DOCSLIB.ORG

Regulation of Nrf2 by a Keap1-Dependent E3 Ubiquitin Ligase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

REGULATION OF NRF2 BY A KEAP1-DEPENDENT E3 UBIQUITIN LIGASE A Dissertation presented to The Faculty of the Graduate School at the University of Missouri-Columbia In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by SHIH-CHING (JOYCE) LO Dr. Mark Hannink, Dissertation Adviser DECEMBER 2007 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled REGULATION OF NRF2 BY A KEAP1-DEPENDENT E3 UBIQUITIN LIGASE Presented by Shih-Ching (Joyce) Lo A candidate for the degree of Doctor of Philosophy And hereby certify that in their opinion it is worthy of acceptance. Mark Hannink Thomas Guilfoyle David Pintel Grace Sun Richard Tsika DEDICATION Okay. Mom, I got you the Ph.D. you asked, at the place you picked. Can I now please do something else? Dad, this is to you, too… you accomplice! ACKNOWLEDGEMENTS Dr. Mark Hannink. This work would never have been possible without his guidance, criticism and encouragement. His sheer enthusiasm and critical thinking of science are the most valuable lessons that I could ever obtain during my graduate studies. I should also extend my appreciation to my committee members, Drs. Thomas Guilfoyle, David Pintel, Grace Sun and Richard Tsika, as well as Drs. Lesa Beamer, Joan Conaway, Beverly DaGue, Marc Johnson, Alan Diehl and Michael Henzl, for their insightful advice and thoughtful comments on this work. I thank the present and previous members in Dr. Hannink’s laboratory, including Drs. Donna Zhang, Rick Sachdev, Sang-Hyun Lee, Xuchu Li, as well as Brittany Angle, Carolyn Eberle, Zheng Sun, Jordan Wilkins, Marquis Patrick, Xiaofang Jin, Casey Williams, Joel Pinkston, Julie Unverferth, and Benjamin Creech. They provided me with technical help and inspiring discussions on this work. Toward completion of my graduate study, I have also received copious amounts of help and encouragement from friends and colleagues both on campus and elsewhere. While it is not possible to list all the names due to space constraint, I would like to express my appreciation to them all. ii TABLES OF CONTENTS ACKNOWLEDGEMENTS ..…………………………………………………………..…ii LIST OF TABLE ..………………………………………………………………………vii LIST OF FIGURE ……………………………………………………………………...viii ABSTRACT …..………………………………………………………………………...xii CHAPTER I. INTRODUCTION …..……………………………………………………………..…...1 A. A snapshot of the ubiquitin/proteasome system (UPS) B. General features of cullin-RING ligases (CRLs) B.1. The cullin family B.2. The RING finger proteins in cullin-RING ligases B.3. Substrate adaptors of cullin-RING ligases B.4. Recognition of degradation signals in substrates by substrate adaptors B.5. Architecture of cullin-RING ligases and the ‘positioning’ model B.6. Polygamy among substrates and substrate adaptors, and the ‘processivity’ model B.7. Cyclic assembly and disassembly of cullin-RING ligase complex, and the major mediator CAND1 C. Modulation of cullin-RING ligase activity toward substrates C.1. Modulation of substrate binding events C.2. Dimerization of culiin-RING ligases C.3. Dynamic subcellular localization of cullin-RING ligases: circuit riders in CRL systems. iii C.4. Degradation of substrate adaptors C.5. Nedd8, and regulation of cullin-RING ligases via neddylation C.6. COP9 signalosome (CSN) and its role in regulation of CRLs C.7. Multilateral interplay among regulatory factors in CRLs D. Nrf2 and Keap1 D.1. The transcription factor Nrf2 D.2. The modular protein Keap1, and its sensor system E. Regulation of Nrf2 by Keap1 E.1. Ubiquitination and degradation of Nrf2 by Keap1-dependent Cul3-RING ligases E.2. Structure of the Nrf2-Keap1 complex, and its binding stoichiometry E.3. Dynamic subcellular localization of the Nrf2-Keap1 system F. A preview of the following chapters in this dissertation II. KEAP1 IS A REDOX-REGULATED SUBSTRATE ADAPTOR PROTEIN FOR A CUL3-DEPENDENT UBIQUITIN LIGASE COMPLEX …………………..………….90 Abstract Introduction Materials and methods Results Discussion References III. UBIQUITINATION OF KEAP1, A BTB-KELCH SUBSTRATE ADAPTOR PROTEIN FOR CUL3, TARGETS KEAP1 FOR DEGRADATION BY A PROTEASOME-INDEPENDENT PATHWAY ……………………………………....135 iv Abstract Introduction Materials and methods Results Discussion References IV. CAND1-MEDIATED SUBSTRATE ADAPTOR RECYCLING IS REQUIRED FOR EFFICIENT REPRESSION OF NRF2 BY KEAP1 ………………………...…...174 Abstract Introduction Materials and methods Results Discussion References V. STRUCTURE OF THE KEAP1;NRF2 INTERFACE PROVIDES MECHANISTIC INSIGHT INTO NRF2 SIGNALING …………………………………………..……..213 Abstract Introduction Materials and methods Results Discussion References v VI. PGAM5, A BCL-XL-INTERACTING PROTEIN, IS A NOVEL SUBSTRATE FOR THE REDOX-REGULAETD KEAP1-DEPENDENT UBIQUITIN LIGASE COMPLEX …………………………………………………………………………….258 Abstract Introduction Materials and methods Results Discussion References VII. REGULATION OF ANTI-OXIDANT GENE EXPRESSION BY A MITOCHONDRIAL-LOCALIZED COMPLEX CONTAINING KEAP1, NRF2 AND PGAM5 ………………………………………………………………………………...302 Abstract Introduction Materials and methods Results Discussion References VIII. SUMMARY AND FUTURE DIRECTIONS ……………………………………341 VITA …………………………………………………………………………………...373 vi LIST OF TABLES Table Page II-1. Half-lives of the HA-Nrf2 proteins in MDA-MB-231 cells …..………………..115 V-1. Table V-1 ……………………………………………………………………….235 V-2. Table V-2 …………………………………………………………………..…...237 V-3. Table V-3 ……………………………………………………………………….238 VI-1. Table VI-1………………………………………………………………………283 VII-1. Table VII-1 ...……...……………………………………………………………324 vii LIST OF FIGURES Figure Page I-1. Architecture of cullin-RING ligases …………………………………………….63 I-2. The conundrum of a 50-60 Å gap in cullin-RING ligases ………………………65 I-3. A dimeric CRL supercomplex ……………………………………………..…….67 I-4. A tentative model for the cyclic assembly and disassembly of a Cul1-RING ligase by neddylation and deneddylation of Cul1 ……………………………………...69 I-5. Domain structures and motifs of Nrf2 and Keap1 ……………………………….71 I-6. Three-dimensional structures of the Kelch β-propeller bound with the Nrf2- derived peptide …………………………………………………………….…….73 II-1. Domain structures of Nrf2 and Keap1 …...…………..…………………………116 II-2. Association of Keap1 with Cul3 mediates Keap1-dependent ubiquitination of Nrf2 …………………………………………………………………….……….118 II-3. Keap1 functions as a substrate adaptor for a Cul3-Rbx1 E3 ubiquitin ligase complex ………………………………………………………………….……...120 II-4. Mutations within BTB domain of Keap1 decrease ubiquitination of Nrf2 but increase ubiquitination of Keap1 ……………………………………….…...…..122 II-5. Identification of lysine residues in Nrf2 targeted for ubiquitination by Keap1 …124 II-6. Oxidative stress results in reduced ubiquitination of Nrf2 by a Keap1-dependent Cul3-RING ligase ………………………………………………………..……...126 II-7. Oxidative stress does not cause dissociation of Nrf2 from Keap1 ………….......128 III-1. Autoubiquitination is a key feature for BTB-Kelch proteins that assemble into viii Cul3-RING ligase complexes …………….………………………………….....159 III-2. Autoubiquitination of Keap1 by a Cul3-Rbx1 subcomplex ………………...…..161 III-3. Increased ubiquitination results in proteasome-independent degradation of Keap1 …………………………………………………………………………….163 III-4. Quinone-induced oxidative stress increases ubiquitination and turnover of Keap1 …………………………………………………………………………….165 III-5. Quinone-induced degradation of Keap1 is enhanced in cells deficient in glutathione Biosynthesis ……………………………………………………………………...167 III-6. Quinone-induced oxidative stress induces a switch in ubiquitination from substrate to substrate adaptor ………………………………………………………………169 IV-1. CAND1 competes with Keap1 for binding to Cul3 ……………………………..195 IV-2. Increased CAND1 expression reduces Keap1-dependent ubiquitination and degradation of Nrf2 ………………………………………………………………197 IV-3. CAND1 is required for efficient repression of Nrf2 by Keap1 ………………….199 IV-4. CAND1 is required for Nrf2 turnover, and contributes to repression of ARE- dependent gene expression ………………………………………………………201 IV-5. Nedd8 is required for ubiquitination of Nrf2 by a Keap1-Cul3 ligase complex ...203 IV-6. Neddylation of Cul3 on Lys 712 is required for efficient ubiquitination and degradation of Nrf2 by a Keap1-Cul3 ligase complex …………………………..205 IV-7. Catalytic activity of a Keap1-Cul3 ligase complex per se does not require neddylation on the Lys 712 of Cul3 ……………………………………………...207 V-1. Sequence alignments among orthologues of Keap1 or Nrf2 ……………………239 V-2. Specific bindings between Keap1 and the Nrf2-derived peptides ………………241 ix V-3. Three-dimensional structure of the complex consisting of a Kelch β-propeller domain and an Nrf2-derived peptide ……………………………………………243 V-4. Intermolecular and intramolecular contacts within the Kelch domain and the bound Nrf2 peptide ……………………………………………………………………..245 V-5. Identification of amino acids within the Kelch domain of Keap1 that are required for binding to Nrf2 ………………………………………………………………247 V-6. Keap1 forms a homodimer capable of binding two Nrf2 molecules ……………249 V-7. Phosphorylation of the conserved threonine within the DxETGE motif in Nrf2 disrupts binding to Keap1 ……………………………………………………….251 VI-1. Identification of PGAM5 as an interaction partner of Keap1 …………………..284 VI-2. Featured domains and motifs in PGAM5, a member in phosphoglycerate mutase family …………………………………………………………………………....286 VI-3. Subcellular localization of N-terminal FLAG-tagged PGAM5 isoforms ………289 VI-4. Identification of the interaction interface between Keap1 and PGAM5 ………..291 VI-5. PGAM5-L is targeted for ubiquitination by a Keap1-dependent Cul3-RING ligase …………………………………………………………………………….293
Recommended publications
  • Kelch Proteins: Emerging Roles in Skeletal Muscle Development and Diseases

    Kelch Proteins: Emerging Roles in Skeletal Muscle Development and Diseases

    Kelch proteins: emerging roles in skeletal muscle development and diseases The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Gupta, Vandana A., and Alan H Beggs. 2014. “Kelch proteins: emerging roles in skeletal muscle development and diseases.” Skeletal Muscle 4 (1): 11. doi:10.1186/2044-5040-4-11. http:// dx.doi.org/10.1186/2044-5040-4-11. Published Version doi:10.1186/2044-5040-4-11 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12406733 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Gupta and Beggs Skeletal Muscle 2014, 4:11 http://www.skeletalmusclejournal.com/content/4/1/11 REVIEW Open Access Kelch proteins: emerging roles in skeletal muscle development and diseases Vandana A Gupta and Alan H Beggs* Abstract Our understanding of genes that cause skeletal muscle disease has increased tremendously over the past three decades. Advances in approaches to genetics and genomics have aided in the identification of new pathogenic mechanisms in rare genetic disorders and have opened up new avenues for therapeutic interventions by identification of new molecular pathways in muscle disease. Recent studies have identified mutations of several Kelch proteins in skeletal muscle disorders. The Kelch superfamily is one of the largest evolutionary conserved gene families. The 66 known family members all possess a Kelch-repeat containing domain and are implicated in diverse biological functions.
  • 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.
  • The C-Terminal GGAP Motif of Hsp70 Mediates Substrate Recognition And

    The C-Terminal GGAP Motif of Hsp70 Mediates Substrate Recognition And

    ARTICLE cro The C-terminal GGAP motif of Hsp70 mediates substrate recognition and stress response in yeast Received for publication, March 2, 2018, and in revised form, August 30, 2018 Published, Papers in Press, September 18, 2018, DOI 10.1074/jbc.RA118.002691 Weibin Gong (宫维斌)‡1, Wanhui Hu (胡万辉)‡§1,2, Linan Xu (徐利楠)¶1, Huiwen Wu (吴惠文)‡§3,SiWu(吴思)‡§, Hong Zhang (张红)‡§, Jinfeng Wang (王金凤)‡, Gary W. Jones¶4, and X Sarah Perrett‡§5 From the ‡National Laboratory of Biomacromolecules, Chinese Academy of Sciences Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, the §University of the Chinese Academy of Sciences, Beijing 100049, China, and the ¶Department of Biology, Maynooth University, Maynooth, W23 W6R7, Kildare, Ireland Edited by Ursula Jakob The allosteric coupling of the highly conserved nucleotide- containing GGAP and GGAP-like tetrapeptide repeats, can and substrate-binding domains of Hsp70 has been studied directly bind to Ure2, the Hsp40 co-chaperone Ydj1, and ␣-sy- intensively. In contrast, the role of the disordered, highly vari- nuclein, but not to the SUMO-like protein SMT3 or BSA. Dele- Downloaded from able C-terminal region of Hsp70 remains unclear. In many tion or substitution of the Ssa1 GGAP motif impaired yeast cell eukaryotic Hsp70s, the extreme C-terminal EEVD motif binds tolerance to temperature and cell-wall damage stress. This study to the tetratricopeptide-repeat domains of Hsp70 co-chaper- highlights that the C-terminal GGAP motif of Hsp70 is impor- ones. Here, we discovered that the TVEEVD sequence of Sac- tant for substrate recognition and mediation of the heat shock charomyces cerevisiae cytoplasmic Hsp70 (Ssa1) functions as a response.
  • Transcriptomic and Proteomic Profiling Provides Insight Into

    Transcriptomic and Proteomic Profiling Provides Insight Into

    BASIC RESEARCH www.jasn.org Transcriptomic and Proteomic Profiling Provides Insight into Mesangial Cell Function in IgA Nephropathy † † ‡ Peidi Liu,* Emelie Lassén,* Viji Nair, Celine C. Berthier, Miyuki Suguro, Carina Sihlbom,§ † | † Matthias Kretzler, Christer Betsholtz, ¶ Börje Haraldsson,* Wenjun Ju, Kerstin Ebefors,* and Jenny Nyström* *Department of Physiology, Institute of Neuroscience and Physiology, §Proteomics Core Facility at University of Gothenburg, University of Gothenburg, Gothenburg, Sweden; †Division of Nephrology, Department of Internal Medicine and Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan; ‡Division of Molecular Medicine, Aichi Cancer Center Research Institute, Nagoya, Japan; |Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and ¶Integrated Cardio Metabolic Centre, Karolinska Institutet Novum, Huddinge, Sweden ABSTRACT IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type–specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell–positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell–positive standard genes.
  • DIPPER, a Spatiotemporal Proteomics Atlas of Human Intervertebral Discs

    DIPPER, a Spatiotemporal Proteomics Atlas of Human Intervertebral Discs

    TOOLS AND RESOURCES DIPPER, a spatiotemporal proteomics atlas of human intervertebral discs for exploring ageing and degeneration dynamics Vivian Tam1,2†, Peikai Chen1†‡, Anita Yee1, Nestor Solis3, Theo Klein3§, Mateusz Kudelko1, Rakesh Sharma4, Wilson CW Chan1,2,5, Christopher M Overall3, Lisbet Haglund6, Pak C Sham7, Kathryn Song Eng Cheah1, Danny Chan1,2* 1School of Biomedical Sciences, , The University of Hong Kong, Hong Kong; 2The University of Hong Kong Shenzhen of Research Institute and Innovation (HKU-SIRI), Shenzhen, China; 3Centre for Blood Research, Faculty of Dentistry, University of British Columbia, Vancouver, Canada; 4Proteomics and Metabolomics Core Facility, The University of Hong Kong, Hong Kong; 5Department of Orthopaedics Surgery and Traumatology, HKU-Shenzhen Hospital, Shenzhen, China; 6Department of Surgery, McGill University, Montreal, Canada; 7Centre for PanorOmic Sciences (CPOS), The University of Hong Kong, Hong Kong Abstract The spatiotemporal proteome of the intervertebral disc (IVD) underpins its integrity *For correspondence: and function. We present DIPPER, a deep and comprehensive IVD proteomic resource comprising [email protected] 94 genome-wide profiles from 17 individuals. To begin with, protein modules defining key †These authors contributed directional trends spanning the lateral and anteroposterior axes were derived from high-resolution equally to this work spatial proteomes of intact young cadaveric lumbar IVDs. They revealed novel region-specific Present address: ‡Department profiles of regulatory activities
  • Omics Technologies: Insights Into the Transmission of Insect Vector-Borne Plant Viruses

    Omics Technologies: Insights Into the Transmission of Insect Vector-Borne Plant Viruses

    Looking Through the Lens of ‘Omics Technologies: Insights into the Transmission of Insect Vector-borne Plant Viruses Jennifer R. Wilson1,2, Stacy L. DeBlasio1,2,3, Mariko M. Alexander1,2 and Michelle Heck1,2,3* 1Plant Pathology and Plant Microbe Biology Section, School of Integrative Plant Sciences, Cornell University, Ithaca, NY, USA. 2Boyce Tompson Institute, Ithaca, NY, USA. 3Emerging Pests and Pathogens Research Unit, United States Department of Agriculture – Agricultural Research Service, Ithaca, NY, USA. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.034.113 Abstract interactions, and the development of novel control Insects in the orders Hemiptera and Tysanoptera strategies. transmit viruses and other pathogens associated with the most serious diseases of plants. Plant viruses transmited by these insects target similar Advances in whole genome tissues, genes, and proteins within the insect to sequencing of insect vectors of facilitate plant-to-plant transmission with some plant viruses fuels discovery degree of specifcity at the molecular level. ‘Omics Major advances in next generation sequencing tech- experiments are becoming increasingly important nologies and their increased afordability has led to and practical for vector biologists to use towards an explosion in the availability of whole-genome beter understanding the molecular mechanisms sequence data for each biological player in the and biochemistry underlying transmission of virus–vector–host relationship: virus, vector and these insect-borne diseases.
  • The Genome of Swinepox Virus

    The Genome of Swinepox Virus

    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Virology Papers Virology, Nebraska Center for 1-1-2002 The Genome of Swinepox Virus C. L. Afonso Agricultural Research Service, U.S. Department of Agriculture E. R. Tulman Agricultural Research Service, U.S. Department of Agriculture Z. Lu Agricultural Research Service, U.S. Department of Agriculture L. Zsak Agricultural Research Service, U.S. Department of Agriculture Fernando A. Osorio University of Nebraska-Lincoln, [email protected] See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/virologypub Part of the Virology Commons Afonso, C. L.; Tulman, E. R.; Lu, Z.; Zsak, L.; Osorio, Fernando A.; Balinsky, C.; Kutish, G. F.; and Rock, D. L., "The Genome of Swinepox Virus" (2002). Virology Papers. 71. https://digitalcommons.unl.edu/virologypub/71 This Article is brought to you for free and open access by the Virology, Nebraska Center for at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Virology Papers by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors C. L. Afonso, E. R. Tulman, Z. Lu, L. Zsak, Fernando A. Osorio, C. Balinsky, G. F. Kutish, and D. L. Rock This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/ virologypub/71 JOURNAL OF VIROLOGY, Jan. 2002, p. 783–790 Vol. 76, No. 2 0022-538X/02/$04.00ϩ0 DOI: 10.1128/JVI.76.2.783–790.2002 The Genome of Swinepox Virus C. L. Afonso,1*E.R.Tulman,1 Z. Lu,1 L. Zsak,1 F.
  • Coordination Between Autophagy and the Heat Shock Response: Evidence from Exercise in Animals and Humans Nathan H

    Coordination Between Autophagy and the Heat Shock Response: Evidence from Exercise in Animals and Humans Nathan H

    University of New Mexico UNM Digital Repository Health, Exercise, and Sports Sciences ETDs Education ETDs 9-1-2015 Coordination Between Autophagy and the Heat Shock Response: Evidence From Exercise in Animals and Humans Nathan H. Cole Follow this and additional works at: https://digitalrepository.unm.edu/educ_hess_etds Part of the Health and Physical Education Commons Recommended Citation Cole, Nathan H.. "Coordination Between Autophagy and the Heat Shock Response: Evidence From Exercise in Animals and Humans." (2015). https://digitalrepository.unm.edu/educ_hess_etds/52 This Thesis is brought to you for free and open access by the Education ETDs at UNM Digital Repository. It has been accepted for inclusion in Health, Exercise, and Sports Sciences ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. Nathan H. Cole Candidate Health, Exercise, & Sports Sciences Department This thesis is approved, and it is acceptable in quality and form for publication: Approved by the Thesis Committee: Christine M. Mermier, Chairperson Karol Dokladny Orrin B. Myers i COORDINATION BETWEEN AUTOPHAGY AND THE HEAT SHOCK RESPONSE: EVIDENCE FROM EXERCISE IN ANIMALS AND HUMANS by NATHAN H. COLE B.S. University Studies, University of New Mexico, 2013 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE PHYSICAL EDUCATION CONCENTRATION: EXERCISE SCIENCE The University of New Mexico Albuquerque, New Mexico July, 2015 ii Acknowledgments I would like to thank Dr. Christine Mermier for her unwavering guidance, support, generosity, and patience (throughout this project, and many that came before it); Dr. Orrin Myers for all his assistance in moving from the numbers to the meaning (not to mention putting up with my parabolic model); Dr.
  • Unique Integrated Stress Response Sensors Regulate Cancer Cell

    Unique Integrated Stress Response Sensors Regulate Cancer Cell

    RESEARCH ARTICLE Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised Sara Sannino1*, Megan E Yates2,3,4, Mark E Schurdak5,6, Steffi Oesterreich2,3,7, Adrian V Lee2,3,7, Peter Wipf8, Jeffrey L Brodsky1* 1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, United States; 2Women’s Cancer Research Center, UPMC Hillman Cancer Center, Magee- Women Research Institute, Pittsburgh, United States; 3Integrative Systems Biology Program, University of Pittsburgh, Pittsburgh, United States; 4Medical Scientist Training Program, University of Pittsburgh School of Medicine, Pittsburgh, United States; 5Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, United States; 6University of Pittsburgh Drug Discovery Institute, Pittsburgh, United States; 7Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, United States; 8Department of Chemistry, University of Pittsburgh, Pittsburgh, United States Abstract Molecular chaperones, such as Hsp70, prevent proteotoxicity and maintain homeostasis. This is perhaps most evident in cancer cells, which overexpress Hsp70 and thrive even when harboring high levels of misfolded proteins. To define the response to proteotoxic challenges, we examined adaptive responses in breast cancer cells in the presence of an Hsp70 inhibitor. We discovered that the cells bin into distinct classes based on inhibitor sensitivity. Strikingly, the most resistant cells have higher autophagy levels, and autophagy was maximally activated only in resistant cells upon Hsp70 inhibition. In turn, resistance to compromised Hsp70 *For correspondence: function required the integrated stress response transducer, GCN2, which is commonly associated [email protected] (SS); with amino acid starvation. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating [email protected] (JLB) PERK.
  • Senescence Inhibits the Chaperone Response to Thermal Stress

    Senescence Inhibits the Chaperone Response to Thermal Stress

    SUPPLEMENTAL INFORMATION Senescence inhibits the chaperone response to thermal stress Jack Llewellyn1, 2, Venkatesh Mallikarjun1, 2, 3, Ellen Appleton1, 2, Maria Osipova1, 2, Hamish TJ Gilbert1, 2, Stephen M Richardson2, Simon J Hubbard4, 5 and Joe Swift1, 2, 5 (1) Wellcome Centre for Cell-Matrix Research, Oxford Road, Manchester, M13 9PT, UK. (2) Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, M13 9PL, UK. (3) Current address: Department of Biomedical Engineering, University of Virginia, Box 800759, Health System, Charlottesville, VA, 22903, USA. (4) Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, M13 9PL, UK. (5) Correspondence to SJH ([email protected]) or JS ([email protected]). Page 1 of 11 Supplemental Information: Llewellyn et al. Chaperone stress response in senescence CONTENTS Supplemental figures S1 – S5 … … … … … … … … 3 Supplemental table S6 … … … … … … … … 10 Supplemental references … … … … … … … … 11 Page 2 of 11 Supplemental Information: Llewellyn et al. Chaperone stress response in senescence SUPPLEMENTAL FIGURES Figure S1. A EP (passage 3) LP (passage 16) 200 µm 200 µm 1.5 3 B Mass spectrometry proteomics (n = 4) C mRNA (n = 4) D 100k EP 1.0 2 p < 0.0001 p < 0.0001 LP p < 0.0001 p < 0.0001 ) 0.5 1 2 p < 0.0001 p < 0.0001 10k 0.0 0 -0.5 -1 Cell area (µm Cell area fold change vs. EP fold change vs.
  • Structural–Functional Interactions of NS1-BP Protein with the Splicing and Mrna Export Machineries for Viral and Host Gene Expression

    Structural–Functional Interactions of NS1-BP Protein with the Splicing and Mrna Export Machineries for Viral and Host Gene Expression

    Structural–functional interactions of NS1-BP protein with the splicing and mRNA export machineries for viral and host gene expression Ke Zhanga, Guijun Shangb, Abhilash Padavannilb, Juan Wanga, Ramanavelan Sakthivela, Xiang Chenc,d, Min Kime, Matthew G. Thompsonf, Adolfo García-Sastreg,h,i, Kristen W. Lynchf, Zhijian J. Chenc,d, Yuh Min Chookb,1, and Beatriz M. A. Fontouraa,1 aDepartment of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; bDepartment of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; cDepartment of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; dHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390; eDepartment of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX 75390; fDepartment of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; gDepartment of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029; hGlobal Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; and iDivision of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 Edited by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved November 13, 2018 (received for review October 23, 2018) The influenza virulence factor NS1 protein interacts with the Viral mRNA splicing requires the cellular spliceosome. Most cellular NS1-BP protein to promote splicing and nuclear export of of cellular splicing occurs in the nucleoplasm, where splicing the viral M mRNAs. The viral M1 mRNA encodes the M1 matrix factors are recruited to nascent transcripts through interaction protein and is alternatively spliced into the M2 mRNA, which is with polymerase II (Pol II).
  • HSPA1B Antibody Product Data Sheet Tested Species Reactivity Details Human (Hu) Catalog Number: PA5-28369

    HSPA1B Antibody Product Data Sheet Tested Species Reactivity Details Human (Hu) Catalog Number: PA5-28369

    Lot Number: RI2273253R HSPA1B Antibody Product Data Sheet Tested Species Reactivity Details Human (Hu) Catalog Number: PA5-28369 Size: 100 µL Tested Applications Dilution * Class: Polyclonal Western Blot (WB) 1:500-1:3000 Type: Antibody Immunofluorescence (IF) 1:100-1:1000 Clone: Immunocytochemistry (ICC) 1:100-1:1000 Host / Isotype: Rabbit / IgG Immunohistochemistry (Paraffin) 1:100-1:1000 Recombinant protein fragment (IHC (P)) corresponding to a region within Immunogen: amino acids 377 and 569 of Human * Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own experiment using appropriate negative and positive controls. HSP70 1B Form Information Form: Liquid Concentration: 0.55mg/ml Purification: Antigen affinity chromatography 0.1M tris glycine, pH 7, with 10% Storage Buffer: glycerol Preservative: 0.01% thimerosal Storage Conditions: -20° C, Avoid Freeze/Thaw Cycles Product Specific Information General Information PA5-28369 targets HSP70 1B in IF, IHC (P), and WB applications and This intronless gene encodes a 70kDa heat shock protein which is a member shows reactivity with Human samples. of the heat shock protein 70 family. In conjuction with other heat shock proteins, this protein stabilizes existing proteins against aggregation and The PA5-28369 immunogen is recombinant protein fragment corresponding mediates the folding of newly translated proteins in the cytosol and in to a region within amino acids 377 and 569 of Human HSP70 1B. organelles. It is also involved in the ubiquitin-proteasome pathway through For Research Use Only. Not for use in diagnostic procedures. Not for interaction with the AU-rich element RNA-binding protein 1.