana.
3-PGA + Pi + NAD+ ATP + 3-PG PGK ADP + 1,3-PG NADH GAPDH
The recombinant clones of L. mexicana PGK (PGKB and PGKC) in E. coli available to us have been checked by isolating the plasmid and sequencing to confirm the presence of the PGK genes in the correct reading frame. The enzymes were characterized in terms of kinetic parameters.
Phosphoglycerate Kinase Assay for ADP binding followed a protocol using pyruvate Kinase/PEP for ATP generation.
ADP ATP + 3-PG ADP + 2,3-PG 2 -PGA+ Pi + NAD+ PEP PGK NADH Pyruvate kinase GAPDH
PGK in leishmania cultures: The PGK activity was measured using the standard assay, in the amastigote cultures. Suramin dependent inhibition of activity was also observed as expected from a known inhibitor of PGK.
The specific activity of PGKB-Lmex and PGKC-Lmex was found to be different, PGKB- Lmex being more active than PGKC-Lmex. The ATP binding of PGKC-Lmex is stronger as compared to PGKB-Lmex whereas 3-PG binding is stronger in the case of the latter. ADP binding is stronger in the case of PGKB-Lmex. When compared with data published by others on yeast PGK we find the ATP affinity is higher with the Leishmania enzymes whereas yeast has a higher affinity for the other ligands 3-PG and ADP. When trying to understand the basis of protein function one is inevitably led to the structure of the protein (if it is known) or biochemical studies based on structure or sequence of the protein. No structure of Leishmania PGK is available so far and only two sequences published that of L. major and L.mexicana. Our goal is also to determine the structure of these enzymes by NMR and in conjuction do dynamic conformational studies.
Based on the results of computational analysis 3 synthetic peptides derived from the C- terminal sequence of L. mexicana PGKC, were complexed with lipids or micelles and studied by circular dichroism spectra and NMR. Proton NMR spectra of the peptide complexes reconstituted in SDS micelles were recorded. Structure of the 26-mer peptide derived from C-terminus of PGKC-Lmex was determined in MeOH and deuterated SDS. This revealed that the peptide adopted a complete helical structure in the membrane-like environment provided by these solvents (see publication). In plain aqueous media the peptide was insoluble. Further structural studies outlined below will help us understand the structural biochemistry of the 62 residue c-terminal domain of PGKC-Lmex.
172
Progress of work during the current reporting year (2013-2014)
In lieu of the structure of the peptide as determined by NMR in solution [S. Kaushik, B. Krishnarjuna, S. Raghothama, S. Aggarwal, V. Raghunathan, A. Ganjiwale. Theoretical and in vitro studies of a C-terminal peptide from PGKC of Leishmania mexicana mexicana. 185 (2012) 27-35], we have launched into looking at the structure of the entire C-terminal domain extension of PGKC-Lmex by cloning in E. coli. The 62 residue domain of the C-terminus of PGKC has been cloned in E. coli BL21 (RP) strain. The protein expression is good and western blotting shows the presence of a strong clean band after purification by metal affinity chromatography.
We have already initiated structural as well as biochemical studies using peptides from the 62r domain of C-terminus of PGKC-Lmex.
Future Plans this we will generate PGKB [13C, 15N-Trp] and [13C, 15N-Trp] PGKC for experimental studies by high resolution NMR. Position of L. mexicana tryptophan in PGKB is W334 and in PGKC are W334 and W447. NMR with these labeled proteins will be used to determine the conformation. We are also cloning the PGKB-Lmex and PGKC-Lmex in E. coli. Once we are able to do change in the protein upon binding of substrate. All other structural studies listed in objectives will become possible too. Complete all structural studies.
Action taken on the RAP/SAC 2013 recommendations
No specific suggestions were made on the scientific aspects of the work. The committee asked me general questions on the feasibility of using in-cell NMR and its adaptability to the present set-up.
173 Elucidating the molecular mechanisms of aging and innate immunity using Caenorhabditis elegans as a model system
Principal Investigator Arnab Mukhopadhyay
Research Associate Neeraj Kumar
Ph.D. Students Manish Chamoli Anupama Singh Syed Shamsh Tabrez Sonia Verma Anita Goyala
Collaborators Sagar Sengupta, NII Mahesh Kulkarni, NCL, Pune Kausik Chakraborty, IGIB, New Delhi Subhashini Srinivasan, IBAB, Bangalore
Theme of research
We are interested in understanding the molecular basis of aging using the nematode, Caenorhabditis elegans as a model system. Our laboratory uses a combination of genetics, molecular biology and genomics coupled to Next Generation sequencing (NGS) to decipher the underlying mechanisms of aging. We are trying to understand the complex interplay of transcription factors and co-regulators downstream of the Insulin-IGF-1 signalling (IIS) pathway in regulating gene expression required for longevity, metabolism, stress- and pathogen resistance. Since Dietary Restriction (DR) is the only intervention that can increase life span and delay age-onset diseases, we are trying to decipher the molecular events that follow initiation of DR. We are also using chemical genetics to find novel longevity extending compounds and are studying their mechanism of action.
Objectives
1. Deciphering coordinate regulation of genes downstream of IIS pathway 2. Involvement of miRNA-Transcription factor networks in dietary restriction 3. Involvement of novel kinases in dietary restriction 4. Drugs that can extend C. elegans life span
174 Work reported in 2012-2013
1. Deciphering coordinate regulation of genes downstream of IIS pathway
The IIS pathway controls aging, development, stress- and pathogen resistance in C. elegans as well as other organisms. Several transcription factors like FOXO/DAF-16, SKN-1/NRF2 and HSF-1/HSF1 function downstream of the IIS pathway to couple modulations in nutrient/stress conditions to gene expression changes. We are interested in profiling these changes at the level of TF-DNA interactions under different physiological conditions using ChIP-sequencing. We have standardized Chromatin immunoprecipitation (ChIP) coupled to Next Generation Sequencing (NGS) to generate a profile for genome-wide FOXO/DAF-16 recruitment under conditions of low insulin signalling. We also performed transcriptomics analysis to determine gene expression changes depending on the TFs when IIS pathway signalling is lowered.
2. Involvement of miRNA-Transcription factor networks in Dietary Restriction
DR is the only intervention that can increase life span and delay age-related diseases across the animal kingdom. DR will require large changes in the transcriptome to support reprogramming of metabolism required for long life. In order to study changes in miRNA profile during DR, we performed NGS of the enriched miRNA fraction. We used the eat- 2(ad1116) strain that has defective pharyngeal pumping and as a result consume considerably less food; it is a genetic model of DR in C. elegans. The sequencing was performed both on day 1 and day 8 of adulthood. We found that the expression 81 miRNA was upregulated while none was downregulated on day 1. Most of the upregulated miRNA expression went back to wild-type levels on day 8.
3. Involvement of novel kinases in dietary restriction
Very little is known about the signalling events downstream of DR initiation. We are characterizing a series of kinases that qualify as components of a nutrient sensing pathway. We have shown that a novel kinase idr-1 initiates a process similar to DR, when knocked down using RNAi. Following idr-1 knockdown, worms shift metabolism towards using beta oxidation as a source of energy. This transcriptional shift requires the HNF4 homolog, NHR- 49. This shift leads to decreased Reactive Oxygen Species (ROS) production and a concomitant upregulation of xenobiotic detoxification genes. We found that transcription factors known to control xenobiotic detoxification genes (NHR-8 and AHR-1) are required for this process.
4. Drugs that can extend C. elegans life span
We reported that a FDA-approved drug was able to increase C. elegans life span under euglycemic as well as hyperglycaemic conditions. This drug is able to decrease formation of advanced glycation endproducts or AGEs in vitro. We showed that the drug activates the FOXO transcription factor DAF-16 and leads to transcription of a different subset of FOXO target genes.
175 Progress of work during the current reporting year (2013-2014)
1. Deciphering coordinate regulation of genes downstream of IIS pathway
We have analyzed the AF-16/FOXO ChIP-Seq data is details. We used the low insulin signaling mutant daf-2(e1370) and compared it to daf-16(mgdf50);daf-2(e1370), where daf- 16 is deleted. The data was normalized to their respective input samples. We analyzed more than 20 million reads for each sample with Q score of 30 or more. More than 50% of the reads mapped in case of the ChIP DNA while more than 80% mapped in case of input DNA. Out of the total of 6852 peaks, DAF-16 was found to be bound to the 5098 coding genes and 216 non-coding genes. About 35% of the peaks mapped within 0.5 kb of the transcription start sites (TSS) of the genes while around 20% was found in the region 0.5-2.5 kb upstream of TSS. DAF-16 peaks were also found in the 3’ UTR, introns as well as in intergenic regions. Interestingly, while the distribution of DAF-16 peaks is uniform over chromosomes I-V, the number of peaks in the X chromosome is considerably higher. We compared the ChIP-seq data with existing microarray, ChIP and proteomics data that compared mRNA or proteins between daf-2(e1370) and daf-16(mgdf50);daf-2(e1370). We found an overlap of ~35% with microarray data, 50% with proteomics data and about 40-60% with related ChIP- seq data. These show that microarray data has captured events that are not directly related to DAF-16 direct recruitment. Additionally, since we performed ChIP for endogenous DAF-16 protein, ChIP-seq experiments with overexpression strain of DAF-16, reported by other groups may have produce artifacts. About 32% of the DAF-16 peaks have consensus DAF- 16 binding sites while 42% possess the related GATA-like site. Additionally, predictive algorithms could detect other novel TF binding sites in the peaks where DAF-16 binds indicating that this TF may be part of a large transcription initiation complex.
2. Involvement of miRNA-Transcription factor networks in dietary restriction
MiRNA sequencing revealed that a total of 81 miRNA were upregulated during day 1 of DR in C. elegans while none was significantly downregulated. In order to identify transcription factors that are responsible for upregulating miRNA during DR, we analyzed the promoters of these miRNA for transcription factor binding. We used the ChIP-seq data available at MODENCODE for 28 transcription factors (www. http://deepbase.sysu.edu.cn/chipbase). We found that the FOXA transcription factor PHA-4 has the highest number of binding sites in these promoters. This was interesting as DR is known to require PHA-4 to extend life span. We found that 66 of the 81 promoters are bound by PHA-4 in the ChIP-seq data. Next, we validated the expression of 10 miRNA, both primary miRNA as well as matured miRNA for dependence on PHA-4. We also found that another zinc finger protein has high number of binding sites in the promoters of upregulated miRNA. We validated 10 target primary and mature miRNAs and found them to be dependent on this zinc-finger TF. Both FOXA/PHA-4 as well as the zinc-finger TF are upregulated in eat-2(ad1116) on day 1 showing that they are responsive to nutrient availability. Finally, we showed that like PHA-4/FOXA, the increased life span of eat-2(ad1116) is dependent on presence of the zinc-finger TF. Together, we showed that TF-miRNA interactions are important for dietary restriction-induced longevity.
176 3. Involvement of novel kinases in dietary restriction
We have earlier shown that PHA-4 is required for idr-1 knockdown to increase life span; however, we did not know why. In order to answer this question and better understand the process of DR, we performed a microarray analysis to study changes in gene expression profiles when idr-1is knocked down. Genes involved in beta oxidation of fatty acid and xenobiotic detoxification were upregulated. Using the MODENCODE ChIP-seq data for PHA-4, we asked whether PHA-4 binding is enriched in the promoters of the upregulated genes. We found that 40% of the genes are indeed PHA-4 direct targets. We used DAVID to categorize these common genes and found that xenobiotic detoxification genes were most enriched. We validated this using qRT-PCR to show that PHA-4 regulates the xenobiotic detoxification genes following initiation of the DR-like phase on idr-1 knockdown. Earlier we have shown that DR produces low levels of ROS. We hypothesized that during DR, fatty acid oxidation shifts the balance of NADH/FADH2 in such a way that more of mitochondrial electron chain (ETC) complex II is used, compared to when worms are fed ad libitum. If that is the case, idr-1 knockdown-mediated life span extension will not be able to tolerate perturbations in the complex II of ETC. We showed that idr-1 cannot increase life span in a complex II defective mev-1 mutant, but can do so in a complex I defective gas-1 mutant. Additionally, we showed that in a mutant strain defective in beta oxidation, DR cannot decrease ROS production. Thus, we have now established that during DR, specifically in case of idr-1 knockdown and eat-2(ad1116) mutants, metabolic reprogramming is the reason for low ROS generation and increased longevity. This, however, is a different paradigm of DR as it does not require induction of the hormesis machinery as proposed for DR-like state brought about by glucose restriction.
Further, we have begun a detailed characterization of a gene that is homologous to idr-1. We call this gene idr-2. Knocking down the gene either by mutation or by RNAi leads to depletion of fat storage and increased life span. We showed that the life span is dependent on PHA-4 and not on DAF-16, hinting to the fact that the gene knockdown may be bringing about a DR-like state similar to idr-1 knockdown. But interestingly, this mutant increases life span only when they are grown on a particular strain of bacteria (HT115, a K12 strain) and not on another (OP50, a B strain). We are currently trying to decipher the reason for this food-type-dependent life span extension.
4. Drugs that can extend C. elegans life span
We confirmed the requirement for the JNK signalling pathway in the life span extension brought about by the FDA-approved drug. We found that the drug failed to increase life span when either jnk-1 or its upstream kinase jkk-1 was mutated. JNK-1 was also found to be required for the protective role of the drug under hyperglycaemic conditions. Additionally, we identified two variants of the drug; one had similar anti-glycating activity while the other did not have significant activity in vitro. We found that the life span extension and protection against polyglutamine aggregation was conferred by the drug with anti-glycating activity and not by the other one. This showed that anti-glycating activity of the drug is the reason for its effect on life span.
177 Next we used LC-MSE, a Data-Independent Acquisition (DIA) strategy to identify AGEs targeted by RIF in vivo. LC-MSE is a unique approach wherein all eluted peptides are fragmented and the fragment ions are time-aligned with the retention time of the peptides. This method allows analysis of low-intensity AGE-modified peptides as well as heterogenous AGEs. A total of 24 proteins carrying 59 AGE modifications were detected in samples treated with DMSO; the number of modifications decreased to 25 on drug treatment. We found that VIT-6, a vitellogenin that is reported to be heavily carbonylated in worms, had the most number of modifications (eleven) followed by ALDO-2, a fructose bisphosphate aldolase (seven); modifications on both these proteins decreased on drug treatment. The modifications on mitochondrial citrate synthase, malate dehydrogenase, aconitase, as well as two fatty acid and retinol binding proteins also decreased. We also found lesser number of modifications on a calreticulin and a beta hexoaminidase A protein after drug treatment. While the aldolase, aconitase, malate dehydrogenase, and citrate synthase are important rate- limiting enzymes of glycolysis and Kreb’s cycle, calreticulins prevent misfolded proteins from exiting the endoplasmic reticulum. On the other hand, beta-hexosaminidase A deficiency causes Tay-Sachs and Sandhoff diseases due to accumulation of its substrate, GM2 ganglioside, in neuronal lysosomes. Modifications on these important proteins may disrupt normal cellular functions leading to aging. Drug treatment may help in retaining proper functioning of these proteins and thereby affect life span positively.
Future plans
1) Identify signal transduction pathways involved in idr-1 knockdown-mediated DR. 2) Detailed characterization of molecular functions of idr-2. 3) Detailed molecular genetics characterization of some of the direct targets of DAF- 16/FOXO that we obtained by ChIP-seq. 4) Efforts will be made to further characterize the molecular mechanisms of drug action and evaluate effects in mouse model
Action taken on the RAP/SAC 2013 recommendations
No specific recommendation was received
Publications Original peer-reviewed articles
1. Ritter AD, Shen Y, Fuxman BJ, Jeyaraj S, Deplancke B, Mukhopadhyay A, Xu J, Driscoll M, Tissenbaum HA, Walhout AJ* (2013) Complex expression dynamics and robustness in C. elegans insulin networks. Genome Res. 23: 954-965.
2. Chamoli M, Singh A, Malik Y, Mukhopadhyay A* (2014) A novel kinase regulates Dietary Restriction-mediated longevity in C. elegans. Aging Cell (in press).
*Corresponding author
178 Molecular biology of infectious diseases
Principal Investigator Lalit C. Garg
PhD Students Shweta Chatrath Vineet Gupta Bharti Bhatia Amit Kumar Solanki Suresh Kumar
Research Fellows Himani Kaushik (till Feb. 2014) Sachin Deshmukh Gagan Chhabra (Since November 2013) Sweta (till August 2013) Pragati Sharma (Since November 2013) Smriti Sahu (Since November 2013) Abhishek Acharya (Since November 2013)
Collaborators S. A. Wani, SKUAST-K, Srinagar A. Dixit, JNU, New Delhi P. K. Sahoo, CIFA, Bhubaneswar
Theme of research
In global surveys, infectious diseases rank among the leading causes of death of both humans and animals. Vaccination against infectious agents continues to be one of the most effective methods of limiting the cost of management of many infectious diseases. The goal of this study is to clone and express genes of biomedical importance with an emphasis on the development of vaccines against pathogens and to unravel the molecular mechanisms of infectious diseases to explore new drug targets.
Objectives
1. Development of recombinant and DNA based vaccine against Clostridium perfringens
Gram positive Clostridium perfringens is a major cause of human and veterinary enteric diseases largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. Epsilon toxin (Etx), produced by C. perfringens types B and D, is the key antigen implicated in the Enterotoxaemia and Pulpy kidney disease of domestic animals. C. perfringens type B and C produce beta toxin which is responsible for necrotizing enteritis and enterocolitis, being the most common causes of cattle mortality, these bacteria are of great economic importance. The project aims to develop recombinant and DNA vaccine
179 against the toxins produced by C. perfringens. We also aim to assess the role of various residues within the toxin for its toxicity, oligomerization, binding to host cell and immunogenicity.
2. Studies on functional characterization of PE_PGRS and PE_PPE proteins of Mycobacterium tuberculosis H37Rv
Sequence analysis of the Mtb H37Rv genome resulted in identification of novel multigene families- the PE (proline-glutamic acid) and the PPE (proline-proline-glutamic acid). These families account for much of the genomic difference between the pathogenic and nonpathogenic mycobacterial genomes. Therefore, they may play role in Mtb's virulence and host specificity. However, their exact role in Mtb biology is not clearly understood. We aim to explore the possible role of the PPE and PE-PGRS proteins in the biology of Mtb.
Work reported in 2012-2013
1. Development of recombinant and DNA based vaccine against Clostridium perfringens
The efficacy of intranasal immunization of BALB/c mice with recombinant epsilon toxin (Etx) was evaluated. Intranasal immunization of the recombinant Etx generated a very good immune response. The anti-Etx antisera was able to neutralize the toxic effect of the Etx in vitro. Further, incubation of the antisera with the lethal dose of the Etx prior to injection rendered the toxin non-toxic as no mortality was observed upon its administration to the naïve mice. One hundred percent protection of the intra-nasally immunized mice upon challenge with the 50× LD50 dose of Etx was observed. Secretory expression of the immunodominant B cell epoitopes of Etx in translational fusion with LTB was achieved. The anti-sera generated against the fusion proteins was able to confer protection against epsilon toxin toxicity in MDCK cells.
2. Studies on functional characterization of PE_PGRS and PE_PPE proteins of Mycobacterium tuberculosis H37Rv
Over expression of PE_PGRS30 and PE_PGRS35 in M. smegmatis resulted in prolonged lag phase of growth profile. Sequence comparison of PE_PGRS30 and PE_PGRS35 proteins was performed revealing that PE_PGRS30 possesses a much larger PGRS domain than PE_PGRS35 with an additional highly similar C-terminal domain. Three other PE_PGRS proteins (PE_PGRS11, PE_PGRS16 and PE_PGRS62) possessing the C-terminal domain when expressed in M. smegmatis did not affect the growth of recombinant mycobacteria, indicating that the change in growth profile is not solely due to C-terminal domain.
Proteomic analysis of recombinant M. smegmatis expressing PE_PGRS30 showed differential expression of 8 proteins in the cytosol and 12 proteins in the cell wall of PE_PGRS30 recombinant M. smegmatis. The up-regulated proteins were identified using MS/MS analysis. These were DNA-binding response regulator, putative F420-dependent
180 oxidoreductase and probable forkhead associated protein, a putative pyrimidine permease and an ABC transporter. Further, deletion variants of the PE_PGRS30 were expressed in M. smegmaatis to dissect the functional properties of different domains of PE_PGRS30 (PE only, PE+PGRS and C-term only). All the 3 mutants localized in the cell wall. Fluorescence microscopy data illustrated that the PGRS domain of PE_PGRS30 is responsible for association of the protein with cell poles since only PE+PGRS mutant showed polar localization. It was established that PE_PGRS30 is exposed on mycobacterial surface through its PGRS domain. Delay in the lag phase of growth of M. smegmatis was due to PGRS domain. PE+PGRS mutant rendered a more pronounced effect on growth of M. smegmatis on solid media as compared to the complete protein.
Progress of work during the current reporting year (2013-2014)
1. Development of recombinant and DNA based vaccine against Clostridium perfringens . For the development of vaccine against beta toxin produced by the C. perfringens type B and C, the protein is needed to be produced in large amounts. Earlier attempts to express the beta toxin in E. coli resulted in very poor expression and extremely low yields. Therefore, in order to optimize expression in E. coli, mature beta toxin gene was synthesized with codon optimization for E. coli expression. The beta toxin thus obtained was subcloned in pET 22b+ vector and transformed in E. coli DH5α cells. The transformants were selected on ampicillin plate and confirmed by restriction digestion and automated DNA sequencing. Subsequently, the pET 22b+ plasmid containing beta toxin gene was transformed in E.coli BL21 strain. The transformed cells were induced for expression with 1 mM IPTG for 6 hours. SDS-PAGE analysis of cell lysates prepared from both the induced and uninduced cultures revealed high level expression of the recombinant protein in the induced cultures only, as a band of the expected size was present in the induced cultures. The authenticity of the expressed product was established by immunoblot analysis using anti-His antibody. Subcellular fractionation of the induced cultures was carried out to determine localization of expression and it was established that the recombinant beta toxin was present predominantly as inclusion bodies. Beta toxin was purified from inclusion body fraction using batch purification with Ni-NTA beads and protein was eluted with 100 mM Imidazole. The protein was refolded by pulse refolding method. The biological activity of the refolded protein was evaluated in HL 60 cells in vitro and in vivo using BALB/c mice.
BALB/c mice were immunized with the purified recombinant beta toxin . After first booster dose, serum was collected and assessed by immunoblotting and ELISA. The anti-beta toxin sera were able to detect the recombinant beta toxin in immunoblot analysis as a band of ~34 kDa could be detected in the Western blot. ELISA was carried out to check the antibody titers. The end-point titer (maximum serum-dilution which generated an absorbance at 490 nm above the background level) was determined to be 100000. Splenocyte proliferation assay using splenocytes isolated from the immunized mice showed significantly higher proliferation in comparison to those isolated from the control mice upon stimulation with the recombinant beta toxin.
181 In vitro antibody neutralization assays indicated that serum obtained from beta toxin immunized mice significantly neutralized the toxin in vitro using HL-60 cells. The toxin- antisera formulation reduced the HL60 cell death compared to serum from the PBS immunized mice. Complete protection (100% survival) was observed against MLD dose (minimal lethal dose) of beta toxin in animal group immunized with heat inactivated recombinant beta toxin. The animals were observed upto 8 week post challenge. No protection was observed in case of PBS immunized mice.
2. Studies on the functional characterization of PE_PGRS and PE_PPE proteins of Mycobacterium tuberculosis H37Rv
Mtb employs numerous intelligent strategies to persist inside host and evade the host defence mechanisms. One such strategy involves the modulation of cytokine profile of macrophages to deviate anti-microbial T-cell responses. Therefore, a dynamic interplay between host immunity and mycobacterial survival strategies determines the course of tuberculosis disease. The pro-inflammatory cytokines, IL-12, TNF-α and IL-6, play an important role in protective immunity to tuberculosis infection by regulating T-cell activation and stimulating macrophage-mediated microbicidal mechanisms. In order to assess the role of PE_PGRS30 in modulation of host immune response, M. smegmatis cells expressing PE_PGRS30 were used as this mycobacterial species naturally lacks PE_PGRS family of genes. Infection of PMA-differentiated human THP-1 macrophages with M. smegmatis expressing PE_PGRS30 resulted in reduced production of inflammatory cytokines, including IL-12, TNF-α, and IL-6 as compared to those infected with vector control M. smegmatis. However, no change was recorded in the survival ability of PE_PGRS30-recombinant M. smegmatis in macrophages or in macrophage viability. We also analysed the levels of IL-10 in the culture supernatant of infected macrophages and found no significant difference in the IL10 levels of culture supernatant of macrophages infected with pVV16 M. smeg or pVV1651c M. smeg was detected, suggesting that down-regulation of pro-inflammatory cytokines by PE_PGRS30 is not mediated through IL-10 and may involve a distinct mechanism.
Further, to dissect the PE_PGRS30 protein into distinct functional domains, we infected THP-1 macrophages with M. smegmatis expressing deletion mutants of the protein. Analysis of macrophage viability using LDH release assay revealed that PE + PGRS mutant prevented the death of infected macrophages. Effect on cell cytotoxicity might be masked in the full length PE_PGRS30 protein due to conformational changes brought about by the C-term domain. Profiling of pro-inflammatory cytokines illustrated that, similar to the full length protein, only PE + PGRS mutant caused reduction in the levels of IL-12, TNF-α and IL-6. However, greater reduction in the TNF-α levels by PE + PGRS mutant as compared to complete PE_PGRS30 might be attributed to the anti-inflammatory effect of the C-term domain. As a subset of PE proteins is displayed on the bacterium's cell surface, can elicit an immune response, and may be a source of antigenic diversity for Mtb. PPE proteins have also been found on the cell surface, may be secreted, and can confer virulence suggesting a possible role for the PE and PPE gene families in pathogenesis.
182 To understand the function of these proteins, PPE14 protein were cloned and expressed as recombinant protein. When PPE14 protein was over expressed it mainly localised to inclusion bodies. These proteins were purified under denaturing condition and refolded. Purified protein was subsequently used for antibody generation in rabbit. The role of PPE14, a mycobacterial secretory protein, in modulating host macrophage effector functions was evaluated. The data demonstrated that the purified recombinant- PPE14 stimulated the production of pro- inflammatory cytokines particularly TNF α (required for granuloma formation) and IL12 by PMA-differentiated THP-1 macrophages and human Peripheral Blood Mononuclear Cell (PBMCs). The induction of pro- inflammatory cytokines by PPE14 was found to be TLR2- and MyD88- dependent. Inhibition of PPE14-induced TNF- α production in the presence of ERK1/2 (Extracellular signal regulated kinase 1/2) inhibitor suggested that the ERK1/2 pathway was involved in TNF- α production. Thus, the present study defines an immunomodulatory role for PPE14.
Future plans
Deletion/substitution mutant clones of beta toxin will be generated to identify the residues important for its toxicity, oligomerization and binding to the host cell. Non-toxic mutants will be evaluated for their vaccine potential. Functional significance of the PE_PGRS35 of M. tuberculosis in modulating host cell effector function will be investigated.
Action taken on the RAP/SAC 2012 recommendations
No specific recommendations were made.
Publications Original peer-reviewed articles
1. Chatrath S, Gupta VK, Garg LC* (2014) The PGRS domain is responsible for translocation of PE_PGRS30 to cell poles while the PE and the C-terminal domains localize it to the cell wall. FEBS Lett (doi: 10.1016/j.febslet.2014.01.059).
2. Kaushik H, Deshmukh S, Mathur DD, Tiwari A, Garg LC* (2013) Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein. Bioinformation 9: 617-621.
Patents
1. Garg LC, Bhatia B (2014) Recombinant subunit vaccine against beta toxin of Clostridium perfringens. Indian Patent application # 58/DEL/2014 (January 08, 2014).
*Corresponding author
183 Epigenetic regulation of the eukaryotic genome: Role of transcriptional insulators in organizing chromatin
Principal Investigator Madhulika Srivastava
Ph.D. Students Pratishtha Rawat Manisha Jalan Abhilasha Kanaujia Faizan Uddin (Since January 2014)
Research Fellows Ananya Sadhu (until July 2013) Garima Varma (until October 2013) Ambica Paul (since January 2014)
Collaborator Karl Pfeifer, NIH, Bethesda, USA
Theme of Research
The mechanisms by which cis-acting regulatory elements interact with each other in context of chromatin are incompletely understood even though such interactions are crucial for appropriate regulation of nuclear processes like transcription and VDJ recombination. CTCF dependent insulators play an important role in the functional organization of the mammalian genome as they can coordinate intrachromosomal and interchromosomal contacts and thus influence cis-DNA interactions. A large number of CTCF binding sites have been identified genome-wide suggesting their extensive involvement in governing cis DNA interactions among regulatory elements. Our efforts are directed towards understanding how the mammalian insulators influence chromatin domain organization and contribute to regulation of nuclear processes. A combination of genetic, molecular and biochemical approaches are being utilized for investigations.
Objectives
To explore the mechanisms underlying insulator activity, antigen receptor loci like IgH, TCRα/δ, TCRβ etc, are particularly interesting. Transcription as well as RAG mediated recombination is exquisitely regulated during development at these loci and underscores the importance of appropriate enhancer-promoter interactions. Further, recombination requires physical interaction between RSS elements associated with the V, D and J segments. These segments are located at large distances from each other on the chromosome and higher order chromatin reorganization is necessary to bring them together prior to recombination. By exhibiting long range interactions between different types of elements, the antigen receptor loci present a useful framework to explore the role of CTCF in defining independently regulated chromatin domains. Taking advantage of this, we are currently investigating the
184 chromatin structure and organization of the wild type and genetically manipulated TCRβ loci to understand various aspects of insulator function as well as VDJ recombination.
Work reported in 2012-2013
Organization of an ectopic CTCF dependent insulator at the TCRβ locus was observed to impair enhancer Eβ dependent transcription and D-to-J recombination in the genetically manipulated TCR-ins allele. Consistent with the position dependence of insulator activity, the abrogation was position dependent. Consequently, in TCR-ins allele, promoter PDβ1 regulated transcription and recombination of DJCβ1 gene segments was severely curtailed but promoter PDβ2 driven transcription and recombination of DJCβ2 gene segments was comparable to the wild type TCRβ allele.
To understand these observations, analysis of several aspects pertinent to chromatin organization were initiated in Rag1 deficient thymocytes. Allele specific ChIP analysis to detect the presence of H3K4me3 and H3K9Ac demonstrated that the acquisition of these activating histone modifications at the PDβ1 promoter was almost completely abolished in the mutant TCR-ins allele compared to the wild type TCRβ allele. The enrichment of these marks was also enormously reduced in the gene segments Jβ1 and Cβ1 that do not rely on Eβ-PDβ1 interaction. Further, PDβ1 was observed to be associated with H3K27me3 and methylation of CpG residues suggesting a complete repression of PDβ1 promoter. H3K4- me3 has been reported to act as a signal for recruitment of Rag2, an important cofactor of Rag1. Hence, the presence of Rag2 on the wild type and mutant alleles was analysed by allele specific ChIP and was observed to correlate well with the histone modifications. By an entirely independent assay relying on restriction enzyme accessibility, we confirmed that the Eβ mediated accessibility is significantly reduced at the DJCβ1 gene segments. Thus, it forms the molecular basis of the altered transcription and recombination profiles observed earlier at the DJCβ1 cluster. As predicted by the position dependence of insulator activity, the PDβ2-DJCβ2 region of TCR-ins allele did not exhibit any significant differences when compared to the wild type TCRβ allele.
We have earlier reported that in addition to blocking the enhancer activity at TCRβ locus, the inserted H19-ICR also altered the choice of Vβ segments used for VDJ recombination in a CTCF dependent manner suggesting the ability of CTCF to modulate interactions between cis-regulatory elements other than promoters and enhancers. We have identified a few regions of the TCRβ locus that bind CTCF and standardized chromosome conformation capture (3C) assay to investigate the higher order chromatin organization defined by CTCF binding.
Progress of the work during current reporting year (2013-2014)
Chromatin structure at the nucleosomal level is the crucial determinant of transcription and recombination. However, it is the specific interactions between cis-regulatory elements i.e.
185 enhancers, promoters and insulators which are necessary to define the chromatin structure at the nucleosomal level. We have analysed these interactions at the wild type TCRβ locus as well as in context of the ectopic insulator insertion in TCR-ins. Some of the observations are delineated below:
A. Enhancer Eβ employs facilitated tracking to activate PDβ1 and PDβ2
Paucity of clear understanding of enhancer mechanisms makes it difficult to understand the mechanisms underlying insulator activity. Although there is substantial evidence demonstrating the enhancer-promoter contact (loop) as captured by 3C analysis, enhancers might also initiate “tracking signals.” While the nature of “tracking signal” for enhancer action remains elusive, both RNAPolII/CBP and histone modifications were suggested to be the “tracking signals” that are initiated at the enhancer and travel to the cognate promoter. According to the “facilitated tracking” model, the activators bound at the enhancer move along the chromatin while retaining their contact with the enhancer until a cognate promoter is found suggesting that the enhancers can also “track.”
Eβ has been proposed to regulate the locus by “looping” as well as by some form of “tracking.” In this context, we observed, as described in the previous year, the ability of the inserted insulator to abrogate Eβ-PDβ1 interaction dependent acquisition of activating histone modifications and chromatin accessibility at the PDβ1. Interestingly, the insulator also abrogated acquisition of activating histone modifications in the DJCβ1 region which is Eβ dependent but PDβ1 independent suggesting its interference in some form of “tracking.”
Allele specific ChIP analysis of RNAPolII and CBP binding on the wild type TCRβ demonstrated the presence of these factors not only at Eβ, PDβ1 and PDβ2 but also in the region nearly 2kb upstream to PDβ2 as if the Eβ loaded with RNAPolII was tracking. Similar analysis in TCR-ins and TCR-mut alleles suggests that the functional H19-ICR insulator prevented such a tracking. These observations were complemented by the 3C analysis. It was evident that Eβ makes a physical contact with PDβ1 as well as PDβ2 on the wild type TCRβ locus. However, presence of a functional CTCF dependent insulator in TCR-ins prevents the formation of Eβ-PDβ1 loop while leaving the Eβ-PDβ2 loop unaffected.
Together, our analysis of enhancer-promoter-insulator interactions suggests that Eβ employs facilitated tracking to eventually form a loop with the target promoters PDβ1 and PDβ2 and the CTCF dependent H19-ICR insulator has the ability to abrogate several aspects of facilitated tracking.
B. CTCF mediates higher order chromatin organization necessary for VDJ recombination at TCRβ locus
Our functional analysis has earlier demonstrated that the insertion of ectopic CTCF binding sites dramatically influenced V-to-DJ recombination at TCRβ locus. It is interesting to note that analysis of CTCF at IgH and Igk loci suggests its role in curtailing enhancer activity at
186 some of the CTCF binding sites while a large majority of the multiple intergenic CTCF binding sites appear to be important for locus contraction. In contrast, at the TCRα/δ locus, CTCF was observed to bind the enhancer Eα as well as promoters for Jα and Vα such that Eα and CTCF cooperate to facilitate the formation of a chromatin hub necessary for synapsis of recombining segments. Thus, in keeping with the complexity of CTCF as an organizer, it is likely to play overlapping as well as distinct roles on different antigen receptor loci. The role of CTCF on TCRβ locus has not been delineated. Our ChIP analysis in the previous years has demonstrated that CTCF as well as cohesin are bound at the endogenous TCRβ locus at several sites. Using 3C followed by qPCR, the interactions amongst these CTCF binding sites (C2, C3, C4, C5, C6, C8) were analysed.
The 3C was carried out in thymocytes derived from Rag1 deficient mice i.e. on TCRβ alleles prior to VDJ recombination. ProB cells from Rag1 deficient mice were used as controls. Clearly, the interactions of C3 with any of the other CTCF binding sites (C4, C5, C6, C8 etc) were specific to T cell lineage and were significantly lower in ProB cells. The interactions of C3 (located amidst the upstream V segments) with distantly located C5 and C6 (located at almost the 3’ end of the locus) were much higher compared to its interactions with proximally located C2 and C4. A similar analysis using C6 as the anchor demonstrated interactions between C6-C2, C6-C3, C6-C4, C6-C5 and C6-C8. The C6-C5 interaction was nearly 10 fold higher in magnitude than any others.
A 3C based interaction map was created to examine if the insertion of ectopic CTCF binding sites as H19-ICR (as created in TCR-ins mutant mice) alters the pattern of interactions. For this analysis, allele specific-3C-qPCR strategy was designed and the interactions selectively scored from the maternally inherited wild type, TCR-ins and TCR-mut alleles in Rag1 deficient thymocytes while the paternal allele was prevented from contributing to the analysis due to single nucleotide differences in the amplification primers. Each of the interactions investigated i.e. C3-C5, C3-C6 and C6-C5 were reduced 2-3 fold in the TCR-ins allele compared to the wild type allele. This effect was CTCF dependent as the TCR-mut allele, unable to bind CTCF at the ectopic locations, did not show such a reduction.
Our analysis suggests that CTCF based chromatin loops are relevant for V-to-DJ recombination at TCRβ locus and get altered under the influence of ectopic CTCF binding sites. The alteration has a significant functional outcome as observed earlier.
Future plans
TCRβ enhancer Eβ has been shown to exert its influence on the chromatin accessibility limited to 25kb region encompassing PDβ1-DJCβ1 and PDβ2-DJCβ2 regions. However, a recent study has demonstrated that Vβ regions interact with the Eβ plausibly due to their simultaneous presence in the recombination hub. The details of the chromatin hub organization at TCRβ locus are not known and need further exploration. Efforts will be made to dissect this aspect in context of endogenous TCRβ locus as well as TCRins and TCRmut alleles.
187
Action taken on the RAP/SAC 2013 recommendations
The projects being pursued were discussed in detail. It was suggested that the role of CTCF should be analysed for wild type TCRβ locus organization. The ChIP and 3C studies are ongoing to examine the role of CTCF in wild type as well as mutant contexts.
188 Role of cell signaling in eukaryotic development
Principal Investigator Pushkar Sharma
Research Associates Prashant Kumar Modi Anuj Tripathi Aparna Dixit
Ph.D. Students Praveen Kumar Surbhi Jaiswal Sudhir Kumar Priyanka Bansal
Collaborators Tim Gilberger, BNI, Hamburg Christian Doerig, Monash University, Melbourne Manoj Duraisingh, Harvard School of Public Health Keshav Prasad/Akhilesh Pandey, IOB, Bangalore Dominique Soldati, University of Geneva
Theme of Research
It is well known that extracellular signals control biological responses in most eukaryotic cells by regulating specific intracellular signaling and trafficking cascades. We are interested in signaling and trafficking events in two diverse cell types: 1) Apicomplexan parasites like Plasmodium falciparum and 2) mammalian neurons.
Objectives
1. Dissection of intracellular signaling and trafficking cascades in apicomplexan parasites like Plasmodium falciparum.
Characterization of signaling pathways that operate in malaria parasite may help unravel novel mechanisms involved in its development. We are interested in the role and regulation of parasite signaling by second messengers like calcium, phosphoinostides and their effectors in the life cycle of Plasmodium falciparum. Since Toxoplasma gondii is an excellent model for studying obligate intracellular parasitism, we plan to study some of the signaling pathways relevant to apicomplexans in this parasite.
2. Molecular mechanisms that regulate Cell cycle Related Neuronal Apoptosis (CRNA)
While apoptosis of neurons is critical for brain development, it also results in neuronal loss which leads to several neurological disorders. A subset of neurons upon encountering neurotoxic insults attempt to re-enter the cell cycle, which is reflected by the aberrant
189 modulation of cell cycle proteins like cyclins and cyclin dependent kinases (cdks). We are interested in molecular mechanisms that regulate Cell Cycle Related Neuronal Apoptosis (CRNA).
Work Reported in 2012-2013
A. Signal transduction and trafficking in Plasmodium falciparum.
i) Cross talk between cAMP and calcium in the blood stage development of malaria parasite. We reported the regulation of glideosome associated protein 45 (PfGAP45) by calcium effectors like PfCDPK1 and PfPKB. In addition, the modulators of cAMP like cAMP phospodiesterase inhibitor IBMX and 8-BrcAMP caused a decrease in phosphorylation of GAP45 at PfCDPK1/PfPKB target sites. These observations suggested that cAMP may either modulate calcium levels in the parasite or regulate the down stream effector cAMP- dependent protein kinase (PfPKA).
ii) Role of phosphoinositides in parasite signaling and trafficking A phosphoinositide-binding kinase ((PLASMODB ID: PF11_0242), which has a pleckstrin homology (PH) domain, was identified and was named PH-domain containing kinase (PfPHDK). Since this protein is now referred in literature and various databases as Calcium Dependent Kinase 7 (CDPK7), we will henceforth refer to it as PfCDPK7. Immunolocalization studies suggested that PfCDPK7 may be present in the ER and vesicles. PfCDPK7 interacts with PI(4,5)P2 via its PH domain, which may also be critical for its sub cellular localization. Gene disruption studies suggested that PfCDPK7 may be important for asexual development of P. falciparum and may regulate parasite division.
B. Role of cyclins and cyclin dependent kinases in neuronal apoptosis
We identified a novel mechanism via which beta amyloid peptide Aβ42 causes Cell Cycle Related Neuronal apoptosis (CRNA) by elevating the expression of cyclin D1. The role of CDK inhibitor (CKI) p27 and cdk5 in this process was further explored. The inhibition of cdk5 or its knockdown lead to CRNA via hyperactivation of the MEK-ERK pathway, which suggested that one of the roles of cdk5 is to keep the neuronal cell cycle arrested. Furthermore, the treatments of neuronal cells with Aβ42 lead to the formation of inactive cyclin D1-cdk5 complex. p27 further enhanced the formation of this complex by interacting with cdk5 and cyclin D1. In contrast, p27 did not interact with p35 and cdk5. The role of microRNA34a (miR34a) in CRNA was also investigated. The overexpression of miR34a in cortical neurons prevented the expression of cyclin D1 induced by Aβ42 and also blocked the S-phase entry and cell death.
190
Progress of work in the current reporting year ( 2013-2014)
1. Dissection of intracellular signaling and trafficking cascades of Plasmodium falciparum.
a) cAMP and calcium signaling in the blood stage development of malaria parasite. Calcium Dependent Protein Kinases (CDPKs) are important calcium effectors that regulate diverse parasitic processes. Some CDPKs are essential for parasite, therefore, are refractory to gene disruption. As mentioned above, PfCDPK1 may be regulated by PLC-mediated calcium release. In order to unequivocally establish the role of PfCDPK1 in asexual blood stage development, we used an approach which involved a FKBP or DD domain. We have a parasite line in which PfCDPK1 is fused to DD domain by single crossover homologous recombination. In the absence of DD domain ligand shield-1, which stabilizes the DD domain fusion protein, almost 60-80% PfCDPK1 knock down was achieved. PfCDPK1 knock down resulted in a significant decrease in erythrocyte invasion by the parasite providing a strong evidence for a role of this kinase in invasion. In order to decipher the mechanism via which calcium signaling and PfCDPK1 may regulate invasion, we need to identify substrates of this kinase. A comparative phosphoproteomics is being used to identify PfCDPK1 substrates. Preliminary studies lead to the identification several differentially phosphorylated proteins, which needs validation. We have further dissected the mechanism via which cAMP and calcium signaling pathways crosstalk. Parasitic cAMP levels seem to modulate calcium levels in the parasite, thereby may regulate calcium signaling. In this context, the role of CDPKs was investigated; the modulation of cAMP levels resulted in altered CDPK activity.
b) Role of phosphoinositides in parasite signaling and trafficking Some of the results related to a PH domain containing kinase (PfPHDK) were reported last year. Since this kinase clusters with Calcium Dependent Kinase (CDPK) family, it has been named CDPK7 in various databases. We will subsequently refer to the P. falciparum orthologue of this kinase as PfCDPK7. PfCDPK7 is an atypical CDPK as it does not possess the classical CDPK architecture and in addition has a PH domain via which it interacts with PI(4,5)P2. The preliminary studies reported last year had suggested that it is important for the development of asexual parasites as the growth rates of PfCDPK7 knockout (PfCDPK7-KO) were significantly attenuated in comparison to the 3D7 parasite line. Next, we investigated the stage at which PfCDPK7 was involved in parasite development. Strikingly, the transition of a significant number of PfCDPK7-KO rings failed to mature to trophozoites. These aborted parasites exhibited distorted morphology, which explained the slower growth rate PfCDPK7-KO line. Furthermore, comparison of the number of merozoites per segmenter/schizonts revealed that PfCDPK7-KO parasites had significantly fewer nuclear bodies per schizont/segmenter. These observations suggest that PfCDPK7 may be important for the development of rings and may also have a role in schizogony.
PfCDPK7 may not be a major player in the process of erythrocyte invasion. However, the parasite development post-invasion was markedly abrogated in PfCDPK7-KO parasites. A significant number of PfCDPK7-KO parasites exhibited abnormal parasitophorous vacuole
191 (PV)/parasitophorous vacuole membrane (PVM) morphology and failed to mature to trophozoites. In addition, rhoptry bulb protein (RAP1), which is typically transferred to the PV post invasion, was found in the cytoplasm of RBCs infected with PfCDPK7-KO parasites. It is possible that PfCDPK7 may directly or indirectly be involved in PV/PVM formation. The tubulovesicular membrane network (TVN), which is an extension of the PVM in the host RBC, facilitates nutrient uptake from the host and extracellular milieu. To determine the effect of PfCDPK7 in the formation of TVN, iRBCs were labeled with membrane-permeable BODIPY-Ceramide. Tubular structures and vesicles labeled with BODIPY-Ceramide, which are typically observed in trophozoites, were present in 3D7. Strikingly, the continuous membranous staining of tubules was lacking in a significant number of PfCDPK7-KO parasites. Instead, punctate spots and vesicles suggestive of residual and stunted membranous structures in the iRBC cytoplasm were observed in these parasites. These observations suggested that TVN formation may be disrupted in the absence of PfCDPK7. Since TVN and PVM facilitate the import of nutrients like lipids to the parasite, internalization of a membrane-impermeable lipid FM4-64 was monitored. The uptake of the lipid was observed in 3D7 parasites and the host. In comparison, a significantly smaller number of PfCDPK7-KO parasites exhibited FM4-64 staining and the amount of FM4-64 internalized was markedly reduced. These data suggested that the stalled TVN formation may prevent lipid uptake by the parasites, which may explain the defects in development of PfCDPK7-KO parasites during ring to trophozoite transition.
Toxoplasma gondii, an apicomplexan parasite, shares several similarities with Plasmodium in processes like host cell invasion. Because of its simplicity and versatility for in vitro and in vivo studies and its accessibility to genetic manipulation, Toxoplasma gondii ranks among the best experimental models to study obligate intracellular parasitism. The CDPK family, which is absent in the mammalian host, is conserved in apicomplexan parasites. TgCDPK7 is an orthologue of PfCDPK7 in Toxoplasma gondii. To study the function of TgCDPK7, gene disruption studies were carried out which suggested that it may be essential for parasite growth. As indicated above, conditional or inducible gene knock out (iKO) is possible in Toxoplasma. Attempts to generate TgCDPK7-iKO using tetracycline (Tet)-repressor based system were successful. Preliminary studies suggested that TgCDPK7 may be involved in parasite replication. The ongoing analyses of Pf/Tg-CDPK7-KO parasites may shed light on molecular mechanisms via which CDPK7 may regulate parasite development
2. Molecular mechanisms that regulate Cell Cycle Related Neuronal Apoptosis (CRNA)
The cell cycle of terminally differentiated cells like neurons is arrested in response to neurotrophic factor mediated signaling. However, the cell cycle of neurons is reactivated in response to neurotoxic insults, which leads to their apoptosis. Recently, we identified a novel mechanism via which the levels of cyclin D1 are upregulated, which results in Cell Cycle Related Neuronal Apoptosis (CRNA). We are interested in investigating the role of miRNA in CRNA. Our studies suggest that miR34a expression is elevated during neuronal differentiation and may promote it. While the knock down of miR34a caused terminally differentiated neurons to de-differentiate and re-enter the cell cycle a resulting in CRNA. In contrast, miR34a over expression caused apoptosis independent of cell cycle re-entry. These observations suggest that the levels of miR34a need to be optimal for neuronal differentiation
192 and survival. In silico analysis suggested that miR34a may target the 3’UTR of cyclin D1, which was validated by performing relevant experiments. Inhibition of miR34a expression elevated cyclin D1 expression, which is present at very low levels in neurons, confirming that miR34a may target cyclin D1 in neurons. Based on these findings, we proposed that miR34 a may have a role in CRNA. A series of experiments were carried out to test this hypothesis. The levels of miR34a upon treatment with neurotoxic amyloid peptide Aβ42, which causes CRNA, were measured. An initial increase in miR34a expression was followed by a significant reduction in its levels. When miR34a was over expressed in neurons, Aβ42 induced CRNA was significantly reverted. Interestingly, the levels of cyclin D1, which increase upon Aβ42 treatment, reduced significantly upon miR34a overexpression. To explore if the observed protection of CRNA by miR34a was via cyclin D1, cyclin D1 was overexpressed using adenovirus along with miR34a. The decrease in CRNA caused by miR34a was significantly reverted by cyclin D1 over expression. These data suggested that the decrease in miR34a expression contributes to CRNA and it may achieve this by targeting cyclin D1. Further studies to decipher the mechanisms involved in the regulation of miR34a in CRNA are in progress.
Future Plans
The knock out of PfCDPK7 and TgCDPK7 in P. falciparum and Toxoplasma gondii exhibited an interesting phenotype. Studies are in progress to gain further insights into the function of CDPK7 in these apicomplexan parasites. The substrates of these and other kinases of our interest will be identified by using approaches like phosphoproteomics. Subsequently, the identified targets will be validated and the role of their phosphorylation and its relevance to parasite biology will be elucidated. While some progress in understanding the cross-talk between cAMP and calcium cross talk has been made, we need to work out the molecular mechanisms. These and other studies will help in achieving the long term goal of making contributions to the signaling “map” in the parasite, which can provide novel insights into parasite biology.
We have identified miR34a as a regulator of neuronal cell cycle and CRNA. However, the mechanisms via which miRA34a levels are regulated during CRNA need to be elucidated. The role of other miRNAs in neuronal differentiation and CRNA is also being investigated. In addition, we are making attempts to dissect the role of CDK inhibitor p27 in CRNA.
Action taken on RAP-SAC recommendations
No specific suggestions were made.
193 Publications Reviews
1. Sharma P*, Chitnis CE* (2013) Key molecular events during host cell invasion by Apicomplexan pathogens. Curr Opin Microbio 16: 432-437.
2. Sreenivasamurthy SK, Dey G, Ramu M, Kumar M, Gupta MK, Mohanty AK, Harsha HC, Sharma P, Kumar N, Pandey A, Kumar A, Keshava Prasad TS* (2013) A compendium of molecules involved in vector-pathogen interactions pertaining to malaria. Malaria J 12: 216.
*Corresponding author(s)
194 Reconstructing the chemico-cellular trestleto decipher biology of Tuberculosis and Vitiligo
Principal Investigator Rajesh S. Gokhale
Ph.D. Student Parul Ganju
Collaborators Chetan Gadgil, CSIR-NCL Rajni Rani, NII K. Natarajan, JNU
Theme of Research
Our group is interested to elucidate mechanistic and spatiotemporal coherence of cellular processes that result in two distinct pathological diseases – Tuberculosis (TB) and Vitiligo. Although unrelated, both disorders involve decisive role of unusual metabolites- complex lipids with very-long branched acyl chains produced by the TB pathogen Mycobacterium underlying implications in disease outcome.
Objectives
To summarize, the tuberculosis (Mtb) and heteropolymeric structurally uncharacterized melanins produced by melanocytes. Both these diseases are also characterized by unpredictable disease progression profiles. TB manifests in active, latent, reactivated and dissemination phases. Vitiligo is a chronic unstable depigmenting disorder that often shows symmetry in its manifestation. While 1/6th of the Mtb genome encodes genes involved in lipid metabolism; the only well-characterized function of melanocytes is to produce melanin. Our endeavor is to understand how small molecule metabolic networks are elaborately tuned in nature and how these pathways provide distinct advantages to the specific biological system and finally their objectives of the studies proposed are:
1. Delineating networks and pathways underlying biosynthesis or degradation/recycling of lipidic metaboilites in mycobacteria 2. Identify factors involved in melanogenesis and decode spatiotemporal coherence associated with melanocyte-keratinocyte biology
Work reported in 2012-2013
We reported identification of an evolutionarily conserved biosynthetic cluster that produces redox-active molecules – bifilmoquinones and bifilmopyrones, specifically during biofilm development. We also showed that altered intracellular redox changes could be the crucial trigger that dictates the process of aggregated morphogenesis. In order to maintain cellular
195 respiration, Msmeg produces such alkyl benzoquinones specifically in the oxygen-depleted zones of biofilm.
Progress of work during the current reporting year (2013-2014)
IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation
In response to external stimuli, biological systems exhibit a variety of complex behaviors attributed to interplay between signaling, metabolic, and regulatory pathways that function over a broad range of timescales. Coordinated functionality of these events is critical in maintaining physiological homeostasis. Epidermal pigmentation represents one such central mechanism operative in the body’s largest organ, skin, that protects the genome from external damage. Pigmentation is an outcome of the interplay between two epidermal cell types: melanocytes and keratinocytes. Several paracrine and autocrine factors are known to regulate this intricate process. Although immune cells recruited to the skin protect this organ from various infections, it is not clear whether these cells could also influence skin pigmentation. We developed a cell autonomous cyclical oscillator model of pigmentation and depigmentation that is known to sustain its oscillations through a feedback loop to identify negative regulatory pathways involved in melanogenesis. Unbiased genome-wide transcriptional profiling of this pigmentation oscillator revealed a dominant IFN-γ signature inversely correlated with pigmentation. Although addition of IFN-γ to depigmented B16 melanoma cells prevents cells from pigmentation in the low-density state, cultured primary melanocytes showed a distinct hypopigmentation in 4–5 days of IFN-γ treatment. We then showed a clear association of increased IFN-γ signaling to a decreased number of stage III and IV melanosomes in the lesional skin of leprosy patients. Although other immune factors can also perturb skin pigmentation, the identification of a specific IRF1-mediated regulation of pigmentation genes provided confidence that IFN-γ must have a significant role in controlling melanosome maturation and thus, skin pigmentation. Further, we show that chronic overexpression of IFN-γ in both mouse and human skin can result in hypopigmentation phenotype. On UV exposure, a clear delay in regaining the basal pigmentation of skin can be observed in the IFN-γ-knock out mouse. Together, our studies support the conjecture that the strength, durability and temporal response of the IFN-γ response could be a crucial factor for maintaining epidermal pigmentation homeostasis and this mechanism may have important implications in the detanning of human skin.
Future Plans
Selective degradation of dysfunctional and damaged organelles through autophagy-related pathways is a vital homeostatic process. Melanosomes in melanocytes are known hubs of intense free-radical melanin synthesis, however, it is not understood how these specialized organelles maintain their integrity. While autophagy-related proteins colocalize on melanosomes, intrinsic cellular pathways regulating melanosome turnover are not known. We had recently reported a novel cell-autonomous pigmentation oscillator that can undergo
196 repetitive cycles of pigmentation-depigmentation and thus provides a platform to investigate into melanosome biogenesis and degradation.
DISCOVERY MODEL PATHOPHYSIOLOGY
With IFN-γ Th1 Th2 response IFN-γ removed High IFN-γ Low IFN-γ
Transcriptional Profiling
1 1 Skin Other skin 0.9 0.9
0.8 0.8 y t
ue depigmentation i changes 0.7 l 0.7 s a n
Mathematical v f te 0.6 f 0.6 n e Melanin-devoid melanosomes i
o c d 0.5 0.5 d
ze i l ze
0.4 i a 0.4 l a m r m o 0.3 Analysis r 0.3 o N N 0.2 0.2
0.1 0.1
0 0 0 5 10 15 20 25 30 35 40 45 0.02 0.04 0.06 0.08 0.1 0.12 time (days) frequency (1/day) Normal
+++ IFN-γ
Action taken on the RAP/SAC 2008 recommendations
No specific recommendation was made.
Publication Original peer-reviewed article
1. Natarajan VT, Ganju P, Singh A, Vijayan V, Kirty K, Yadav S, Puntambekar S, Bajaj S, Dani PP, Kar HK, Gadgil CJ, Natarajan K, Rani R, Gokhale RS* (2014) Proc Natl Acad Sci, USA 11: 2301-2306.
*Corresponding author
197 Determining the signaling and repair pathways that are altered in human cancer using RecQ helicases as the model system
Principal Investigator Sagar Sengupta
Research Associate Vivek Tripathi
Ph.D. students Suhas Sampat Kharat Jyoti Kumari Raina Priyadarshini Swati Priya Himanshi Agarwal
Research Fellows Vinoth Madhavan Mansoor Hussain Arun Pr
Collabotators K. Muniyappa, IISc, Bangalore Grant Stewart (University of Birmingham, UK Avinash Bajaj, RCB, Gurgaon Gaelle Legube (LBCMCP-CNRS, Toulouse, France Arnab Mukhopadhyay, NII, New Dellhi Shantanu Chowdhury, IGIB, Delhi
Theme of Research
Our research program evolves around the understanding of the cellular processes that are altered in neoplastic transformation leading to human cancer. Towards this aim, we focus my research endeavor on the RecQ helicases. BLM and RECQL4 are members of the RecQ family of DNA helicases. Germline mutations in both BLM and RECQL4 helicase result in autosomal-recessive disorders, Bloom syndrome (BS) and Rothmund-Thomson syndrome (RTS) respectively. BS afflicted individuals are predisposed to almost all types of cancers while RTS individuals are predominantly predisposed towards osteosarcomas. Since RecQ helicases are intimately involved in the many vital cellular processes, they are ideal candidates to investigate the reasons for neoplastic transformation.
Objectives
In the current year the work in the lab was aimed to dissect the in vivo functions of BLM and RECQL4 helicases. Specifically the aims were:
1. Decipher the role of BLM in DNA damage response 2. Determine the functions of RECQL4 and p53 in mitochondria
198 Work reported in 2012-2013
1. Decipher the role of BLM in DNA damage response
Various post-translational modifications on BLM have been reported which possibly influence its diverse functions during DNA damage response. Recruitment of proteins to the site of DNA damage is a highly ordered, hierarchical process controlled by certain key elements. In the last few years it has become increasingly obvious that two E3 ligases, RNF8 and RNF168, play very important roles in the regulation of the DNA damage response by catalyzing the recruitment of two important downstream factors – RAP80/Abraxas/BRCA1 complex and 53BP1 to the sites of DNA damage. Based on the facts about the role of BLM in DNA damage response, it was hypothesized that like BRCA1 complex and 53BP1, BLM may also be recruited to the sites of DNA in an RNF8/RNF168 mediated ubiquitylation- dependent manner, which might affect its downstream roles in multiple steps of recombination. Last year we had provided evidence that BLM is recruited to sites of replication stress in an ubiquitin-dependent manner. It was also demonstrated that BLM is targeted by RNF8 and RNF168 for ubiquitylation, both under in vitro and in vivo conditions.
Progress of work during the current reporting year (2013-14)
1. Decipher the role of BLM in DNA damage response
N-terminal ubiquitylation of BLM is required for its relocalisation to sites of DNA damage In order to ascribe a biological function to the RNF8/RNF168-dependent ubiquitylation of BLM, initially the sites of ubiquitylation needed to be identified. To narrow down the lysine residues within BLM potentially targeted for ubiquitylation, two independent ubiquitylation site prediction programs (Ubpred and Ubipred) were used, which both highlighted lysines at 105, 225 and 259 (K105, K225 and K259) as being high confidence target resides. To investigate whether these lysine residues were ubiquitylated by RNF8/RNF168, recombinant BLM containing each individual lysine mutated to arginine or all three sites mutated in combination (3K-R) was purified, tested for their ability to perform helicase activity to equal extent and subsequently used as the substrates during in vitro ubiquitylation reactions. Loss of any of the three predicted lysine residues individually resulted in a reduction in the level of in vitro BLM poly-ubiquitylation. The RNF8/RNF168-dependent ubiquitylation was completely abrogated in 3K-R BLM mutant indicating that RNF8/RNF168 can target multiple lysine residues within BLM for ubiquitin chain conjugation in vitro.
To determine whether the three sites of BLM ubiquitylation identified in vitro also mediated the conjugation of poly-ubiquitin chains in vivo, extracts derived from 293T cells transfected with either WT or mutant 3K-R BLM expression constructs were subjected to immunoprecipitation using an antibody specific for K63-linked poly-ubiquitin chains and Western blotted for the presence of BLM. Loss of these three critical N-terminal lysine residues resulted in a significant decrease in the overall level of K63-linked BLM ubiquitylation after HU-treatment, supporting the notion that they represent the major sites of K63-linked BLM ubiquitylation in vivo.
199
It is conceivable that the RNF8/RNF168-dependent ubiquitylation of BLM at specific residues is required for the relocalisation BLM to sites of replication stress following HU exposure. To test this hypothesis, cells transfected with either a WT or 3K-R mutant EGFP- tagged BLM expression constructs were treated with HU and the focal recruitment of BLM monitored by fluorescence microscopy. In stark contrast to the WT BLM, the single mutants substantially compromised the ability of the exogenous BLM to form HU-induced foci. This defect was exacerbated when all three sites of ubiquitylation on BLM were lost. A similar lack of BLM 3K-R localization was also observed after ionizing radiation. Combined, these data support the concept that RNF8/RNF168-dependent ubiquitylation of BLM promotes its recruitment to sites of DNA damage.
RNF8/RNF168-dependent ubiquitylation and recruitment of BLM is required to suppress HR at stalled replication forks Since we have demonstrated that RNF8/RNF168 are required to localize BLM to sites of HU-induced DNA damage, it can be hypothesized that cells lacking these E3 ubiquitin ligases would display an elevated level of HR in response to HU, as observed in BS cells. In keeping with the proposed role for these enzymes in regulating BLM recruitment, cells depleted of either RNF8 or RNF168 exhibited an increased level of HR as judged by either a plasmid-based HR assay or sister chromatid exchange (SCEs). The elevation in levels of SCEs induced by HU treatment was also evident in RIDDLE syndrome cells complemented with empty vector but not with WT RNF168. Importantly, in keeping with an essential requirement for the RNF8/RNF168-dependent ubiquitylation of BLM after DNA damage for its function, wild type BLM but not the BLM 3K-R mutant was able to suppress HR in BS cells.
Given that the underlying cause of the elevated HR in cells lacking RNF8/RNF168 could be inpart explained by a inability of these cells to properly recruit BLM to regions of chromatin proximal to a stalled replication fork, it is conceivable that this could be corrected by forcing BLM onto chromatin and bypassing the need for the UbS-DDR. Based on this premise, two BLM fusion proteins were created, one that was fused to histone H2AX (H2AX-BLM) and the other that was fused to the FHA domain of MDC1 (MDC1-FHA-BLM). The rationale for the creation of these mutants is that H2AX-BLM should be constitutively bound to chromatin whereas the MDC1-FHA-BLM should only be relocalised to chromatin following the induction of DNA damage through its ability to bind to phosphorylated MDC1 located at sites of DNA damage. However, both these BLM fusion proteins circumvent the need for RNF8/RNF168-dependent ubiquitylation to promote chromatin binding. Strikingly, both the H2AX-BLM or MDC1-FHA-BLM fusions completely suppressed the exacerbated levels of HR caused by depletion of either RNF8 or RNF168. Importantly, both the fusions, which cannot be ubiquitylated due to the lack of the E3 ligases, could however restore the nuclear relocalisation of BLM to sites of DNA damage. Combined, these data strongly support a role of RNF8 and RNF168 in preventing excessive HR at sites of stalled or compromised replication forks through their ability to stimulate the RAP80-dependent recruitment of ubiquitylated BLM.
200 2. Determine the functions of RECQL4 and p53 in mitochondria
Germline mutations in RECQL4 and p53 lead to cancer predisposition syndromes, Rothmund Thomson Syndrome (RTS) and Li–Fraumeni syndrome (LFS), respectively. We have earlier provided evidence that RECQL4 is essential for the transport of p53 to the mitochondria under unstressed conditions.
RECQL4 and p53 regulate the binding of PolγA/B2 to the mitochondrial control region known to form D-loop structures We have recently demonstrated that RECQL4 and p53 are required for optimal de novo mtDNA replication. To further characterize the interaction of RECQL4-p53-PolγA with mitochondrial control region in vivo, we carried out mitochondrial chromatin immunoprecipitation (mtChIP) assays with a combination of five primer sets spanning the entire control region. In contrast to NHF shRECQL4 cells, PolγA in NHF shControl cells showed distinct and consistent binding within certain specific regions of the control region. Binding of PolγA was observed only with PCR products I and IV. PCR product I is adjacent to the cytochrome B coding sequence, whereas product IV which spans the C-tract or D310 region, is known to be a mutation hot-spot in a wide variety of cancers and is involved in mtDNA replication and transcription. This region also serves as one of the origins of mtDNA replication. Decreased binding of PolγA to the PCR product IV was observed in multiple RTS patient fibroblasts, LFS patient fibroblasts and in NHF E6 cells lacking p53 expression. These results indicate that both RECQL4 and p53 are required for optimal binding of mitochondrial polymerase to this specific region. Importantly, both p53 and RECQL4 also bind to the PCR product IV. To confirm concomitant binding of PolγA-RECQL4 to this region, sequential and reciprocal mtChIP (re-mtChIP) was carried out with PolγA and RECQL4. We observed consistent increase in signal intensity for PCR products I and IV (but not for the PCR product III which served as the negative control). Altogether, the results indicated that the presence of RECQL4 and p53 were both required for optimal binding of PolγA to specific regions within the control region and thereby regulate mtDNA replication.
RECQL4 and p53 physically interact with Polγ in vivo To elucidate the role of RECQL4 and p53 in mtDNA replication, we first determined whether RECQL4 and p53 were complexed in vivo with the PolγA/B2 holoenzyme. For this purpuse, co-immunoprecipitations of endogenous proteins was carried out in two isogenic pairs of cell lines, namely RTS patient fibroblast AG05013 and its isogenic counterpart expressing wild-type RECQL4 namely AcGFP-RECQL4 (1-1208) Clone 1. In addition we also performed co-imunoprecipitations of the endogenous proteins from cell lines HCT116 and HCT116 p53-/-. Immunoprecipitation with anti-RECQL4 antibody revealed the presence of a complex consisting of RECQL4, p53, PolγA and PolγB in AG05013 AcGFP-RECQL4 (1-1208) Clone 1 cells. Such a complex was absent in the co-IPs of AG05013 cells, which do not express RECQL4, indicating the specificity of the reaction. A similar complex was also observed in HCT116 cells when immunoprecipitated with PolγA antibody. The PolγA immunoprecipitate in HCT116 p53-/- cells contained only RECQL4 and PolγB (and not p53), thereby again indicating the specificity of the complex formation. The lack of any specific
201 signal when the immunoprecipitations were carried out with IgG beads further confirmed the presence of the RECQL4-p53-PolγA/B2 complex in vivo.
Future plans
We will continue to understand how BLM helicase functions during DNA damage response. Effort will be made to further decipher the role of RECQL4 and p53 in the mitochondria.
Action taken on the RAP/SAC 2013 recommendations
The data presented was appreciated by the RAPSAC committee members.
Publications Original peer-reviewed articles
1. Sreekanth V, Bansal S, Motiani RK, Kundu S, Muppu SK, Majumdar TD, Panjamurthy S, Sengupta S, Bajaj A* (2013) Design, Synthesis, and Mechanistic Investigations of Bile acid- Tamoxifen Conjugates for Breast Cancer Therapy. Bioconjug Chem 24: 1468-1484.
2. Tikoo S, Madhavan V, Hussain M, Miller ES, Arora P, Zlatanou A, Modi P, Townsend K, Stewart GS, Sengupta S* (2013) Ubiquitin-dependent recruitment of the Bloom Syndrome helicase in response to replication stress is required to suppress homologous recombination. EMBO J 32: 1778-1792.
3. Gupta S, Chandra S, Priyadarshini R, Madhavan V, Tikoo S, Hussain M, Mudgal R, Modi P, Srivastava V, Sengupta S* (2013) Enhancement of c-Myc degradation by BLM helicase leads to delayed tumor initiation. J Cell Sci 126(Pt 16): 3882-3795.
4. De S, Srivastava V, Hussain M, Kumari J, Muniyappa K, Sengupta S* (2014) RECQL4 and p53 potentiate the activity of polymerase γ and maintain the integrity of the human mitochondrial genome. Carcinogenesis 35: 34-45.
*Corresponding author
202 Understanding the regulation of DNA replication
Principal Investigator Sandeep Saxena
Research Associates Akhil Varshey Ananya Kar
Ph.D. Students Manpreet Kaur Tanushree Ghosh Ritu Shekhar Raksha Devi Praveen Kumar
Research Fellows Md. Muntaz Khan Mahendra Sahu Koganti Praveen
Theme of research
DNA replication is a vital process of life and must be completed precisely during each cell cycle. When mammalian cell experiences DNA damage, it activates checkpoint mechanisms to stall the progression of cell cycle and DNA replication. Our laboratory is working towards understanding the mechanisms by which microRNA and checkpoint proteins stall the cell cycle, preventing genomic instability and cancer.
Objectives
We are studying the regulation of replication machinery during stress in order to identify underlying mechanisms responsible for inhibition of essential replication factors during stress. We are trying to understand the role of ubiquitination machinery in regulating replication proteins under normal and stressed conditions. Further, we are investigating the cellular response to aberrations in replication complexes. The objective is to identify yet unknown checkpoint pathways that monitor the replication apparatus. Emerging evidences suggest that miRNAs target genes that regulate DNA replication and cell cycle progression and we aim to determine the role of miRNA in regulating the DNA replication machinery as the cell progresses from one phase to the next. This would provide an insight into the mechanisms by which miRNAs regulate mammalian cell cycle and DNA replication during normal and pathological conditions. Summing up, we are attempting to unravel the protective regulatory control of mammalian cells, failure of which is likely to cause genomic instability.
203 Work reported in 2012-2013
We previously reported that Mcm10, an essential human replication factor, is selectively proteolyzed after UV-irradiation to inactivate the replication machinery. During the last year, we demonstrated that E3 ubiquitin ligase comprising of VprBP as the Substrate recognition subunit (SRS) has a vital role in regulating replication by mediating the stress-induced turnover of replication factor, Mcm10. Single-stranded DNA generated at stalled replication forks and during DNA repair serves as an intermediate for activating the Ataxia telangiectasia and Rad3-related protein (ATR) checkpoint kinase which phosphorylates Chk1, initiating a signal transduction cascade. It is believed that binding of Replication protein A to the single- stranded DNA is essential for the recruitment of ATR-ATRIP complex to the sites of DNA damage facilitating the initiation of checkpoint response. During the last year, we reported that ATR can phosphorylate Chk1 independent of RPA. We observed that depletion of human single-stranded binding protein 1 (hSSB1) or its partner protein called INTS3 prevented RPA- depletion triggered ATR phosphorylation, suggesting a role of hSSB1 and INTS3 in ATR mediated signaling cascade. During the last year, we determined the stress triggered changes in the levels of individual microRNA by Real-time PCR amplification and reported that many microRNAs are induced during stress. Therefore, we had initiated our efforts to understand the regulatory mechanisms that mediate the stress-triggered proteolysis of the replication machinery.
Progress of work during the current reporting year (2013-2014)
Role of alternate single-stranded DNA-binding proteins in checkpoint signaling
ATR–ATRIP mediated DNA damage pathway is triggered in response to generation of single- stranded DNA as a result of replication stalling or genomic insults like exposure to UV radiation or hydroxyurea. The nascent single strands are rapidly coated with the primary single strand binding protein complex, Replication Protein A (RPA) which enables the recruitment of ATR and its interacting partner, ATRIP to the sites of DNA damage. We attempted to look into the fate of this process when the RPA complex is not present. We observed that in the absence of RPA complex, an alternate single-stranded DNA-binding protein complex, hSSB1- INTS3 associates with the single-stranded DNA and elicits ATR–ATRIP mediated checkpoint response (Figure 1). Furthermore, similar to the canonical checkpoint pathway this alternate pathway requires the sensors Rad17-9-1-1 complex, co-activator Topbp1 as well as the adaptor protein claspin for activating the ATR kinase. Therefore, we identified an alternate pathway for the activation of ATR-ATRIP complex, modifying the fundamental understanding of checkpoint activation where recruitment of RPA is considered obligatory.
204
Figure 1: ATRIP foci formation after RPA depletion. HeLa cells were transfected with siRNA duplexes against GL2, RPA1 alone or in combination with ATRIP or INTS3 and the cells were visualized for ATRIP foci. Scale bar is 10 μM.
Role of non-coding RNAs in regulation of cell-cycle and DNA replication
The mechanism of regulation of cell cycle and DNA replication genes during quiescence is not completely understood and we aim to elucidate the role of non-coding RNAs in regulating the transition between proliferation and quiescence. We have determined the changes in the levels of miRNAs after serum starvation by miRNA Arrays (Exiqon). We have standardized two miRNA assays in our laboratory: hemi-nested and poly-A-tailing assay and we have utilized them to confirm the changes in the level of individual miRNA (Figure 2A). Similarly, we have also analyzed the changes in gene expression during serum starvation and cataloged the regulated cell cycle and DNA replication genes. The targets of the upregulated miRNAs have been predicted using TargetScan (Figure 2B). We will now establish the targets of stress- regulated miRNAs by assaying the activity of firefly luciferase expressed in fusion with 3’ UTR of target genes. Next, we will manipulate the expression of stress-regulated miRNAs and assay the effect on cell cycle and replication factors. We will also address if disruption of miRNA targeting of replication factors alters the cell cycle block and release. Therefore, in this part we aim to determine the role of non-coding RNAs in regulating cell-cycle and DNA replication genes during transition between proliferation and quiescence.
205
Figure 2: MiRNAs targeting cell cycle and DNA replication genes. (A) HCT116 cells were grown in the absence (Serum Starved; S) or presence of serum (Asynchronous; A) for 48 h and the levels of hsa-miR-1d# and RNU48 control RNA were determined by Real-time PCR amplification. Serum starved cells were later released into the cell cycle by moving cells into a medium containing serum to evaluate microRNA levels (Serum released; R). (B) The table displays examples of microRNAs that are upregulated and the cell cycle genes that are downregulated during serum starvation. Based on TargetScan, the targets of microRNAs are predicted (denoted by •). # referred as per DRCC laboratory index.
Role of replication proteins during mitosis
Recent studies have shown that some replication proteins localize to centrosomes, kinetochores and other mitotic structures such as spindle poles and midbody. Depletion of many replication factors have been shown to result in many mitotic aberrations such as spindle multipolarity, abnormally condensed chromosomes and amplification of centrosomes. Depletion of some replication proteins also results in cell division defects such as delay in daughter cell abscission and failure of cytokinesis. We systematically depleted replication proteins by RNAi and assayed for mitotic defects: we identified that depletion of a GINS subunit results in multipolar spindle formation and increased centrosome number in mitotic
206 cells (Figure 3). By utilizing two different antibodies we also established that the GINS subunit localizes to the centrosomes (Figure 4). Controlled depletion of GINS subunit did not trigger a DNA damage checkpoint response indicating that the observed phenotypes are not due to DNA damage induced cell cycle checkpoint. Moreover the aberrant centrosome number was not observed in interphase cells indicating that the GINS subunit localizes to the centrosomes and is required for mitotic progression. We would now like to determine the role of GINS subunit in coordinating the mitotic events.
Figure 3: RNAi depletion of GINS subunit induces mitotic aberrations. HeLa cells were transfected with GL2 or GINS subunit siRNA followed by immunofluorescence with mouse anti-α-tubulin antibody. Nuclei were stained with DAPI. Note that depletion of GINS subunit results in multipolar spindles.
Figure 4: GINS subunit localizes to centrosomes. HeLa cells were fixed followed by co-immunofluorescence with rabbit anti-GINS antibodies (Ab1 and Ab2) and mouse anti-gamma-tubulin antibody. Nuclei were stained with DAPI. Note that the single focal plane does not display out-of-focus centrosomes.
207 Future Plans
Future plans have been incorporated in the main section. In brief, we will evaluate the role of the alternate SSBs in the activation of ATR-ATRIP after different genomic stresses. We will determine the role of non-coding RNAs in regulating cell-cycle and DNA replication genes during proliferation and quiescence. And we will ascertain the role of replication proteins during mitosis.
Action taken on the RAP/SAC 2013 recommendation
No specific comment was received.
208 The role of tumor suppressors in stress response
Principal Investigator Sanjeev Das
Ph.D. Students Yatendra Kumar Satija Abhishek Bhardwaj Rajni Kumari Ruhi Deshmukh Saishruti Kohli (from December 2013)
Research Assistants Shakti Verma (till August 2013) Ankit Chauhan (till August 2013)
Theme of research
In response to various intracellular and extracellular stresses p53 is rapidly stabilized and activated thereby inducing cell cycle arrest, apoptosis or senescence depending upon the extent of cellular damage. Temporal regulation of diverse sets of target genes is thought to be achieved by post-translational modifications and through its interaction with other cellular proteins. To investigate p53 regulation, we performed a proteomics screen to identify p53 interacting proteins. We are now trying to understand the function of HDAC5 as a p53 interacting protein.
p73 is one of the tumor suppressors of the p53 family of nuclear transcription factors. p73 exhibits many p53-like properties: it can bind to p53 DNA target sites, transactivate p53- responsive genes and induce cell cycle arrest or apoptosis. However the molecular mechanisms underlying p73 regulation remain unanswered. To address the lacunae in the understanding of p73 stability and function, we carried out a proteomics screen to identify p73 interacting proteins under normal and genotoxic stress conditions. We have now identified TRIM28 and MED15 as potential p73 interacting proteins.
Objectives
1. Characterization of HDAC5 as a key modulator of p53-mediated transactivation: Our previous studies have led to characterization of HDAC5 as a bonafide p53 deacetylase. We plan to investigate the effect of HDAC5 on p53 function.
2. Characterization of novel p73 interacting proteins involved in regulation of p73 stability and function: We have now identified an ubiquitin ligase TRIM28 and a transcriptional coactivator Med15 as potential p73 interacting proteins. Since TRIM28 is a RING-type E3 ligase, we plan to investigate the role of TRIM28 in p73 homeostasis. Since MED15 serves as a key coactivator in various transcriptional complexes, we plan to investigate whether MED15 can serve as a p73 coactivator.
209 Work reported in 2012-2013
A. Characterization of HDAC5 as a key modulator of p53-mediated transactivation
We performed a proteomics screen to identify novel p53 interacting proteins. We carried out further studies to understand the function of one of the novel interacting proteins viz. HDAC5. HDAC5 belongs to the class IIa HDAC (histone deacetylase) subfamily. By performing coimmunoprecipitations as well as in vitro binding assays we found that HDAC5 directly interacts with p53. Since HDAC5 is a member of class II family of deacetylases, we examined the effect of HDAC5 on p53 acetylation upon genotoxic stress. Using immunoblotting as well as mass spectrometry based assays we showed that HDAC5 deacetylates p53 with specificity for lys120 site of p53. Since our results indicate that HDAC5 undergoes nuclear-cytoplasmic shuttling upon genotoxic stress, we further investigated the underlying molecular mechanism. We found that with increased accumulation of ROS at extended periods of genotoxic stress, there is a concomitant activation of CAMKII (Ca2+/calmodulin-dependent protein kinase II), which phosphorylates HDAC5 at Ser259 and Ser498. Previous studies report that CaMKII phosphorylates HDAC5 at Ser259 and Ser498 leading to its nuclear export. We also found that at early time points of genotoxic stress when HDAC5 is not phosphorylated, it is nuclear but at extended periods of genotoxic stress concomitant to HDAC5 phosphorylation, it becomes cytoplasmic.
B. Identification and characterization of novel p73 interacting proteins involved in regulation of p73 stability and function
We carried out a proteomics screen to identify novel p73 interacting proteins. For this purpose a recombinant adenovirus expressing HA and Flag tagged p73 was generated. To generate a replication deficient recombinant adenovirus which expresses p73 tagged with HA and FLAG epitope we followed the strategy described previously. p73 expression from this adenovirus was confirmed by western blotting. This virus was used to infect HCT116p53-/- cells followed by tandem immunoprecipitations using HA and FLAG antibodies and mass spectrometry to identify the p73 interacting proteins. The LC-MS/MS analysis results showed many novel interactors along with known interactors of p73. We are now trying to understand the function of two of the novel interacting proteins viz. TRIM28 and MED15. Among the novel interactors, TRIM28 was of particular interest because it interacts under unstressed condition and there was no visible interaction after DNA damage. To validate our proteomics screen results, immunoprecipitations were carried out to check physical interaction with p73. We found that both of them coimmunoprecipitated with p73. To confirm that p73 directly interacts with TRIM28 and MED15, GST pull down was performed in vitro using bacterially expressed and purified proteins. Our results show that these are direct interactions. Further we found that RNAi-mediated abrogation of TRIM28 expression significantly increases p73 protein levels but not the transcript levels.
210 Progress of work during the current reporting year (2013-2014)
A. Characterization of HDAC5 as a key modulator of p53-mediated transactivation
Our previous results indicate that HDAC5 is a deacetylase with specificity for K120 site of p53. As previous studies report that K120 acetylation plays an important role in determining p53 target selection, we next determined the effect of HDAC5 on p53 transcriptional activity. Thus HCT116 control and HDAC5 knockdown cells were subjected to different forms of genotoxic stress including etoposide treatment and ionizing radiation, and the transcript and protein levels of different p53 target genes including proarrest, antioxidant and proapoptotic genes, were analyzed. Our results indicate that HDAC5 is required for the selective induction of p53 proarrest and antioxidant target genes at early phase of genotoxic stress while at extended periods HDAC5 undergoes nuclear export resulting in downregulation of p53 proarrest and antioxidant target genes and induction of proapoptotic target genes. These finds were further corroborated by analyzing the DNA binding activity of p53 on the promoters of its target genes by carrying out quantitative chromatin immunoprecipitation assay (ChIP).
We next studied the effects of HDAC5 on key p53 functions viz. cell cycle regulation and apoptosis. In HCT116 control cells, DNA damage leads to a pronounced G1 arrest at 6-12 hours, after which there was a marked increase in the apoptotic population. In presence of KN-93 (CAMKII inhibitor which inhibits HDAC5 phosphorylation and prevents HDAC5 nuclear export), there was prominent G1 arrest and no induction of apoptosis at late time points (24 and 36 hours). In case of HCT116 HDAC5 knockdown cells, there was onset of apoptosis from early time points (6 and 12 hours), which increased further at later time points. KN-93 had no effect on p53-mediated genotoxic stress response in the absence of HDAC5. TUNEL staining also confirmed these observations. We further investigated the role of HDAC5 in p53-mediated senescence. For this purpose HCT116 cells stably expressing constitutively nuclear HDAC5 double mutant (HDAC5 S259/498A) were subjected to prolonged genotoxic stress extending up to 5 days. In HCT116 control cells, prolonged DNA damage leads to p53 K120 acetylation resulting in apoptosis. However, in presence of KN-93, which abrogates p53 K120 acetylation, prolonged genotoxic stress leads to senescence. In HCT116 cells expressing HDAC5 double mutant (HDAC5 S259/498A), prolonged genotoxic stress can not trigger p53 K120 acetylation resulting in senescence and KN-93 had no effect on the cellular outcome. Since senescence is conversely associated with transcript levels of proliferation markers, we also assessed the levels of well known proliferation markers in these cells upon prolonged genotoxic stress. Both HCT116 control and HDAC5 double mutant (HDAC5 S259/498A) expressing cells showed reduced levels of the proliferation markers including PCNA, Cyclin A2 and MCM6. Furthermore, previous studies suggest that senescence associated repressive chromatin mark H3K9me3 (trimethylated lys9 form of histone H3) accumulates at the promoters of proliferation markers. We found an increased accumulation of H3K9me3 at the promoters of the proliferation markers in HCT116 control cells upon DNA damage only in the presence of KN-93. HCT116 HDAC5 double mutant (HDAC5 S259/498A) expressing cells also showed an increased accumulation of H3K9me3 at the promoters of proliferation markers upon DNA damage. These results indicate that at extended periods of stress concomitant to HDAC5
211 nuclear export p53-dependent apoptosis is induced, and in case HDAC5 nuclear export is repressed it leads to senescence.
Our previous results show that at extended periods of genotoxic stress there is accumulation of high levels of ROS which coincides with HDAC5 phosphorylation and nuclear export. Hence we examined the effect of ROS on HDAC5-mediated regulation of p53 functions. Thus HCT116 control and HDAC5 knockdown cells were subjected to genotoxic stress and the ROS levels were regulated by addition of ROS scavenger, tiron. Our data demonstrates that at early periods of genotoxic stress HDAC5 prevents p53 K120 acetylation and modulates its transactivation function thereby promoting its proarrest and antioxidant functions. But upon prolonged genotoxic stress, accumulation of high levels of ROS promotes phosphorylation-dependent nuclear export of HDAC5 which facilitates p53 K120 acetylation and selective transactivation of proapoptotic genes, thereby triggering the onset of apoptosis.
To corroborate the role of HDAC5 in genotoxic stress response in vivo, we used RNAi to knockdown HDAC5 expression in mice which were then subjected to genotoxic stress. We next measured the extent of apoptosis and the ROS levels in the intestine and spleen of these mice. In control mice apoptosis was minimal at early time point of DNA damage but increased substantially at later time points. However, in case of HDAC5 knockdown mice the extent of apoptosis at early time point was much higher as compared to control mice which increased further at later time points. In control mice no significant amount of ROS was detected at early time point of DNA damage but at later time points there was a sharp increase in ROS levels concomitant to p53 K120 acetylation and onset of apoptosis. The ROS levels were significantly higher in HDAC5 knockdown mice at early point of DNA damage as compared to control mice, which increased further at later time points. We also measure the levels of different p53 target genes. In control mice the levels of proarrest and antioxidant target genes were significantly induced at early point of DNA damage (1day) but at later time points (2 and 3 days) there was a sharp decrease. In the case of HDAC5 knockdown mice no induction of p21 and Sestrin 2 levels were observed at any point of stress period. In control mice Bax levels were induced only at extended periods of DNA damage (2 and 3 days) but in the case of HDAC5 knockdown mice significant induction of Bax was seen from early point of DNA damage itself which increased further at later time points. Taken together, these results demonstrate that HDAC5 plays a key role in modulating p53-mediated genotoxic stress response in vivo that augments prosurvival functions of p53 over apoptosis.
These findings provide novel insights into the role of HDAC5 in regulating p53-mediated transactivation and have been published as a research article in the journal Molecular Cell.
B. Characterization of novel p73 interacting proteins involved in regulation of p73 stability and function
TRIM28 encodes an 835-amino acid polypeptide containing a RING finger, B boxes, and a PHD finger. TRIM28 has been shown to exhibit E3 ubiquitin ligase activity. Since our
212 coimmunoprecipitation experiments show that p73 directly interacts with TRIM28, we mapped the domains involved. Our results indicate that coiled coil domain of TRIM28 binds to the N-terminal transactivation domain of p73. We also examined this interaction under genotoxic stress conditions. Our results show that TRIM28-p73 interaction is curtailed with increased duration of genotoxic stress and is completely abolished at late time points. Previous reports suggest that tyrosine kinase c-abl phosphorylates p73 at Tyrosine 99 upon genotoxic stress which is critical for p73 stabilization. Our results showed that there is no accumulation of p73 protein in absence of c-abl but under such conditions if TRIM28 expression is abrogated there is significant accumulation of p73. Thus we concluded that upon genotoxic stress p73 gets phosphorylated in c-Abl-dependent manner leading to abrogation of its interaction with TRIM28.
MED15 is a component of the activator-recruited cofactor (ARC) complex or the Mediator complex. The Mediator complex is one such multiprotein complex that functions as a bridge between regulatory proteins and Pol II, thereby regulating Pol II-dependent transcription. Since MED15 serves as a key coactivator in various transcriptional complexes, we investigated whether MED15 can serve as a p73 coactivator. Using luciferase reporter assays we found that the p73 transactivation function upon genotoxic stress was significantly increased in presence of Med15. Further, we determined the effect of MED15 on p73- mediated transactivation of its target genes including proarrest (p21 and GADD45), proapoptotic (Bax and PUMA) and anti-metastatic targets (S100A2 and TIMP3). We found out that upon genotoxic stress the activation of p73 downstream targets is severely compromised in the absence of MED15. These results establish that MED15 is an indispensable coactivator of p73.
Future plans
Since TRIM28 is an E3 ligase, we plan to investigate whether TRIM28 can ubiquitylate p73 thereby regulating p73 protein levels in cells and how is this temporally regulated under genotoxic stress conditions. We also plan to carry out xenograft studies to investigate the effect of TRIM28 on p73-mediated tumor suppression.
Since MED15 serves as a key coactivator in various transcriptional complexes, we plan to investigate the effect of MED15 on p73-mediated transactivation of its target genes including proarrest, proapoptotic and anti-metastatic target genes will also be investigated. For in vivo validation of these observations, we plan to carry out orthotopic transplantation in nude mice and luciferase chemiluminescence based live-animal imaging to study the role of Med15 in p73-mediated tumor suppression.
Action taken on the RAP/SAC 2013 recommendations
The scientific and technical clarifications sought during the presentation were provided. No specific recommendations were made.
213 Publications Original peer-research articles
#1. Satija YK, Bhardwaj A, Das S* (2013) A portrayal of E3 ubiquitin ligases and deubiquitylases in cancer Inter J Cancer 133: 2759-2768.
2. Sen N, Kumari R, Singh MI, Das S* (2013) HDAC5, a key component in temporal regulation of p53-mediated transactivation in response to genotoxic stress (2013). Molecular Cell 52: 406-420.
*Corresponding author # In press last year, since published
214 Molecular analysis of the human and animal genome(s)
Principal Investigator Sher Ali
Research Associates Monal Sharma (until February 28, 2014)
Ph.D. Students Anju Kumari (Till June 30, 2013) Sandeep K Yadav Md. Qudratullah Subranshu Sekhar Sahoo Priyank Singhavi Suresh Kumar
Research Fellow Leena Rawal
Collaborators Lalit C. Garg, NII Jamal Ahmad, JN Medical College, AMU, Aligarh
Theme of research
Our focus has been on genome analysis of human and animal systems in the context of functional and comparative genomics. In the present study, we screened vital 51 STSs corresponding to MSY region of the human Y chromosome, sequenced SRY gene and assessed copies of DYZ1 arrays in prostate cancer cell lines DU145 and LNCaP. The MSY remained intact whereas SRY showed no sequence variation in both the cell lines. Significantly, DU145 and LNCaP cell lines were found to have 742 and 1945 copies of DYZ1, respectively and showed sequence variations.
Objectives
1. Status of several Y linked genes and loci and copy number estimation of DYZ1 arrays using real time PCR in DU145 and LNCaP cell lines. 2. Sequencing of DYZ1 array and the coding region of SRY gene from these cell lines and in silico analysis.
Work reported in 2012-2013
Expression of proto-oncogene KIT is up regulated in subset of human meningiomas KIT is a proto-oncogene involved in diverse neoplastic processes. Aberrant kinase activity of the KIT receptor has been targeted by tyrosine kinase inhibitor (TKI) therapy in different neoplasias. In all the earlier studies, KIT expression was reported to be absent in meningiomas. However, we observed KIT mRNA expression in some meningioma cases. This prompted us
215 to undertake its detailed analyses in meningioma tissues resected during 2008–2009. Tumor tissues and matched peripheral blood samples collected from meningioma patients were used for detailed molecular analyses. KIT expression was ascertained immunohistochemically and validated by immunoblotting. KIT and KITLG transcript levels were discerned by reverse transcription quantitative real-time PCR (RT-qPCR). Similarly, KIT amplification and allele loss were assessed by quantitative real-time (qPCR) and validated by fluorescence in situ hybridization (FISH) on the neoplastic tissues. Possible alterations of the gene at the nucleotide level were analysed by sequencing. Contrary to earlier reports, KIT expression, was detected immunohistochemically in 20.6% meningioma cases (n = 34). Receptor (KIT) and ligand (KITLG) transcripts monitored by RT-qPCR were found to co-express (p = 0.048) in most of the KIT immunopositive tumors. 1/7 KIT positive meningiomas showed allele loss corroborated by reduced FISH signal in the corresponding neoplastic tissue. Sequence analysis of KIT showed M541L substitution in exon 10, in one of the immunopositive cases. However, its biological consequence remains to be uncovered.
Progress of work during the current reporting year (2013-2014)
Fate of the human Y chromosome linked genes and loci in prostate cancer cell lines DU145 and LNCaP
The incidence of prostate cancer (PC) is increasing worldwide though show variation amongst different ethnic groups. This is a common malignancy affecting global population of men becoming significant cause of morbidity and mortality. The involvement of several Y linked genes and loci have been reported to be associated with the initiation and progression of PC. The male sex determining gene SRY is down regulated in PC and is a negative regulator of the androgen receptor. SRY does not express in any adult male tissue except the adult testis. However, SRY is expressed in DU145, androgen untreated LNCaP cells and prostate adenocarcinoma cell lines. Thus, SRY gene seems to play an important role for the development and progression of PC. We studied several Y linked genes and loci and assessed copy number variation of DYZ1 arrays using real time PCR in DU145 and LNCaP cell lines. In addition, we sequenced the 3564 bp unit of DYZ1 array and the coding region of SRY gene from these cell lines.
Status of the male-specific region of the Y chromosome (MSY) region in DU145 and LNCaP The genomic DNA from DU145 and LNCaP was screened for the presence or absence of 51 STSs specific to MSY. These STSs in both the cell lines were found to be intact.
HERV15 provirus sequence recombination Human AZFa region contains HERV15 provirus A and B sequences and homologous recombination between these two regions accounts for most of the AZFa deletions. The AZFa regions of DU145 and LNCaP Y chromosomes were found to remain intact showing the presence of both USP9Y and DBY genes (Figure 1).
216
Figure 1: Deletion breakpoints mapping of AZFa region in DU145 and LNCaP based on STS analysis. Schematic diagram showing positions of proviruses A and B. Light and dark grey blocks are the regions of identical sequences in the proviruses A and B involved in recombination. 12 STSs specific to proviruses A and B are shown along with the corresponding positive controls.
Interstitial deletion mapping There are known deletion observed in different clinical conditions such as AZFa, P5- proximal P1, P5- distal P1, AZFc, gr/gr, b1/b2 and b2/b3 and TSPY-TSPY deletion. We have screened STSs encompassing these regions which were found to remain intact in DU145 and LNCaP cells (Figure 2).
Figure 2: STS map showing common interstitial deletions of the human Y chromosome based on different studies conducted earlier. Plus sign and blank spaces denote the presence and absence of a particular STS, respectively. Last row shows the results of the present study denoted as PS.
Status of the DYZ1 array For ascertaining structural status of the DYZ1 array, 6 sets of primers were designed to amplify 19 fragments from a single array of DYZ1 (Figure 3). The analysis of all the 19 combinations showed intact DYZ1 arrays both in DU145 and LNCaP.
217
Figure 3: Diagrammatic illustration showing end point PCR for the amplification of different sub-fractions of the DYZ1 array in DU145 and LNCaP cells. A single array of DYZ1 is shown on top. Forward and reverse primers are shown above and below the bars. Similarly, amplification products and their corresponding sizes are shown below.
Copy number calculation of DYZ1 using real time PCR We calculated DYZ1 copy number in DU145 and LNCaP by Real Time PCR using SYBR green chemistry. The amplification plot, dissociation curve and standard curve are given in Figures 4A, B, and C, respectively. Figure 4D represents the number of DYZ1 copies. DU145 contains 742 copies, while LNCaP contains 1945 copies per haploid genome.
218 Figure 4: Copy number estimation of DYZ1 in DU145 and LNCaP. (A) represents the amplification plot, (B), the corresponding dissociation curve (C), the standard plot and (D) shows the number of DYZ1 copies in DU145 and LNCaP.
3564 bp array sequence of DYZ1
Four PCR amplified fragments (Figure 3, dark bar) were cloned and sequenced. Sequencing results showed insertion, deletion and several point mutations. Compared to normal sequences of the DYZ1, the number of highly abundant “TTCCA” repeats and its derivatives per 3564 bp (HaeIII fragment) were found to be different amongst normal, DU145 and LNCaP cells. The DYZ1 sequences from both DU145 and LNCaP cells were aligned with the reference sequence from NCBI database (Accession no. AC068123.5, Gene ID- 100499443) using ClustalW software. Between DU145 and the reference sequence; 23 point mutations, between DU145 and LNCaP; 11 point mutations and between LNCaP and the reference sequence; 27 point mutations were detected. Compared to the reference DYZ1 sequence, DU145 showed insertion of 16 bp in between the position of 1654and 1655 bp, deletions of 1 bp at position 792 bp and 15 bp between 2606-2622 position, respectively. LNCaP showed insertion of 15 bp between 1654- 1655bp position and deletion of 114 bp between 2541 -2657 bp. The regions showing major deletions are highlighted in Figure 5. A comparison of LNCaP with DU145 showed a net deletion of 99 bp in LNCaP. Upon virtual restriction mapping of 3.56 Kb unit of DYZ1 using Restriction Mapper version 3 software, loss and gain of several restriction sites were detected. Differences in the restriction site frequency were compared amongst DU145, LNCaP and the reference sequence. The result showed DYZ1 arrays are uniquely affected in each category of cells.
Figure 5: DYZ1 sequence showing insertion and deletion. Partial aligned DYZ1 sequence from DU145 and LNCaP showing insertion and deletion compared to that of the reference sequence AC068123.5.
219
FISH analysis To ascertain the presence or the absence of Y chromosome in DU145 cells, we conducted FISH experiment using a labelled DYZ1 probe. Several nuclei and metaphases in DU145 were found to be devoid of DYZ1 fluorescence signals. We found approximately 48% of DU145 cells had no Y chromosome (Figure 6).
Figure 6: Localization of DYZ1 in DU145 cells by FISH. DAPI (4’, 6-diamidino-2-phenylindole) stained metaphases and interphase nuclei are shown having green signal of DYZ1. Nuclei and metaphases lacking DYZ1 are indicated by red arrow. Note the variation in the DYZ1 signal intensities across nuclei reflecting copy number variation.
In this study, we detected 742 copies of DYZ1 in DU145 and that of 1945 in LNCaP cells. Significant difference in the copy number of DYZ1 between DU145 and LNCaP could be due to the unequal number of Y chromosome(s) per cell. A normal array of 3564 bp harbours approximately 80 different restriction enzyme recognition sites. We detected more loss of restriction sites in both the cell lines than that of their gains. Startlingly, a single Eco57I site was gained in both the cell types though its biological significance remained unclear. These results clearly suggest that DYZ1 is indeed affected either during the process of cells becoming cancerous or as a result of prostate cancer. Y chromosome was found to survive only in about 58% of DU145 cells (Figure 6). This suggests that individual cells are unable to maintain and sustain in the cancerous environment. We construe that sustenance of DU145 cells depend largely if not exclusively on the critical number of the DYZ1 copies with near normal sequences.
DYZ1 arrays were never ever thought to be an important milestone in any cancer cell lines. Since several cancers show gender preponderance, it is relevant to assay the intactness of the Y chromosome. Based on our work, we construe that DYZ1 analysis may be fine tuned to eventually use it as a marker along with other available ones.
220 Future plans
Clearly, the DYZ1 is maximally affected in both the cell lines. Since, DYZ1 region is maximally affected both in terms of sequence and array’s copy number; this may be exploited as possible bio-marker for DNA based diagnosis of PC together with other marker systems. Work on additional cell lines and biopsied samples would augment our understanding about the susceptibility of this region in diseased human genome.
Action taken on the RAP/SAC 2013 recommendations No recommendation was received.
Publications Original peer-reviewed articles
1. Yadav SK, Kumari A, Ali S* (2013) Fate of the human Y chromosome linked genes and loci in prostate cancer cell lines DU145 and LNCaP. BMC Genomics 14: 323-330.
2. Kumar SK, Kumari A, Javed S, Ali S* (2014) DYZ1 arrays show sequence variation between the monozygotic males. BMC Genetics 15: 19-31.
Reviews/Proceedings/Etc.
1. Rawal L, Sehgal N, Ali S* (2013) Genome Analysis and Human Health: A Critical Appraisal. Global Journal of Human Genetics and Gene Therapy 1: 16-37.
2. Kamle S, Ali S* (2013) Mutations in H-ras gene and Costello syndrome. The Journal of Genetics- Photon 114: 124-129.
3. Kamle S, Ali S* (2013) Reverberations on Biosafety Issues pertaining to Genetically Modified Crops. Biosafety 2: 112- 114.
4. Kamle S, Ali S* (2013) Genetically Modified Crops: Detection Strategies and Biosafety Issues. Gene 522: 123-132.
*Corresponding author
221 Deciphering the role of cell signalling in M. tuberculosis biology and in the function and dynamics of nucleoporins
Principal Investigator Vinay K. Nandicoori
Research Associates Gagan Jhingan Kalpana Rajanala Sangeeta Kumari
Ph.D. Students Sandeep Upadhyay Yogesh Chawla Divya Arora Prabhjot Kaur Preeti Jain Basanti Malakar
Research Fellows Sathya Narayanan Nagarajan Vijay Soni
Collaborators D. Sriram, BITS, Hyderabad Balaji Prakash, IIT, Kanpur Yogendra Singh, IGIB, Delhi Sanjeev Khosla, CDFD, Hyderabad Debasis Mohanty, NII, New Delhi
Theme of research
A. Elucidating serine/threonine kinase ( STPK) mediated signalling pathways in M. tuberculosis and their role in the survival of pathogen in the host: Analysis of the Mycobacterium tuberculosis genome sequence suggested the presence of 11 putative eukaryotic-like STPKs and 3 protein phosphatases. The M. tuberculosis STPKs affect key mycobacterial processes. Our aim is to decipher the signalling pathways in M. tuberculosis and investigate their role in modulating the host signalling network and the survival of pathogen in the host.
B. Deciphering the role of signalling in modulating the function of nucleoporins: Nucleocytoplasmic transport between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPC). Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Our aim is to decipher the role of phosphorylation on the functions of nucleoporin Tpr.
222 Objectives
1. Elucidating serine/threonine kinase mediated signalling pathways in M. tuberculosis and their role in the survival of pathogen in the host a) To generate gene replacement mutants and investigate the kinase mediated signalling pathways. b) Investigate modulation of host signalling pathways upon infection with M. tuberculosis H37Rv wild type or kinase gene replacement mutants.
2. Deciphering the role of signalling in modulating the function of nucleoporins a) Identification and validation of in vivo phosphorylation sites on Tpr b) Determining the functional significance of identified phosphorylation sites
Work reported in 2012-2013
A. Elucidating serine/threonine kinase mediated signalling pathways in M. tuberculosis and their role in the survival of pathogen in the host
We work on four mycobacterial STPKs. In 2013 report, we described our efforts in generating conditional gene replacement mutants of two essential STPKs pknA and pknB. Growth analysis of these conditional mutants in the presence and absence of inducer unequivocally demonstrated that both PknA and PknB are individually essential for the survival of mycobacteria.
N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), is a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU catalyzes two key reactions; acetyl transfer and uridyl transfer reactions, at two independent domains. Previously, we identified GlmU to be a substrate for PknB and determined the biochemical significance. In 2013 report, we described our collaborative efforts with Dr. Balaji Prakash in solving the GlmUMtb in complex with substrates/products bound at the acetyltransferase active site. We showed that a short helix at the C-terminal extension is uniquely found in GlmUMtb, which provides the highly conserved W460 for substrate binding. We also described our efforts to generate conditional gene replacement mutant of glmU in mycobacteria.
B. Deciphering the role of signalling in modulating the function of nucleoporins
Previously, we demonstrated that Tpr is phosphorylated in vivo in MAP kinase ERK2 dependent and independent fashion. We reported identification of ERK2 independent phosphorylation sites on Tpr. We also reported raising and extensively characterizing phospho-specific antibodies that can recognize phosphorylation on these residues.
223 Progress of work during the current reporting year (2013-2014)
1. Elucidating serine/threonine kinase mediated signalling pathways in M. tuberculosis and their role in the survival of pathogen in the host
Protein phosphorylation/dephosphorylation is a critical regulatory mechanism by which extracellular cues are transduced into cellular responses. Traditionally, two-component systems comprising a histidine kinase sensor and the associated response regulator were thought to be responsible for phosphorylation-mediated signalling in prokaryotes. Two- component pathways are rare in eukaryotes, and phosphotransfer-based signalling predominantly involves phosphorylation/ dephosphorylation on serine, threonine or tyrosine residues, often in a cascade. The presence of a eukaryotic-like serine/threonine protein kinase (STPK) in a prokaryote was first reported in 1991 (pkn1 in Myxococcus xanthus). These ‘eukaryotic-like’ serine/threonine protein kinases play important roles in bacterial cellular processes including cell division, cell wall synthesis, cell metabolism, and dormancy exit. Analysis of the M. tuberculosis genome sequence suggested the presence of 11 putative eukaryotic-like STPKs and 3 protein phosphatases. Except PknG and PknK, all of these kinases were predicted to have a transmembrane domain. All the kinases possessed the protein kinase ‘signature’ motifs including eleven conserved sub-domains as per Hanks criteria, and amino acid sequence alignment of these STPK family members revealed that 15 catalytically important residues were conserved across all of them. The M. tuberculosis STPKs affect key mycobacterial processes - signal transduction mediated by PknA and PknB plays an important role in determining cell shape, morphology and possibly cell division; PknG and PknH influence M. tuberculosis virulence, adaptation and growth within the host; PknF affects cell division, growth rate, morphology and glucose transport.
Protein Kinase B (PknB) of M. tuberculosis is essential for both in vitro growth and in vivo survival of pathogen in the host. Protein kinases A and B, encoded by pknA and pknB respectively, are part of the operon carrying cistrons coding for protein phosphatase pstP, rodA (involved in cell shape control) and pbpA (involved in peptidoglycan synthesis). This locus also includes two forkhead- associated (FHA) domain containing genes, and is found near the origin of replication throughout the Mycobacterium genus. As in most other kinases, PknA and PknB consist of a kinase domain, a juxtamembrane region, a short transmembrane domain, and an extracellular region. While the kinase domain of PknB by itself has been shown to be sufficient for its activity, PknA requires the kinase domain and the adjoining juxtamembrane region for catalytic activity. Both kinases have been found to be essential based on transposon-insertion experiments, and the pknB gene can be disrupted by allelic replacement in M. tuberculosis and M. smegmatis only in the presence of a second functional copy of the gene. Forti et al., in 2009, have generated M. tuberculosis H37Rv-pptr-pknB conditional mutant in which the genomic copy of pknB was converted to pristinamycin-inducible copy, and demonstrated its essentiality, re-affirming the previous findings.
Though a number of substrates of PknB have been identified, structure-function relationships of the various domains of the protein have not been investigated in vivo. We have undertaken a study to extensively analyz the importance of different domains of PknB in modulating its
224 function in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. While the kinase activity of PknB is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants. Results suggest that the intracellular kinase domain has unique functions in the virulent versus the avirulent strain, and cannot be substituted by other kinases. Interestingly, we find that although the presence of the carboxy-terminal PASTA domain is dispensable in M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The notably distinct requirements of the number of PASTA domains, and non-redundant functions of kinase domain in PknB-mediated signalling in M. tuberculosis versus M. smegmatis, reflects differential modulation of signalling events in virulent and avirulent mycobacteria. Finally, macrophage and mouse infection studies reveal that depletion of PknB results in clearance of pathogen from the host, demonstrating its essentiality ex vivo and in vivo.
2. Deciphering the role of signalling in modulating the function of nucleoporins
Nucleoporins are vital components of the nuclear envelope that mediate several critical cellular processes such as transport of macromolecules, progression of cell cycle, gene expression, and chromatin organization. Over the past decade phosphorylation of nucleoporins has been shown to serve as a node to transduce cellular signals, to regulate critical functions such as nucleocytoplasmic transport and cell cycle progression. Many cellular kinases namely CDK1, MAP Kinase ERK2, Protein Kinases A and C have been reported to phosphorylate nucleoporins. Phosphorylation of FG repeat-containing nucleoporins Nup153 and Nup214 has been shown to disrupt the interaction of these nucleoporins with importin-β, thus inhibiting nuclear protein import. In addition, nucleoporins have been shown to be extensively phosphorylated during mitosis both in vitro and in vivo
Nucleoporin Tpr, which is associated with the nuclear basket region, was initially believed to be functioning as a scaffolding element, regulating intranuclear and nucleocytoplasmic transport at the nuclear phase of the NPC. Studies have implicated a role for Tpr in regulating important processes including chromosome segregation, chromatin organization and unspliced RNA export. Tpr associates with Mad1, Mad2 and the members of the dynein complex during mitosis, and these interactions have been found to be crucial for mediating the proper segregation of chromosomes during the anaphase We undertook the study with the aim of investigating the phosphorylation status of Tpr protein, and the significance of specific Tpr phosphorylation events, during cell cycle progression.
We have previously established that Tpr is phosphorylated in both, MAP kinase dependent and independent manner, and found that Tpr acts as both a substrate and as a scaffold for MAP kinase ERK2. We have identified S2059 and S2094 as the major novel ERK- independent phosphorylation sites and T1677, S2020, S2023 and S2034 as the minor ERK independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue, and the site is hyperphosphorylated during mitosis. Further, we find that Tpr phosphorylated at the S2059 residue distinctly localizes with chromatin during the telophase. Abrogation of S2059 phosphorylation
225 abolishes its interaction with Mad1, thus compromising nuclear distribution of Mad2, and results in cell cycle defects. The necessity of S2059 phosphorylation for Tpr-Mad1 interaction and Mad2 nuclear localization suggests that S2059 phosphorylation may modulate Tpr’s scaffolding function. Though S2094 phosphorylation does not seem to have any effect on Tpr-Mad1 interaction or Mad2 subcellular localization, the inability of the mutant to rescue the chromosome lagging phenotype suggests a critical role for S2094 phosphorylation. An indepth study of the mechanistic details of Tpr phosphorylation would assist us in investigating how crucial functions such as spindle assembly and chromatin organization are modulated by Tpr, throwing more light on how various cellular processes are regulated by the nucleopore complex during the different stages of the cell cycle.
Future plans
1. Generation of conditional replacement mutant of PknA and deciphering its role in mycobacterial biology. 2. Determining the role of different domains of M. tuberculosis PknG in modulating its function in the host upon infection. 3. Systems Biology whole proteomics approach to identify differences between different strains of M. tuberculosis.
Action taken on 2013 SAC recommendations
Director, NII forwarded one comment on my work from a SAC member. The work was appreciated and the SAC member expected more progress for the future. We appreciate the comments received and efforts have been made towards better progress.
Publication Original research article
1. Singhal A, Arora G, Sajid A, Maji A, Bhat A, Virmani R, Upadhyay S, Nandicoori VK, Sengupta S, Singh Y* (2013) Regulation of homocysteine metabolism by Mycobacterium tuberculosis S-adenosylhomocysteine hydrolase. Sci Rep 3: 2264.
*Corresponding author
226 Production of transgenic and other animal models for biomedical research
Principal Investigator Subeer S. Majumdar
Research Associate Mohd. Abul Rehan Usmani
Ph.D. Student Mansi Shukla
Research fellow Nirmalya Ganguli Nilanjana Ghosh
Collaborators: Shyamal Goswami, JNU, New Delhi Satyajit Rath, NII Prafulla K. Tailor, NII Suresh B Gokhale, BAIF, Pune Rakesh Tyagi, JNU, New Delhi Sagar Sengupta, NII P. Nagrajan, NII Ana Mendez, IDIBELL, Spain Debi P. Sarkar, University of Delhi
Theme of research
Theme of the research is to generate transgenic animals for using them as a system for the study of functional genomics and mammalian development and other animal models for use in Biomedical research.
Objective
To develop new easier techniques for making transgenic animals. To develop transgenic animal models using genes relevant to human health and diseases as well as to use this technology for making large animals expressing therapeutic products in their milk for increasing affordability of such therapeutics. The other objective is to extend collaborative help in using specific animal models (transgenic or non transgenic) for Biomedical research.
Work reported in 2012-2013
Aif mediated circumvention of T cell defects: Transgenic mice over-expressing Aif generated by us for Dr. Satyajit Rath helped his lab in establishing that Aif rescues T cell developmental defects in harlequin (Hq) strain of mice where naive T cell numbers are low in circulation.
227 Studies with SG2NA: We generated mice overexpressing SG2NA (a cell cycle regulated WD-40 repeat protein with potential scaffolding function) under CMV promoter, however there was a high mortality rate in them. Hence, new construct was being designed in laboratory of Dr. S.Goswami.
SMAR1 mediated susceptibility to infection by M. tuberculosis: Studies with Mycobacterium tuberculosis infected SMAR1 transgenic mice by Dr. S. Chattopadhyay revealed that over expression of SMAR1 decreases resistance towards the pathogen. This might have occurred due to reduced production of IFNg compared to wild type mice in response to infection.
Transgenic mice to study dendritic cell development: This study was initiated from the laboratory of Prafulla Tailor to generate transgenic animals for intervention of different signal transduction pathways involved in dendritic cell subtype development. This would allow to asses role of these molecules in dendritic cell subtype development and function.
Functional genetics to decipher the molecular basis of vision loss in inherited retinal dystrophies through transgenic mice (Indo-Spain): Retinal dystrophies (retinitis pigmentosa and macular degeneration) are a group of genetically heterogeneous disorders that involve the degeneration of photoreceptors, resulting in partial or complete blindness, that affect 1 in 4000 individuals in Western countries and have a considerably higher prevalence in India (up to 1 in 372 individuals reported for rural areas of South India). Gene construct MOP- bovineGCAP2-Mp1, having the mouse opsin promoter was used for making models to study this disease.
Transgenic mice expressing xenoreceptors: Transgenic mice were being generated for Dr. Rakesh Tyagi using mouse PXR gene (with flag tag) which acts as a xenoreceptor and has a major role in drug metabolism.
Transgenic mice over expressing BLM helicase: BLM is a multi-functional protein, acting not only during the resolution of the DNA damage and in fact working as an upstream sensor of the DNA damage signal. Dr. Sagar Sengupta’s laboratory hypothesized that the function of BLM during its role as a sensor to DNA damage could be regulated by post-translational modifications, especially phosphorylation. To address this and other issues, constructs were being designed to generate transgenic animals.
Establishment of a new technique for generation of transgenic mice without any major invasive procedures or surgery: Overcoming caveats of testicular transgenesis, we have established an easier, ethically superior, non-surgical, incision-free alternative procedure for generating transgenic animals at faster pace by exploiting the male germ cell of the testis. The delivery of the transgene was mediated through the hypotonic solution into the testis.
Attempts to generate transgenic buffalo expressing therapeutic protein in the milk: For generation of transgene construct which can express various human proteins of therapeutic value, genes were amplified and coding regions were subcloned in pBucsn2-IRES2-EGFP
228 construct (EGFP under Beta casein promoter) to generate pBucsn2-gene of interest -IRES2- EGFP construct.
Progress of work during current reporting year 2013-2014
Generation of various transgenic mice for other investigators
All collaborative work for making various transgenic animals for other investigators were undertaken as and when the constructs were given and fore founder animals were given to Principal Investigator’s for generating transgenic lines to address their respective scientific goals. Since a lot of them were just initiated last year, no major confirmatory results are generated yet. Work is being continued.
Attempts to generate transgenic buffalo expressing therapeutic protein in the milk
Isolation of buffalo β-casein promoter region and transcriptional regulatory element of this important udder gland specific milk protein gene of Indian river buffalo by us provided an elegant opportunity for guiding the expression of recombinant therapeutic proteins in milk. We have isolated the β-casein genomic region (BuCSN2) which contains promoter region, exon1, intron1 and exon2 (NCBI Accession No. KF612339) from the genome of Indian river buffalo (Bubalus bubalis). We had reported the generation of transgenic mice in which milk glands expressed EGFP when EGFP cloned under this promoter (BuCSN2-EGFP) was used to make the transgene.
There are difficulties and high cost of making transgenic large animals. In present day scenario, transgenic animals may also be blamed to carry potential risk of germ line transmission of introduced transgene to normal traits if not kept isolated, making them as possible biohazard. Keeping all these in view, it is worth trying to transfect Mammary Luminal Epithelial Cells, the cells which express and secrete milk proteins in the time of lactation, in-vivo to have a possible bioreactor by somatic genomic modification. In spite of several attempts using various gene delivery methods people have failed to generate a efficient method for in-vivo gene delivery in mammary gland. It was shown before that virosome mediated targeted delivery of transgene in liver cells is possible in vivo. Taking clue from this, we have used reconstituted Sendai Viral Envelope to generate a gene delivery vehicle for easy, efficient and cost effective delivery of transgene in mammary gland in-vivo. Sendai virus is a negative stranded RNA virus of the family Paramyxoviridae. Sendai viral envelope devoid of its nucleic acid which is known to contain F and HN glycoproteins, was used to entrap gene of Enhanced Green Fluorescent Protein under control of Buffalo Beta- casein Promoter (BuCSN2) which was isolated and functionally characterized by us, for generating Sendai Virosome (HNF-V) for transfection. DNA entrapped Sendai Virosome was prepared as per standard procedure. In brief, membrane of Sendai Virus is solubilized using detergent followed by removal of viral nucleic acid by centrifugation. Desired transgene constructs are mixed into supernatant which contain only viral lipid bilayer along with HN and F glycoprotein. Removal of applied detergent by SM20 Bio Beads start the reconstruction of the viral membrane making gene entrapped HFNV virosome. Dilutions of
229 HNF-V was done in sterile PBS for mammary gland intra ductal delivery. Perfusion of such virosomes through the teat canal in various female mice at different time of mammary development helped us target mammary luminal epithelial cells preferentially. Lactating breast gland of such HNF-V treated female mice were analyzed for detection of EGFP expression. EGFP expression was discerned in the breast gland of some of the treated females but best results were obtained when HNF-V was delivered in mammary glands during advanced stage of pregnancy (Figure 1, right panel). This work can be extrapolated to buffalo or cow which can potentially serve as a rich source of therapeutic proteins when EGFP is replaced with other gene(s) coding therapeutic Proteins. This may avoid the risk of uncontrolled dissemination of gametes. In-vitro, BuCSN2-EGFP transfected buffalo mammary epithelial cells expressed EGFP (Figure 1, left panel)).
Figure1: BuCSN2-EGFP transfected buffalo mammary epithelial cells expressing EGFP (left panel). Breast gland of virosome entrapped BuCSN2-EGFP treated female mice during lactation (right panel). Note: Upper panels represent light microscopic images and lower panels represent same area under UV light.
Generation of transgenic rat through testicular route
Transgenic mice serve as surrogate models of animal and human diseases for evaluating occurrence of the disease and treatment modalities using sufficient number of individuals which may not necessarily be possible in case large/ farmed animals are to be considered or for that matter human beings, who cannot be experimented. Several disorders can be
230 efficiently and meaningfully studied only in rat models which are 10-12 times bigger in size than mice. Mouse cannot be bled frequently for determining humoral changes and it cannot withstand major surgeries under anesthesia unlike, rat. Structure – function relationship of various organs for toxicological evaluation is vastly studied in rats and parameters to evaluate teratogenic effects are better established in rats, mainly due to rat’s extensive use in drug testing in the past. It is important to note that developmental process of fetus is fast ,similar to mice, in rats and 10-12 offspring are born within 21 days of mating in rats making them suitable for teratological studies also. Since rat is considered as more closer to human than mice in terms of genetics, physiology and pathology, rat models bear more significance than mice.
Hence, establishing an easy method of rat transgenesis will open a great scope to study several disease conditions more easily addressing issues of animals and man both. The genetic manipulation in rat model remained limited due to lack of tools for the efficient transgenesis in this system mainly because of inefficient embryo manipulation unlike mice. Therefore, there is a persistent need to develop methods to produce transgenic rat which should be user friendly, less time consuming and relatively inexpensive. In recent past, we reported transgenesis in mice by exploiting the ability of undifferentiated spermatogonia to integrate foreign gene. We are in the process of establishing methodologies to generate the transgenic rat by exploiting the spermatogonial germ cell in the testis for electroporation with transgene. As a proof of principle, we tried to generate α-thalassemia disease model by over expressing the beta globin chain in a physiologically superior transgenic rat system. We found a decrease in hemoglobin concentration, mean cell volume (MCV) and mean cell hemoglobin (MCH) in blood and also detected the HbH inclusion body by Briliant cresyl blue staining of blood of the alpha thalassemic rat model mimicking the silent carrier state of human α-thalassemia. Deformities of RBCs were also noted. Following this new procedure, we wish to interrupt expression of specific genes in vivo by shRNA and evaluate physiological effects of such RNA interference by knock-down in transgenic rats.
Generating a better model for post chemotherapeutic germ cell transplantation
A model for post chemotherapeutic infertility alleviation is made routinely by intraperitonial injection of busulfan. Busulfan doses, less than 40 mg/kg, given to adult mice do not result in prolonged depletion of endogenous spermatogenesis in most tubules; on the other hand, higher doses are often known to induce severe hematopoietic suppression requiring bone marrow transplantation or causing death, as shown before. Data on the effects of busulfan on spermatogonia in other species are limited and the doses used were close to lethal. To overcome such disadvantages of this model, we attempted to standardize busulfan injection directly to testis, locally, in order to deplete the germ cells without compromising animal’s health or animal’s life. F1Bl6SJL mice were used for standardization of busulfan treatment directly in the testis. For this purpose, various doses ranging from 25µg to 100 µg/testis was tested to determine the most effective dose for germ cell depletion of testis. The standardized dose resulted in reduction of the testis size and killing of most of the germ cells of testis within 12-15 days post injection. The animals were observed to be healthy with no signs of health deterioration even after several months. This procedure helped in preparing animal model with less pain and suffering to animal while objective of germ cell depletion was
231 achieved. This also generated such germ cell depleted testis in shorter duration of time (12 days vs 70 days).
Future plans
1. We will continue to serve the scientific community for generation of transgenic mice and other animal models for biomedical research. 2. We will work towards generating farm animals with an objective to produce important rare therapeutic proteins in their milk. 3. Techniques to develop cell specific shRNA expressing transgenic rat will be fine tuned further.
Action taken on the RAP/SAC 2013 recommendations
No specific recommendation was given
List of publications Original peer-reviewed articles
1. Usmani A, Ganguli N, Sarkar H, Dhup S, Batta SR, Vimal M, Ganguli N, Basu S, Nagarajan P, Majumdar SS* (2013) A non-surgical approach for male germ cell mediated gene transmission through transgenesis. Scientific Rep 3: 3430. 2. Shailendra A, Jashdeep B, Jerald MK, Barun D, Upadhyay P, Asif S, Ramesh CJ, Subeer SM, Nagarajan P* (2013) Antigen Peptide Transporter 1 (TAP1-/-) is involved in the development of fructose-induced hepatic steatosis in mice. J Gastroenterol Hepatol 28: 1403-1409.
3. Bhattacharjee J, Jerald MK, Arindkar S, Das B, Upadhya P, Juyal RC, Majumdar SS, Nagarajan P* (2014) Role of immunodeficient animal models in the development of fructose induced NAFLD. J Nut Biochem 25: 219–226.
Patent
1. Subeer S. Majumdar, Abul usmani, Nirmalya ganguli: A shortcut procedure of transgene integration by hypotonic shock into male germinal cells for gene expression and transgenesis. US Patent Application # 14/096,634 (filed on December 4, 2013).
*Corresponding author
232