DOCSLIB.ORG

University of California San Diego

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
University of California San Diego UNIVERSITY OF CALIFORNIA SAN DIEGO Regulation of Ubiquitylation and Ubiquitin-like Post-Translational Modifications A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry by Ryan James Lumpkin Committee in charge: Professor Elizabeth A. Komives, Chair Professor Rommie Amaro Professor Gouri Ghosh Professor Randy Hampton Professor Susan Taylor 2019 Copyright Ryan James Lumpkin, 2019 All rights reserved The dissertation of Ryan James Lumpkin is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California San Diego 2019 iii DEDICATION To my parents who have helped me get this far, my friends who supported me along the way, my PI who believed in me, and my cat who was always waiting for me. iv TABLE OF CONTENTS Signature page.....................................................................................................................iii Dedication............................................................................................................................iv Table of Contents..................................................................................................................v List of Abbreviations............................................................................................................xi Lists of Figures...................................................................................................................xiii Lists of Tables....................................................................................................................xvi Acknowledgements...........................................................................................................xvii Vita...................................................................................................................................xviii Abstract of the Dissertation................................................................................................xix Chapter I Introduction..................................................................................................1 A. Proteome Complexity and Post-Translational Modifications........................2 B. Ubiquitylation................................................................................................2 C. Ubiquitin-like proteins...................................................................................3 D. E3 Ubiquitin Ligases......................................................................................3 E. The ASB family of Protein Receptors...........................................................5 F. References.....................................................................................................6 Chapter II Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.......................................................8 A. Introduction....................................................................................................9 B. Materials and methods.................................................................................12 v 1. WaLP.....................................................................................................12 2. SENP1 and SENP2 deSUMOylation....................................................13 3. Enrichment of KGG-containing peptides..............................................14 4. MS analysis of in vitro deSUMOylation experiment............................15 5. SILAC quantitation of Ub and SUMO..................................................17 6. Preparation of tissue samples................................................................18 7. nLC-MS/MS for SILAC experiments...................................................19 8. Data Availability...................................................................................21 C. Results.........................................................................................................21 1. SUMOylation site profiling by WaLP digestion...................................21 2. Identification of novel SUMOylation sites using WaLP.......................22 3. Validation of SUMOylation sites using in vitro deSUMOylation.........24 4. Parallel Ub and SUMO site identification.............................................29 5. Parallel quantification of Ub and SUMO sites......................................29 6. Identification of SUMOylation in tissue samples..................................34 D. Discussion....................................................................................................35 E. Acknowledgements......................................................................................36 F. References...................................................................................................36 Chapter III DECA, a comprehensive, automatic post-processing program for HDX-MS data.............................................................................................41 A. Introduction..................................................................................................42 B. Methods........................................................................................................44 vi 1. Software Design.....................................................................................44 2. Data Format............................................................................................45 3. Data Analysis.........................................................................................45 4. Sample Dataset.......................................................................................46 5. Fitting of deuterium uptake plots...........................................................46 C. Results..........................................................................................................47 1. Graphical User Interface........................................................................47 2. File Merging...........................................................................................47 3. Back Exchange Correction..............................................................................................48 4. Generation of coverage maps from back-exchange corrected data........51 5. Generation of publication-quality deuterium uptake plots from back-exchange corrected data................................................................51 6. Comparison of overlapping peptides for increased resolution of deuterium uptake data............................................................................52 7. Visualizing Highest Resolution Deuterium Exchange Data..................52 8. Statistical Significance...........................................................................54 9. Data Export............................................................................................56 D. Discussion....................................................................................................57 E. Data Accessibility........................................................................................58 F. Acknowledgements......................................................................................58 G. References....................................................................................................59 vii Chapter IV Structure and dynamics of the ASB9 CUL-RING E3 Ligase................62 A. Introduction..................................................................................................63 B. Materials and methods.................................................................................65 1. Expression Vectors................................................................................65 2. Protein Expression..................................................................................65 3. Protein Purification.................................................................................66 4. Hydrogen Deuterium Exchange.............................................................67 5. Cryo-EM sample preparation and data collection..................................69 6. Homology Modeling..............................................................................74 C. Results..........................................................................................................74 1. The Structure of the CKB-ASB9-ELOB-ELOC Complex by Cryo-EM................................................................................................74 2. Structure of full-length CUL5-RBX2....................................................75 3. Full Model of the ASB9-CRL...............................................................78 4. HDX-MS mapping of the protein-protein interfaces in the CKB-bound ASB9-CRL........................................................................78 5. HDX-MS reveals crosstalk between non-adjacent subunits of the E3 ligase.................................................................................................82 D. Discussion....................................................................................................86 1. Structure of the substrate-bound ASB9-CRL based on Cryo-EM data.........................................................................................................86 2. Allosteric Changes in the ASB9-CRL...................................................89 E. Acknowledgements......................................................................................91
Load more

Recommended publications
  • Transcriptome Analyses of Rhesus Monkey Pre-Implantation Embryos Reveal A
    Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Transcriptome analyses of rhesus monkey pre-implantation embryos reveal a reduced capacity for DNA double strand break (DSB) repair in primate oocytes and early embryos Xinyi Wang 1,3,4,5*, Denghui Liu 2,4*, Dajian He 1,3,4,5, Shengbao Suo 2,4, Xian Xia 2,4, Xiechao He1,3,6, Jing-Dong J. Han2#, Ping Zheng1,3,6# Running title: reduced DNA DSB repair in monkey early embryos Affiliations: 1 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China 2 Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center for Genetics and Developmental Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 3 Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China 4 University of Chinese Academy of Sciences, Beijing, China 5 Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China 6 Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, China * Xinyi Wang and Denghui Liu contributed equally to this work 1 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press # Correspondence: Jing-Dong J. Han, Email: [email protected]; Ping Zheng, Email: [email protected] Key words: rhesus monkey, pre-implantation embryo, DNA damage 2 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press ABSTRACT Pre-implantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA) and cell fate commitment.
    [Show full text]
  • BC-Box Protein Domain-Related Mechanism for VHL Protein Degradation
    BC-box protein domain-related mechanism for VHL protein degradation Maria Elena Pozzebona,1,2, Archana Varadaraja,1, Domenico Mattoscioa, Ellis G. Jaffrayb, Claudia Miccoloa, Viviana Galimbertic, Massimo Tommasinod, Ronald T. Hayb, and Susanna Chioccaa,3 aDepartment of Experimental Oncology, European Institute of Oncology, 20139 Milan, Italy; cSenology Division, European Institute of Oncology, 20141 Milan, Italy; dInternational Agency for Research on Cancer, World Health Organization, 69372 Lyon, France; and bCentre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, United Kingdom Edited by William G. Kaelin, Jr., Harvard Medical School, Boston, MA, and approved September 23, 2013 (received for review June 18, 2013) The tumor suppressor VHL (von Hippel–Lindau) protein is a sub- effects of the wild-type Gam1 protein (18, 20, 21), supporting the strate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), idea that these effects may depend on Gam1 ability to act as containing a BC-box domain that associates to the adaptor Elongin substrate-receptor protein. B/C. VHL targets hypoxia-inducible factor 1α to proteasome- VHL (von Hippel–Lindau) protein is a cellular BC box-con- dependent degradation. Gam1 is an adenoviral protein, which also taining substrate receptor and associates with Cullin2-based E3 possesses a BC-box domain that interacts with the host Elongin B/C, ligases (22–24). VHL is a tumor suppressor, and its loss leads to – thereby acting as a viral substrate receptor. Gam1 associates with the von Hippel Lindau syndrome that often develops into renal both Cullin2 and Cullin5 to form CRL complexes targeting the host clear-cell carcinoma and other highly vascularized tumors (25, 26).
    [Show full text]
  • Protein Biomarkers Analysis Within Muscular Dystrophies
    EXAMENSARBETE INOM BIOTEKNIK, AVANCERAD NIVÅ, 30 HP STOCKHOLM, SVERIGE 2017 Protein biomarkers analysis within muscular dystrophies SANDRA MENA KTH SKOLAN FÖR BIOTEKNOLOGI PROTEIN BIOMARKERS ANALYSIS WITHIN MUSCULAR DYSTROPHIES Master thesis Author: Sandra Carolina Mena Pérez Supervisor: Cristina Al-Khalili Szigyarto Stockholm 2017 Master’s program: Medical Biotechnology Kungliga Tekniska Högskolan Content Abstract .........................................................................................................................................................1 Abstrakt .........................................................................................................................................................1 Introduction .................................................................................................................................................1 Materials and Methods ............................................................................................................................ 3 Results and Discussion ............................................................................................................................ 4 Conclusion .................................................................................................................................................. 11 Future Perspectives ................................................................................................................................. 11 Aknowledgments ....................................................................................................................................
    [Show full text]
  • Integration of the Drug-Gene Interaction Database (Dgidb) with Open Crowdsource Efforts
    bioRxiv preprint doi: https://doi.org/10.1101/2020.09.18.301721; this version posted September 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Integration of the Drug-Gene Interaction Database (DGIdb) with open crowdsource efforts Sharon Freshour1,2,†, Susanna Kiwala2,†, Kelsy C. Cotto1,2,†, Adam C. Coffman2, Joshua F. McMichael2, Jonathan Song1,2, Malachi Griffith1,2,3,4,*, Obi L. Griffith1,2,3,4,*, Alex H. Wagner1,2,5,6,* 1 Department of Medicine, Division of Oncology, Washington University School of Medicine, St Louis, MO, 63110, USA 2 McDonnell Genome Institute, Washington University School of Medicine, St Louis, MO, 63108, USA 3 Department of Genetics, Washington University School of Medicine, St Louis, MO, 63110, USA 4 Siteman Cancer Center, Washington University School of Medicine, St Louis, MO, 63110, USA 5 The Steve and Cindy Rasmussen Institute for Genomic Medicine, Nationwide Children’s Hospital, Columbus, OH, 43215, USA 6 Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH, 43210, USA * To whom correspondence should be addressed. Tel: 1-614-355-1645; Fax: 1-614-355-6833; Email: [email protected] Correspondence may also be addressed to Obi L. Griffith. Tel: 1-314-747-9248; Fax: 1-314-286-1810; Email: [email protected] Correspondence may also be addressed to Malachi Griffith. Tel: 1-314-286-1274; Fax: 1-314-286-1810; Email: [email protected] † The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors ABSTRACT The Drug-Gene Interaction Database (DGIdb, www.dgidb.org) is a web resource that provides information on drug-gene interactions and druggable genes from various sources including publications, databases, and other web-based sources in one resource.
    [Show full text]
  • Anti-CKB Monoclonal Antibody, Clone 904DU30.2.2 (DCABH-6668) This Product Is for Research Use Only and Is Not Intended for Diagnostic Use
    Anti-CKB monoclonal antibody, clone 904DU30.2.2 (DCABH-6668) This product is for research use only and is not intended for diagnostic use. PRODUCT INFORMATION Product Overview Mouse monoclonal to Creatine kinase B type Antigen Description Reversibly catalyzes the transfer of phosphate between ATP and various phosphogens (e.g. creatine phosphate). Creatine kinase isoenzymes play a central role in energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa. Immunogen Recombinant full length protein (His-tag) corresponding to Creatine kinase B type aa 1- 381.Database link: P12277 Isotype IgG Source/Host Mouse Species Reactivity Mouse, Human Clone 904DU30.2.2 Purity Protein G purified Purification This antibody is purified through a protein G column, eluted with high and low pH buffers and neutralized immediately, followed by dialysis against PBS. Conjugate Unconjugated Applications WB Positive Control MDA-MB453; 293 and Y-79 cell lysates; Mouse stomach and Mouse brain tissue lysates Format Liquid Size 400 μl Buffer Preservative: 0.09% Sodium azide; Constituent: 99% PBS Storage Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. Ship Shipped at 4°C. 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 1 © Creative Diagnostics All Rights Reserved GENE INFORMATION Gene Name CKB creatine kinase, brain [ Homo sapiens ] Official Symbol CKB Synonyms CKB; creatine
    [Show full text]
  • JAX-CKB), Which Can Be Queried Readily to Access Comprehensive Data for Clinical Reporting Via Customized Reporting Queries
    Patterson et al. Human Genomics (2016) 10:4 DOI 10.1186/s40246-016-0061-7 PRIMARY RESEARCH Open Access The clinical trial landscape in oncology and connectivity of somatic mutational profiles to targeted therapies Sara E. Patterson, Rangjiao Liu, Cara M. Statz, Daniel Durkin, Anuradha Lakshminarayana and Susan M. Mockus* Abstract Background: Precision medicine in oncology relies on rapid associations between patient-specific variations and targeted therapeutic efficacy. Due to the advancement of genomic analysis, a vast literature characterizing cancer- associated molecular aberrations and relative therapeutic relevance has been published. However, data are not uniformly reported or readily available, and accessing relevant information in a clinically acceptable time-frame is a daunting proposition, hampering connections between patients and appropriate therapeutic options. One important therapeutic avenue for oncology patients is through clinical trials. Accordingly, a global view into the availability of targeted clinical trials would provide insight into strengths and weaknesses and potentially enable research focus. However, data regarding the landscape of clinical trials in oncology is not readily available, and as a result, a comprehensive understanding of clinical trial availability is difficult. Results: To support clinical decision-making, we have developed a data loader and mapper that connects sequence information from oncology patients to data stored in an in-house database, the JAX Clinical Knowledgebase (JAX-CKB), which can be queried readily to access comprehensive data for clinical reporting via customized reporting queries. JAX-CKB functions as a repository to house expertly curated clinically relevant data surrounding our 358-gene panel, the JAX Cancer Treatment Profile (JAX CTP), and supports annotation of functional significance of molecular variants.
    [Show full text]
  • Filamins but Not Janus Kinases Are Substrates of the Asb2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells
    Filamins but Not Janus Kinases Are Substrates of the ASB2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells Isabelle Lamsoul1,2, Monique Erard1,2, Peter F. M. van der Ven3, Pierre G. Lutz1,2* 1 CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne BP64182, F-31077 Toulouse, France, 2 Universite´ de Toulouse, UPS, IPBS, F- 31077 Toulouse, France, 3 Department of Molecular Cell Biology, Institute of Cell Biology, University of Bonn, Bonn, Germany Abstract The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2a substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2a biological effects is crucial for the understanding of ASB2a functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2a induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates. Citation: Lamsoul I, Erard M, van der Ven PFM, Lutz PG (2012) Filamins but Not Janus Kinases Are Substrates of the ASB2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells. PLoS ONE 7(8): e43798. doi:10.1371/journal.pone.0043798 Editor: Zhengqi Wang, Emory University, United States of America Received March 21, 2012; Accepted July 26, 2012; Published August 20, 2012 Copyright: ß 2012 Lamsoul et al.
    [Show full text]
  • Cullin 5 Regulates Cortical Layering by Modulating the Speed and Duration of Dab1-Dependent Neuronal Migration
    5668 • The Journal of Neuroscience, April 21, 2010 • 30(16):5668–5676 Cellular/Molecular Cullin 5 Regulates Cortical Layering by Modulating the Speed and Duration of Dab1-Dependent Neuronal Migration Sergi Simó, Yves Jossin, and Jonathan A. Cooper Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 Themultilayeredmammalianneocortexdevelopsbythecoordinatedimmigrationanddifferentiationofcellsthatareproducedatdistant sites. Correct layering requires an extracellular protein, Reelin (Reln), an intracellular signaling molecule, Disabled-1 (Dab1), and an E3 ubiquitinligase,Cullin-5(Cul5).RelnactivatesDab1,whichisthendegradedbyCul5.HerewetestwhetherCul5regulatesneuronlayering by affecting Dab1 stability or other mechanisms. We find that a stabilized mutant Dab1, which resists Cul5-dependent degradation, causes a similar phenotype to Cul5 deficiency. Moreover, Cul5 has no effect when Dab1 is absent. The effects of Cul5 and Dab1 are cell autonomous, and Cul5 regulates movement of early as well as late cortical neurons. Removing Cul5 increases the speed at which neurons migrate through the cortical plate by reducing the time spent stationary and increasing the speed of individual steps. These results show thatCul5regulatesneuronlayeringbystimulatingDab1degradationandthatCul5controlsmigrationspeedandstoppingpoint,andthey demonstrate the importance of negative feedback in signaling during cortical development. Introduction (Pinto-Lord et al., 1982; Dulabon et al., 2000; Sanada et al., 2004). The mammalian
    [Show full text]
  • Temporal Proteomic Analysis of HIV Infection Reveals Remodelling of The
    1 1 Temporal proteomic analysis of HIV infection reveals 2 remodelling of the host phosphoproteome 3 by lentiviral Vif variants 4 5 Edward JD Greenwood 1,2,*, Nicholas J Matheson1,2,*, Kim Wals1, Dick JH van den Boomen1, 6 Robin Antrobus1, James C Williamson1, Paul J Lehner1,* 7 1. Cambridge Institute for Medical Research, Department of Medicine, University of 8 Cambridge, Cambridge, CB2 0XY, UK. 9 2. These authors contributed equally to this work. 10 *Correspondence: [email protected]; [email protected]; [email protected] 11 12 Abstract 13 Viruses manipulate host factors to enhance their replication and evade cellular restriction. 14 We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a 15 comprehensive time course analysis of >6,500 viral and cellular proteins during HIV 16 infection. To enable specific functional predictions, we categorized cellular proteins regulated 17 by HIV according to their patterns of temporal expression. We focussed on proteins depleted 18 with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be 19 necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the 20 B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). 21 Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent 22 hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. 23 The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral 2 24 lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and 25 conserved Vif function.
    [Show full text]
  • Anti-ARIH1 (Aa 505-554) Polyclonal Antibody (DPABH-17177) This Product Is for Research Use Only and Is Not Intended for Diagnostic Use
    Anti-ARIH1 (aa 505-554) polyclonal antibody (DPABH-17177) This product is for research use only and is not intended for diagnostic use. PRODUCT INFORMATION Antigen Description Might act as an E3 ubiquitin-protein ligase, or as part of the E3 complex, which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, such as UBE2L3/UBCM4, and then transfers it to substrates. Immunogen A region within Synthetic peptide: LSGYLERDIS QDSLQDIKQK VQDKYRYCES RRRVLLQHVH EGYEKDLWEY, corresponding to C terminal amino acids 505-554 of Human ARIH1 Isotype IgG Source/Host Rabbit Species Reactivity Human Purification Protein A purified Conjugate Unconjugated Applications WB Format Liquid Size 100 μg Buffer Constituents: 2% Sucrose, PBS Preservative None Storage Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles. GENE INFORMATION Gene Name ARIH1 ariadne homolog, ubiquitin-conjugating enzyme E2 binding protein, 1 (Drosophila) [ Homo sapiens ] Official Symbol ARIH1 Synonyms ARIH1; ariadne homolog, ubiquitin-conjugating enzyme E2 binding protein, 1 (Drosophila); ariadne (Drosophila) homolog, ubiquitin conjugating enzyme E2 binding protein, 1; E3 ubiquitin- 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 1 © Creative Diagnostics All Rights Reserved protein ligase ARIH1; ARI; ariadne; Drosophila; homolog of; HARI; HHARI; UBCH7BP; ARI-1; MOP-6; H7-AP2; monocyte protein 6; ubcH7-binding protein; protein ariadne-1 homolog; ubcM4- interacting protein; ariadne,
    [Show full text]
  • E3 Ubiquitin Ligase Cullin-5 Modulates Multiple Molecular and Cellular Responses to Heat Shock Protein 90 Inhibition in Human Cancer Cells
    E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells Rahul S. Samant, Paul A. Clarke, and Paul Workman1 Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UK Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 3, 2014 (received for review December 24, 2013) The molecular chaperone heat shock protein 90 (HSP90) is required Given the link between CUL5 and the HSP90 inhibitor- for the activity and stability of its client proteins. Pharmacologic induced degradation of ERBB2 (12), we have investigated the inhibition of HSP90 leads to the ubiquitin-mediated degradation of role of Cullin-RING ligases with respect to HSP90’s protein kinase clients, particularly activated or mutant oncogenic protein kinases. clients in human cancer cell lines. Our initial focused siRNA Client ubiquitination occurs via the action of one or more E3 screen of 28 Cullin-RING ligase family members identified five ubiquitin ligases. We sought to identify the role of Cullin-RING fam- genes, including CUL5, that were required for ERBB2 degra- ily E3 ubiquitin ligases in the cellular response to HSP90 inhibition. dation following treatment with 17-AAG—which we use here as Through a focused siRNA screen of 28 Cullin-RING ligase family a representative HSP90 inhibitor and chemical tool to promote members, we found that CUL5 and RBX2 were required for degra- client protein turnover. We go on to show for the first time to our dation of several HSP90 clients upon treatment of human cancer knowledge that RNAi silencing of CUL5 reduces the 17-AAG– cells with the clinical HSP90 inhibitor 17-AAG.
    [Show full text]
  • Interactions Between HIV-1 Vif and Human Elonginb-Elonginc Are Important for CBF-Β Binding To
    Wang et al. Retrovirology 2013, 10:94 http://www.retrovirology.com/content/10/1/94 RESEARCH Open Access Interactions between HIV-1 Vif and human ElonginB-ElonginC are important for CBF-β binding to Vif Xiaodan Wang1, Xiaoying Wang1, Haihong Zhang1, Mingyu Lv1, Tao Zuo1, Hui Wu1, Jiawen Wang1, Donglai Liu1, Chu Wang1, Jingyao Zhang1,XuLi1, Jiaxin Wu1, Bin Yu1, Wei Kong1,2* and Xianghui Yu1,2* Abstract Background: The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloB- EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-β is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-β-Cul5 E3 ligase complex. Results: The results revealed that: (1) EloB (and EloC) positively affected the recruitment of CBF-β to Vif. Both knockdown of endogenous EloB and over-expression of its mutant with a 34-residue deletion in the COOH-terminal tail (EloBΔC34/EBΔC34) impaired the Vif-CBF-β interaction. (2) Introduction of both the Vif SLQ → AAA mutant (VifΔSLQ, which dramatically impairs Vif-EloB-EloC binding) and the Vif PPL → AAA mutant (VifΔPPL, which is thought to reduce Vif-EloB binding) could reduce CBF-β binding.
    [Show full text]