DOCSLIB.ORG

Functional Reconstruction of a Eukaryotic-Like E1/E2/(RING) E3 Ubiquitylation Cascade from an Uncultured Archaeon

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

ARTICLE DOI: 10.1038/s41467-017-01162-7 OPEN Functional reconstruction of a eukaryotic-like E1/E2/(RING) E3 ubiquitylation cascade from an uncultured archaeon Rory Hennell James1, Eva F. Caceres2, Alex Escasinas3, Haya Alhasan3, Julie A. Howard4, Michael J. Deery4, Thijs J.G. Ettema 2 & Nicholas P. Robinson 3 The covalent modification of protein substrates by ubiquitin regulates a diverse range of critical biological functions. Although it has been established that ubiquitin-like modifiers evolved from prokaryotic sulphur transfer proteins it is less clear how complex eukaryotic ubiquitylation system arose and diversified from these prokaryotic antecedents. The dis- covery of ubiquitin, E1-like, E2-like and small-RING finger (srfp) protein components in the Aigarchaeota and the Asgard archaea superphyla has provided a substantive step toward addressing this evolutionary question. Encoded in operons, these components are likely representative of the progenitor apparatus that founded the modern eukaryotic ubiquitin modification systems. Here we report that these proteins from the archaeon Candidatus ‘Caldiarchaeum subterraneum’ operate together as a bona fide ubiquitin modification system, mediating a sequential ubiquitylation cascade reminiscent of the eukaryotic process. Our observations support the hypothesis that complex eukaryotic ubiquitylation signalling path- ways have developed from compact systems originally inherited from an archaeal ancestor. 1 Department of Biochemistry, The University of Cambridge, Cambridge, CB2 1GA, UK. 2 Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, 751 24, Sweden. 3 Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, LA1 4YG, UK. 4 Department of Biochemistry and Cambridge Systems Biology Centre, Cambridge Centre for Proteomics, The University of Cambridge, Cambridge, CB2 1GA, UK. Correspondence and requests for materials should be addressed to N.P.R. (email: [email protected]) NATURE COMMUNICATIONS | 8: 1120 | DOI: 10.1038/s41467-017-01162-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01162-7 he complex ubiquitylation systems of eukaryotes orches- complex descendants has garnered considerable interest of late. Ttrate diverse regulatory cell signalling pathways that play Indeed, it has been hypothesised that the acquisition of a primitive instrumental roles in maintaining cellular viability. It is well prokaryotic ubiquitylation system may have been a prerequisite to documented that the attachment of the ubiquitin small modifier is permit the endosymbiotic event central to eukaryogenesis9. Fur- central to proteasomal degradation pathways, transcriptional thermore, the diversification of this protein modification system control, DNA repair, cell cycle regulation and a plethora of other has been shown to be concomitant with the increasing cellular – – regulatory pathways1 8. How these ubiquitylation systems were complexity during eukaryotic evolution10 14. acquired by the earliest eukaryotes and then subsequently devel- The canonical eukaryotic ubiquitylation process involves three oped into the sophisticated apparatus employed by the most key biochemical steps, operating in a sequential cascade, which a Aigarchaeota archaeon SCGC AAA471 F17 ASPD01000002.1_68 Aigarchaeota archaeon SCGC AAA471 G05 ASLV01000001.1_69 Aigarchaeota archaeon SCGC AAA471 J08 ASLX01000006.1_25 Thaumarchaeota archaeon JGI OTU 1 AWOE01000006.1_7 Aigarchaeota archaeon SCGC AAA471 D15 AQTB01000052.1_49 Thaumarchaeota archaeon JGI OTU 2 AWOD01000002.1_49 Aigarchaeota archaeon SCGC AAA471 E14 AQTA01000028.1_2 Thaumarchaeota archaeon JGI OTU 3 AWOC01000031.1_2 Cluster I Aigarchaeota archaeon JGI 0000001 A7 ASLY01000016.1_7 Caldiarchaeum subterraneum BAJ51327.1 Candidatus Heimdallarchaeota archaeon AB_125 OLS31900.1 Candidatus Heimdallarchaeota archaeon LC_2 OLS25722.1 Candidatus Heimdallarchaeota archaeon LC_3 OLS27773.1 Candidatus Heimdallarchaeota archaeon LC_3 OLS27494.1 Candidatus Odinarchaeota archaeon LCB_4 OLS18183.1 Candidatus Heimdallarchaeota archaeon AB_125 OLS32357.1 Candidatus Heimdallarchaeota archaeon LC_2 OLS28180.1 Candidatus Heimdallarchaeota archaeon LC_3 OLS27108.1 Cluster II Candidatus Lokiarchaeota archaeon CR_4 OLS16232.1 Lokiarchaeum sp. GC14 _75 KKK43157.1 1 kb Ubiquitin-domain protein - IPR029071, IPR000626 Putative E1-like - IPR000594 Putative E2-like ubiquiting conjugating enzyme - IPR000608 Putative E3-like - IPR013083 Putative deubiquitinating enzyme - IPR000555 Ubiquitin domain - CD Search Predicted ubiquitin / Ubl domain - Phyre2 Search Other CSUB_C1473 ubq E2l E1l srfp rpn11 CSUB_ CSUB_ CSUB_C1476 CSUB_ C1474 C1475 C1477 Fig. 1 Operonic arrangement of the putative archaeal ubiquitylation apparatus. a Gene clusters of the putative ubiquitin-like protein modifier system identified in archaeal species: the ubiquitin, E1-like, E2-like and small RING finger protein (srfp) components are coloured as indicated in the key. The Cluster I and II division is described in Supplementary Fig. 1, which provides a more comprehensive analysis showing an additional Cluster III. See Methods and Supplementary Table 2 for more details on the E1-like C-terminal ubiquitin-like (UBL) domain identification. (boxed inset): Operonic arrangement of the genes encoding the components of the C. subterraneum ubiquitylation cascade investigated in this study. The Rpn11/JAMM domain metalloprotease homologue is juxtaposed to the operon and transcribed right to left, whereas the ubiquitylation operon is transcribed left to right 2 NATURE COMMUNICATIONS | 8: 1120 | DOI: 10.1038/s41467-017-01162-7 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01162-7 ARTICLE ab c –++ – + – – + ATP – + – + ATP Coomassie ––++ ––++ E2 +++E2+ – + Rpn11 ++++++++ E1 +++E1+ ––––++++ Ubq-CLV + + – – Ubq-GG + + Ubq-FL ++++–––– Ubq-FL ––++Ubq-FL 55 kDa E1~Ubq E1~Ubq E1 E1 35 kDa E2~Ubq E2~Ubq 25 kDa Rpn11 20 kDa E2 E2 * Ubq-FL Ubq-FL Ubq-FL Ubq-CLV Ubq-CLV Ubq-GG 12 12345678 1234 Coomassie Coomassie d 2+ y 2+ y16 9 1113.66 y b8 b b 664.63 13 10 11 b b 100 100 2 7 90 90 y16 y15 y14 y10 y8 y7 y6 y5 y4 y y y 80 80 10 9 8 2+ b10 y 2+ 70 10 738.99 70 587.94 60 60 50 50 40 40 2+ y b11 10 30 789.79 30 b 2+ b 8 2+ 8 y y13 Relative abundance 20 6 2+ y 20 2+ y 14 Relative abundance y 5 y 451.28 10 7 y8 y 2+ b 509.24 15 2 b 2+ 2+ 708.18 10 y 1254.59 10 201.14 7 y8 4 385.60 b 0 1434.91 1580.16 1803.72 1960.76 0 7 901.57 971.75 1182.79 1243.77 400 600 800 1000 1200 1400 1600 1800 2000 200 400 600 800 1000 1200 1400 m/z m/z e b11 100 970.82 y14 y13 y12 y11 y10 y9 y8 90 80 70 979.72 60 2+ b11 50 647.59 2+ 2+ y9 y11 40 2+ y10 y 2+ y 2+ 30 8 12 609.95 828.19 2+ 980.62 y14 Relative abundance 20 2+ y13 10 570.04 486.17 1211.47 0 330.46 1332.37 1671.47 200400 600 800 1000 1200 1400 1600 1800 2000 m/z Fig. 2 Rpn11/JAMM metalloprotease cleavage of the C. subterraneum ubiquitin precursor to generate a canonical C-terminal di-glycine motif that is subsequently activated by the E1-like enzyme. a C. subterraneum Rpn11 specific cleavage of the C-terminus of the pro-ubiquitin generates a mature ubiquitin species. Lane 1: pro-ubiquitin only; lane 2: pro-ubiquitin plus Rpn11. (Asterisk: C-terminal truncation of Rpn11). b Auto-ubiquitylation of the E1-like and E2-like components by ATP-dependent conjugation of the cleaved ubiquitin species via the exposed ubiquitin di-glycine motif. Lane 1 and 2: uncleaved, full-length, pro-ubiquitin (Ubq-FL) plus E1 in the presence and absence of ATP, respectively; lanes 3 and 4 as in lanes 1 and 2 but with the inclusion of the E2-enzyme; lanes 5–8 as in lanes 1–4 but using Rpn11-cleaved ubiquitin (Ubq-CLV) (assay at 60 °C for 15 min). c Ubiquitin truncated by a stop-codon after the di-glycine motif (Ubq-GG) also auto-ubiquitylates the E1-like and E2-like proteins. Reactions using Ubq-GG in the absence or presence of ATP are displayed in lanes 1 and 2, respectively (cf Ubq-FL shown in lanes 3 and 4). Reactions were performed as described in b. Products in a, b and c separated by SDS-PAGE and visualised by Coomassie staining. The C-terminal Rpn11-specific cleavage site in pro-ubiquitin is displayed below panel a (solid arrow). Trypsin digestion (dashed arrow) results in the generation of the ‘TVGG’ moiety detected as an isopeptide linkage in the tandem mass-spectrometry analyses. d MS/MS spectra of the auto-ubiquitylated E1 peptides following trypsin digestion to generate the ‘TVGG’ isopeptide linked moiety. m/z values of the precursor ions are shown in the top left of each panel. (2+) indicates doubly charged precursor ions. Spectra show the annotated peaks that are due to C-terminal y (coloured red) and N-terminal b (coloured blue) fragment ions. In each case, the m/z values of the precursor ions and the m/z values of the fragment ions are consistent with lysine residues modified with the ‘TVGG’ moiety. The amino-acid sequence of the trypsin-generated peptide, including the ‘TVGG’ modified lysine, is shown within in each example. e MS/MS spectra of the auto-ubiquitylated E2 peptide, as described in d ultimately results in the covalent attachment of the small ubi- activated modifier is then transferred to the catalytic cysteine of quitin modifier to a target lysine on a substrate. The first protein, the E1 enzyme, forming a covalent thioester intermediate4. Upon referred to as the E1 enzyme, activates the modifier by adeny- interaction with a second protein, the E2 conjugating enzyme, lating the C-terminal residue of the di-glycine motif that is a this activated ubiquitin moiety is then shuttled from the catalytic feature of almost all ubiquitin-like modifier proteins15, 16. The centre of the E1 protein to the catalytic cysteine of the E2 protein NATURE COMMUNICATIONS | 8: 1120 | DOI: 10.1038/s41467-017-01162-7 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01162-7 a K100 K225 Zinc-coordinating C-cluster Catalytic C* Zinc-coordinating Catalytic C* C-cluster H.
Recommended publications
  • Identification of BIRC6 As a Novel Intervention Target For

    Identification of BIRC6 As a Novel Intervention Target For

    Lamers et al. BMC Cancer 2012, 12:285 http://www.biomedcentral.com/1471-2407/12/285 RESEARCH ARTICLE Open Access Identification of BIRC6 as a novel intervention target for neuroblastoma therapy Fieke Lamers1, Linda Schild1, Jan Koster1, Frank Speleman2, Ingrid ra3, Ellen M Westerhout1, Peter van Sluis1, Rogier Versteeg1, Huib N Caron4 and Jan J Molenaar1,5* Abstract Background: Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been identified in neuroblastoma. Methods: Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays) and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform (http://r2.amc.nl). BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by Western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation. Results: We observed frequent gain of the BIRC6 gene on chromosome 2, which resulted in increased mRNA expression. BIRC6 is an inhibitor of apoptosis protein (IAP), that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines.
  • Ubiquitin-Conjugating Enzyme UBE2J1 Negatively Modulates

    Ubiquitin-Conjugating Enzyme UBE2J1 Negatively Modulates

    Feng et al. Virology Journal (2018) 15:132 https://doi.org/10.1186/s12985-018-1040-5 RESEARCH Open Access Ubiquitin-conjugating enzyme UBE2J1 negatively modulates interferon pathway and promotes RNA virus infection Tingting Feng1, Lei Deng1, Xiaochuan Lu1, Wen Pan1, Qihan Wu2* and Jianfeng Dai1,2* Abstract Background: Viral infection activates innate immune pathways and interferons (IFNs) play a pivotal role in the outcome of a viral infection. Ubiquitin modifications of host and viral proteins significantly influence the progress of virus infection. Ubiquitin-conjugating enzyme E2s (UBE2) have the capacity to determine ubiquitin chain topology and emerge as key mediators of chain assembly. Methods: In this study, we screened the functions of 34 E2 genes using an RNAi library during Dengue virus (DENV) infection. RNAi and gene overexpression approaches were used to study the gene function in viral infection and interferon signaling. Results: We found that silencing UBE2J1 significantly impaired DENV infection, while overexpression of UBE2J1 enhanced DENV infection. Further studies suggested that type I IFN expression was significantly increased in UBE2J1 silenced cells and decreased in UBE2J1 overexpressed cells. Reporter assay suggested that overexpression of UBE2J1 dramatically suppressed RIG-I directed IFNβ promoter activation. Finally, we have confirmed that UBE2J1 can facilitate the ubiquitination and degradation of transcription factor IFN regulatory factor 3 (IRF3). Conclusion: These results suggest that UBE2 family member UBE2J1 can negatively regulate type I IFN expression, thereby promote RNA virus infection. Keywords: UBE2J1, Dengue virus, Interferons, IRF3, K48 ubiquitination Background antiviral responses [3]. IFN-α/β regulates the synthesis Dengue virus (DENV), transmitted by Aedes aegypti and of antiviral proteins and immunoregulatory factors Aedes albopicuts, causes an emerging tropical disease through the JAK/STAT signaling pathway [4, 5].
  • Versatile Roles of K63-Linked Ubiquitin Chains in Trafficking

    Versatile Roles of K63-Linked Ubiquitin Chains in Trafficking

    Cells 2014, 3, 1027-1088; doi:10.3390/cells3041027 OPEN ACCESS cells ISSN 2073-4409 www.mdpi.com/journal/cells Review Versatile Roles of K63-Linked Ubiquitin Chains in Trafficking Zoi Erpapazoglou 1,2, Olivier Walker 3 and Rosine Haguenauer-Tsapis 1,* 1 Institut Jacques Monod-CNRS, UMR 7592, Université-Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France; E-Mail: [email protected] 2 Current address: Brain and Spine Institute, CNRS UMR 7225, Inserm, U 1127, UPMC-P6 UMR S 1127, 75013 Paris, France 3 Institut des Sciences Analytiques, UMR5280, Université de Lyon/Université Lyon 1, 69100 Villeurbanne, France; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]. External Editor: Hanjo Hellmann Received: 14 July 2014; in revised form: 14 October 2014 / Accepted: 21 October 2014 / Published: 12 November 2014 Abstract: Modification by Lys63-linked ubiquitin (UbK63) chains is the second most abundant form of ubiquitylation. In addition to their role in DNA repair or kinase activation, UbK63 chains interfere with multiple steps of intracellular trafficking. UbK63 chains decorate many plasma membrane proteins, providing a signal that is often, but not always, required for their internalization. In yeast, plants, worms and mammals, this same modification appears to be critical for efficient sorting to multivesicular bodies and subsequent lysosomal degradation. UbK63 chains are also one of the modifications involved in various forms of autophagy (mitophagy, xenophagy, or aggrephagy). Here, in the context of trafficking, we report recent structural studies investigating UbK63 chains assembly by various E2/E3 pairs, disassembly by deubiquitylases, and specifically recognition as sorting signals by receptors carrying Ub-binding domains, often acting in tandem.
  • The HECT Domain Ubiquitin Ligase HUWE1 Targets Unassembled Soluble Proteins for Degradation

    The HECT Domain Ubiquitin Ligase HUWE1 Targets Unassembled Soluble Proteins for Degradation

    OPEN Citation: Cell Discovery (2016) 2, 16040; doi:10.1038/celldisc.2016.40 ARTICLE www.nature.com/celldisc The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble proteins for degradation Yue Xu1, D Eric Anderson2, Yihong Ye1 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA; 2Advanced Mass Spectrometry Core Facility, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA In eukaryotes, many proteins function in multi-subunit complexes that require proper assembly. To maintain complex stoichiometry, cells use the endoplasmic reticulum-associated degradation system to degrade unassembled membrane subunits, but how unassembled soluble proteins are eliminated is undefined. Here we show that degradation of unassembled soluble proteins (referred to as unassembled soluble protein degradation, USPD) requires the ubiquitin selective chaperone p97, its co-factor nuclear protein localization protein 4 (Npl4), and the proteasome. At the ubiquitin ligase level, the previously identified protein quality control ligase UBR1 (ubiquitin protein ligase E3 component n-recognin 1) and the related enzymes only process a subset of unassembled soluble proteins. We identify the homologous to the E6-AP carboxyl terminus (homologous to the E6-AP carboxyl terminus) domain-containing protein HUWE1 as a ubiquitin ligase for substrates bearing unshielded, hydrophobic segments. We used a stable isotope labeling with amino acids-based proteomic approach to identify endogenous HUWE1 substrates. Interestingly, many HUWE1 substrates form multi-protein com- plexes that function in the nucleus although HUWE1 itself is cytoplasmically localized. Inhibition of nuclear entry enhances HUWE1-mediated ubiquitination and degradation, suggesting that USPD occurs primarily in the cytoplasm.
  • Ubiquitination Is Not Omnipresent in Myeloid Leukemia Ramesh C

    Ubiquitination Is Not Omnipresent in Myeloid Leukemia Ramesh C

    Editorials Ubiquitination is not omnipresent in myeloid leukemia Ramesh C. Nayak1 and Jose A. Cancelas1,2 1Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center and 2Hoxworth Blood Center, University of Cincinnati Academic Health Center, Cincinnati, OH, USA E-mail: JOSE A. CANCELAS - [email protected] / [email protected] doi:10.3324/haematol.2019.224162 hronic myelogenous leukemia (CML) is a clonal tination of target proteins through their cognate E3 ubiq- biphasic hematopoietic disorder most frequently uitin ligases belonging to three different families (RING, Ccaused by the expression of the BCR-ABL fusion HERCT, RING-between-RING or RBR type E3).7 protein. The expression of BCR-ABL fusion protein with The ubiquitin conjugating enzymes including UBE2N constitutive and elevated tyrosine kinase activity is suffi- (UBC13) and UBE2C are over-expressed in a myriad of cient to induce transformation of hematopoietic stem tumors such as breast, pancreas, colon, prostate, lym- cells (HSC) and the development of CML.1 Despite the phoma, and ovarian carcinomas.8 Higher expression of introduction of tyrosine kinase inhibitors (TKI), the dis- UBE2A is associated with poor prognosis of hepatocellu- ease may progress from a manageable chronic phase to a lar cancer.9 In leukemia, bone marrow (BM) cells from clinically challenging blast crisis phase with a poor prog- pediatric acute lymphoblastic patients show higher levels nosis,2 in which myeloid or lymphoid blasts fail to differ- of UBE2Q2
  • Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model

    Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model

    Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 T + is online at: average * The Journal of Immunology , 34 of which you can access for free at: 2016; 197:1477-1488; Prepublished online 1 July from submission to initial decision 4 weeks from acceptance to publication 2016; doi: 10.4049/jimmunol.1600589 http://www.jimmunol.org/content/197/4/1477 Molecular Profile of Tumor-Specific CD8 Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A. Waugh, Sonia M. Leach, Brandon L. Moore, Tullia C. Bruno, Jonathan D. Buhrman and Jill E. Slansky J Immunol cites 95 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2016/07/01/jimmunol.160058 9.DCSupplemental This article http://www.jimmunol.org/content/197/4/1477.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A.
  • Figure S1. DMD Module Network. the Network Is Formed by 260 Genes from Disgenet and 1101 Interactions from STRING. Red Nodes Are the Five Seed Candidate Genes

    Figure S1. DMD Module Network. the Network Is Formed by 260 Genes from Disgenet and 1101 Interactions from STRING. Red Nodes Are the Five Seed Candidate Genes

    Figure S1. DMD module network. The network is formed by 260 genes from DisGeNET and 1101 interactions from STRING. Red nodes are the five seed candidate genes. Figure S2. DMD module network is more connected than a random module of the same size. It is shown the distribution of the largest connected component of 10.000 random modules of the same size of the DMD module network. The green line (x=260) represents the DMD largest connected component, obtaining a z-score=8.9. Figure S3. Shared genes between BMD and DMD signature. A) A meta-analysis of three microarray datasets (GSE3307, GSE13608 and GSE109178) was performed for the identification of differentially expressed genes (DEGs) in BMD muscle biopsies as compared to normal muscle biopsies. Briefly, the GSE13608 dataset included 6 samples of skeletal muscle biopsy from healthy people and 5 samples from BMD patients. Biopsies were taken from either biceps brachii, triceps brachii or deltoid. The GSE3307 dataset included 17 samples of skeletal muscle biopsy from healthy people and 10 samples from BMD patients. The GSE109178 dataset included 14 samples of controls and 11 samples from BMD patients. For both GSE3307 and GSE10917 datasets, biopsies were taken at the time of diagnosis and from the vastus lateralis. For the meta-analysis of GSE13608, GSE3307 and GSE109178, a random effects model of effect size measure was used to integrate gene expression patterns from the two datasets. Genes with an adjusted p value (FDR) < 0.05 and an │effect size│>2 were identified as DEGs and selected for further analysis. A significant number of DEGs (p<0.001) were in common with the DMD signature genes (blue nodes), as determined by a hypergeometric test assessing the significance of the overlap between the BMD DEGs and the number of DMD signature genes B) MCODE analysis of the overlapping genes between BMD DEGs and DMD signature genes.
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
  • The E2 Ubiquitin-Conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice

    The E2 Ubiquitin-Conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice

    The E2 Ubiquitin-conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Koenig, Paul-Albert, Peter K. Nicholls, Florian I. Schmidt, Masatoshi Hagiwara, Takeshi Maruyama, Galit H. Frydman, Nicki Watson, David C. Page, and Hidde L. Ploegh. “The E2 Ubiquitin-Conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice.” Journal of Biological Chemistry 289, no. 50 (October 15, 2014): 34490–34502. © 2014 American Society for Biochemistry and Molecular Biology, Inc. (ASBMB) As Published http://dx.doi.org/10.1074/jbc.M114.604132 Publisher American Society for Biochemistry and Molecular Biology (ASBMB) Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/108038 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/ Male Ube2j1-/- mice are sterile The E2 Ubiquitin Conjugating Enzyme UBE2J1 is Required for Spermiogenesis in Mice Paul-Albert Koenig1, Peter K. Nicholls1, Florian I. Schmidt1, Masatoshi Hagiwara1, Takeshi Maruyama1, Galit H. Frydman2, Nicki Watson1, David C. Page1,3,4, Hidde L. Ploegh1,3 1 Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA 2 Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 3 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA 4 Howard Hughes Medical Institute, Cambridge, MA 02142,
  • Exploring the Metastatic Role of the Inhibitor of Apoptosis BIRC6 in Breast Cancer

    Exploring the Metastatic Role of the Inhibitor of Apoptosis BIRC6 in Breast Cancer

    bioRxiv preprint doi: https://doi.org/10.1101/2021.04.08.438518; this version posted April 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Exploring the metastatic role of the inhibitor of apoptosis BIRC6 in Breast 2 Cancer 3 Corresponding author: Matias Luis Pidre, Pringles 3010, Lanús, Buenos Aires, Argentina, CP 1824 4 [email protected], mobile: +54 9 221 364 6836 5 AUTHORS 6 Santiago M. Gómez Bergna1; Abril Marchesini1; Leslie C. Amorós Morales1; Paula N. Arrías1; Hernán 7 G. Farina2; Víctor Romanowski1; M. Florencia Gottardo2*; Matias L. Pidre1*. 8 *Both authors equally contributed to this work. 9 AUTHOR AFFILIATIONS 10 1Instituto de Biotecnología y biología molecular (IBBM-CONICET-UNLP) 11 2Center of Molecular & Translational Oncology, Department of Science and Technology, 12 National University of Quilmes, Buenos Aires, Argentina. 13 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.08.438518; this version posted April 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 14 Abstract 15 Breast cancer is the most common cancer as well as the first cause of death by cancer in 16 women worldwide. BIRC6 (baculoviral IAP repeat-containing protein 6) is a member of the 17 inhibitors of apoptosis protein family thought to play an important role in the progression or 18 chemoresistance of many cancers. The aim of the present work was to investigate the role of 19 apoptosis inhibitor BIRC6 in breast cancer, focusing particularly on its involvement in the 20 metastatic cascade.
  • Uncovering Ubiquitin and Ubiquitin-Like Signaling Networks Alfred C

    Uncovering Ubiquitin and Ubiquitin-Like Signaling Networks Alfred C

    REVIEW pubs.acs.org/CR Uncovering Ubiquitin and Ubiquitin-like Signaling Networks Alfred C. O. Vertegaal* Department of Molecular Cell Biology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands CONTENTS 8. Crosstalk between Post-Translational Modifications 7934 1. Introduction 7923 8.1. Crosstalk between Phosphorylation and 1.1. Ubiquitin and Ubiquitin-like Proteins 7924 Ubiquitylation 7934 1.2. Quantitative Proteomics 7924 8.2. Phosphorylation-Dependent SUMOylation 7935 8.3. Competition between Different Lysine 1.3. Setting the Scenery: Mass Spectrometry Modifications 7935 Based Investigation of Phosphorylation 8.4. Crosstalk between SUMOylation and the and Acetylation 7925 UbiquitinÀProteasome System 7935 2. Ubiquitin and Ubiquitin-like Protein Purification 9. Conclusions and Future Perspectives 7935 Approaches 7925 Author Information 7935 2.1. Epitope-Tagged Ubiquitin and Ubiquitin-like Biography 7935 Proteins 7925 Acknowledgment 7936 2.2. Traps Based on Ubiquitin- and Ubiquitin-like References 7936 Binding Domains 7926 2.3. Antibody-Based Purification of Ubiquitin and Ubiquitin-like Proteins 7926 1. INTRODUCTION 2.4. Challenges and Pitfalls 7926 Proteomes are significantly more complex than genomes 2.5. Summary 7926 and transcriptomes due to protein processing and extensive 3. Ubiquitin Proteomics 7927 post-translational modification (PTM) of proteins. Hundreds ff fi 3.1. Proteomic Studies Employing Tagged of di erent modi cations exist. Release 66 of the RESID database1 (http://www.ebi.ac.uk/RESID/) contains 559 dif- Ubiquitin 7927 ferent modifications, including small chemical modifications 3.2. Ubiquitin Binding Domains 7927 such as phosphorylation, acetylation, and methylation and mod- 3.3. Anti-Ubiquitin Antibodies 7927 ification by small proteins, including ubiquitin and ubiquitin- 3.4.
  • WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT

    WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT

    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2019/079361 Al 25 April 2019 (25.04.2019) W 1P O PCT (51) International Patent Classification: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, C12Q 1/68 (2018.01) A61P 31/18 (2006.01) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, C12Q 1/70 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/US2018/056167 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, 16 October 2018 (16. 10.2018) TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (26) Publication Language: English GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (30) Priority Data: UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 62/573,025 16 October 2017 (16. 10.2017) US TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, ΓΕ , IS, IT, LT, LU, LV, (71) Applicant: MASSACHUSETTS INSTITUTE OF MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TECHNOLOGY [US/US]; 77 Massachusetts Avenue, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Cambridge, Massachusetts 02139 (US).