DOCSLIB.ORG

Characterization of the E3 Ubiquitin Ligase Pirh2

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Characterization of the E3 Ubiquitin Ligase Pirh2 Characterization of the E3 Ubiquitin Ligase Pirh2 by Elizabeth Tai A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto © Copyright by Elizabeth Tai (2010) Characterization of the E3 Ubiquitin Ligase Pirh2 Elizabeth Tai Doctor of Philosophy Department of Medical Biophysics University of Toronto 2010 Abstract The p53 tumour suppressor gene is inactivated by mutation in over 50% of all human cancers. The p53 protein is activated and stabilized through several post-translational modifications in response to various stresses and promotes cell cycle arrest and apoptosis. Thus, regulation of p53 is critical for normal cellular function. Pirh2 is a p53-regulated gene recently identified in our laboratory which encodes an E3 RING-finger ubiquitin ligase that binds to p53 and negatively regulates p53 by targeting it for ubiquitin- mediated proteolysis. Pirh2 is similar to another well-characterized E3 RING finger ubiquitin ligase, Mdm2, which also participates in a similar negative feedback loop with p53. At least seven E3 ubiquitin ligases are known to target p53 for degradation and the reason for this functional redundancy is unclear. The purpose of this study is to characterize Pirh2 activity. This study has two aims the first is to identify additional interacting proteins for Pirh2, and the second is to delineate Pirh2 regulation of p53. Using several tandem affinity purification strategies and a GST-pull down approach, we have identified PKC as a candidate interacting protein. II The second aim is to further characterize Pirh2 regulation of p53. Splenocytes and thymocytes from Pirh2-/- mice demonstrate a subtle increase in total p53 levels after irradiation when compared to wild-type controls. Phosphoserine 15 p53 levels are significantly higher in splenocytes and thymocytes from Pirh2 -/- mice relative to wild- type counterparts. Cells stably transfected with Pirh2 have decreased levels of phosphoserine 15 p53 and decreased induction of p21 relative to vector control and Mdm2 expressing cells. The stability of the p53 protein is primarily regulated through ubiquitin mediated proteolysis, and there are multiple ubiquitin ligases targeting p53 for degradation. Here we are able to address the question of functional redundancy by indicating that Pirh2 can target serine 15 phosphorylated p53 which is reported to not be regulated by Mdm2. III Acknowledgements This thesis arose from many years of work previously completed in the Benchimol lab. During my time working on the project, I was fortunate to receive several contributions from many individuals; without their assistance, I would not have been able to complete this thesis. I would now like to take the time to humbly acknowledge those individuals. I would like to thank my supervisor Dr. Sam Benchimol for his advice, guidance and patience in helping me to develop scientific skills; it has been a fantastic learning experience. Above all, he has instilled in me a strong sense of integrity, and enthusiasm in the pursuit of science. I would also like to acknowledge my committee members Dr. Cheryl Arrowsmith, Dr. Razqallah Hakem, and Dr. Linda Penn. Their advice and support have been instrumental in the development of my project and in my development as a researcher. I would also like to thank the past and present members of the Benchimol lab for useful and critical discussion. I would like to thank Weili Ma for her unwavering support and for sharing her extensive technical expertise. I would also like to acknowledge Dr. Keith Wheaton, Dr. Wissam Assaily, Dr. Christina Berube and Dr. Jenny Ho for encouragement and scientific discussion. They are my scientific role models, and set a high standard for which I strived to achieve. I would also like to thank Dr. Yi Sheng, Shili Duan, and Dr. Anne Hakem for sharing their technical expertise. I would like to thank my mother, father, brother and friends for their steadfast support. Their encouragement has been instrumental to the completion of my thesis. IV Table of Contents CHAPTER 1: Introduction...............................................................................................1 The p53 Network .................................................................................................................2 p53 as a tumour suppressor......................................................................................2 p53-dependent cell cycle arrest................................................................................3 p53-dependent apoptosis..........................................................................................5 Choice of response...................................................................................................7 Mouse models of p53 tumour suppression ..............................................................8 Regulation of p53 function ....................................................................................11 Phosphorylation of p53 at serine 15.......................................................................16 Ubiquitination ....................................................................................................................17 Ubiquitin ................................................................................................................17 The ubiquitination mechanism...............................................................................18 Ubiquitination and signalling.................................................................................24 Ubiquitination and protein degradation .................................................................26 Deubiquitination ....................................................................................................28 Ubiquitination and regulation of p53.................................................................................30 Pirh2.......................................................................................................................35 Thesis Hypothesis ..............................................................................................................37 CHAPTER 2: Identification of Pirh2 Interacting Proteins .........................................38 Abstract..................................................................................................................39 Introduction............................................................................................................39 Experimental Procedures .......................................................................................41 Cell Culture................................................................................................41 Plasmids .....................................................................................................41 Generation of Clones Expressing pCMV TAP hPirh2 ..............................42 Generation of Clones Expressing pBabe neo Pirh2-SPA ..........................42 Generation of Clones Expressing pBabe puro Pirh2-HF...........................42 Ubiquitination Assay .................................................................................43 Coimmunoprecipitation .............................................................................43 Western blots .............................................................................................44 TAP Purification ........................................................................................44 SPA Purification ........................................................................................45 His-Flag Purification..................................................................................46 GST-pull down...........................................................................................47 Results....................................................................................................................47 Generation and characterization of TAP-hPirh2 clones ............................47 Purification of TAP and TAP-hPirh2 ........................................................51 Generation of clones and purification of Pirh2-SPA .................................51 Generation of clones and purification of Pirh2-HF ...................................54 Identification of Pirh2 interactors using GST-pull down ..........................56 Discussion..............................................................................................................57 V CHAPTER 3: Pirh2 targets phosphoserine 15 p53 for ubiquitination......................61 Abstract..................................................................................................................62 Introduction............................................................................................................62 Experimental Procedure.........................................................................................64 Cell Culture................................................................................................64 Splenocyte and Thymocyte Culture Preparation .......................................65 Preparation of MCF-7 p53shRNA Cells....................................................65 Western Blotting ........................................................................................66 Immunohistochemistry ..............................................................................66 Coimmunoprecipitation .............................................................................66
Load more

Recommended publications
  • 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.
    [Show full text]
  • PGC-1A Protects from Notch-Induced Kidney Fibrosis Development
    BASIC RESEARCH www.jasn.org PGC-1a Protects from Notch-Induced Kidney Fibrosis Development † ‡ ‡ Seung Hyeok Han,* Mei-yan Wu, § Bo Young Nam, Jung Tak Park,* Tae-Hyun Yoo,* ‡ † † † † Shin-Wook Kang,* Jihwan Park, Frank Chinga, Szu-Yuan Li, and Katalin Susztak *Department of Internal Medicine, Institute of Kidney Disease Research, Yonsei University College of Medicine, Seoul, Korea; †Renal Electrolyte and Hypertension Division, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; ‡Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University College of Medicine, Seoul, Korea; and §Department of Nephrology, The First Hospital of Jilin University, Changchun, China ABSTRACT Kidney fibrosis is the histologic manifestation of CKD. Sustained activation of developmental pathways, such as Notch, in tubule epithelial cells has been shown to have a key role in fibrosis development. The molecular mechanism of Notch-induced fibrosis, however, remains poorly understood. Here, we show that, that expression of peroxisomal proliferation g-coactivator (PGC-1a) and fatty acid oxidation-related genes are lower in mice expressing active Notch1 in tubular epithelial cells (Pax8-rtTA/ICN1) compared to littermate controls. Chromatin immunoprecipitation assays revealed that the Notch target gene Hes1 directly binds to the regulatory region of PGC-1a. Compared with Pax8-rtTA/ICN1 transgenic animals, Pax8-rtTA/ICN1/Ppargc1a transgenic mice showed improvement of renal structural alterations (on his- tology) and molecular defect (expression of profibrotic genes). Overexpression of PGC-1a restored mi- tochondrial content and reversed the fatty acid oxidation defect induced by Notch overexpression in vitro in tubule cells. Furthermore, compared with Pax8-rtTA/ICN1 mice, Pax8-rtTA/ICN1/Ppargc1a mice exhibited improvement in renal fatty acid oxidation gene expression and apoptosis.
    [Show full text]
  • The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression
    International Journal of Molecular Sciences Review The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression Tingting Zou and Zhenghong Lin * School of Life Sciences, Chongqing University, Chongqing 401331, China; [email protected] * Correspondence: [email protected] Abstract: The cell cycle is a collection of events by which cellular components such as genetic materials and cytoplasmic components are accurately divided into two daughter cells. The cell cycle transition is primarily driven by the activation of cyclin-dependent kinases (CDKs), which activities are regulated by the ubiquitin-mediated proteolysis of key regulators such as cyclins, CDK inhibitors (CKIs), other kinases and phosphatases. Thus, the ubiquitin-proteasome system (UPS) plays a pivotal role in the regulation of the cell cycle progression via recognition, interaction, and ubiquitination or deubiquitination of key proteins. The illegitimate degradation of tumor suppressor or abnormally high accumulation of oncoproteins often results in deregulation of cell proliferation, genomic instability, and cancer occurrence. In this review, we demonstrate the diversity and complexity of the regulation of UPS machinery of the cell cycle. A profound understanding of the ubiquitination machinery will provide new insights into the regulation of the cell cycle transition, cancer treatment, and the development of anti-cancer drugs. Keywords: cell cycle regulation; CDKs; cyclins; CKIs; UPS; E3 ubiquitin ligases; Deubiquitinases (DUBs) Citation: Zou, T.; Lin, Z. The Involvement of Ubiquitination Machinery in Cell Cycle Regulation and Cancer Progression. 1. Introduction Int. J. Mol. Sci. 2021, 22, 5754. https://doi.org/10.3390/ijms22115754 The cell cycle is a ubiquitous, complex, and highly regulated process that is involved in the sequential events during which a cell duplicates its genetic materials, grows, and di- Academic Editors: Kwang-Hyun Bae vides into two daughter cells.
    [Show full text]
  • Folding, Function and Subcellular Localization of Parkin
    Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universtität München Folding, function and subcellular localization of parkin Julia Schlehe aus München 2008 Erklärung Diese Dissertation wurde im Sinne von §13 Abs. 3 der Promotionsordnung vom 29. Januar 1998 von PD Dr. Winklhofer betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, am 07.10.2008 …………………………………….. (Julia Schlehe) Dissertation eingereicht am 09.10.2008 1. Gutachter PD Dr. Konstanze Winklhofer 2. Gutachter Prof. Dr. Ulrich Hartl Mündliche Prüfung am 10.11.2008 Summary ...............................................................................................................................................................1 Introduction ..........................................................................................................................................................3 Parkinson’s Disease........................................................................................................................................3 History......................................................................................................................................................3 Clinical characteristics, symptoms and treatment ....................................................................................4 Neuropathological characteristics ............................................................................................................6
    [Show full text]
  • 1 the Functional Interplay Between the HIF Pathway and the Ubiquitin System
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2017 The functional interplay between the HIF pathway and the ubiquitin system – more than a one-way road Günter, Julia ; Ruiz-Serrano, Amalia ; Pickel, Christina ; Wenger, Roland H ; Scholz, Carsten C Abstract: The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue func- tions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIF฀ subunits leads to the recruitment ofan E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIF฀ subunits, demonstrating the regulation of the HIF pathway by the ubiq- uitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the beststudied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway.
    [Show full text]
  • The Ubiquitin System for Protein Degradation and Some of Its Roles in the Control of the Cell Division Cycle*
    Cell Death and Differentiation (2005) 12, 1191–1197 & 2005 Nature Publishing Group All rights reserved 1350-9047/05 $30.00 www.nature.com/cdd Review: Nobel Lecture The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle* A Hershko*,1 further molecular mechanisms that regulate the expression of specific genes. Owing to the intensive research activity on 1 The B. Rappaport Faculty of Medicine and the Rappaport Institute for protein synthesis, little attention was paid at that time to the Research in the Medical Sciences, Technion-Israel Institute of Technology, fact that many proteins are rapidly degraded to amino acids. Haifa, Israel This dynamic turnover of cellular proteins had been previously * Corresponding author: A Hershko, Unit of Biochemistry, the B. Rappaport known by the pioneering work of Schoenheimer and co- Faculty of Medicine and the Rappaport Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, 1 Efron Street, P.O. Box workers, who were among the first to introduce the use of 9649, Haifa 31096, Israel. Fax: 9724 853 5773; isotopically labeled compounds to biological studies. They 15 E-mail: [email protected] administered N-labeled L-leucine to adult rats, and the distribution of the isotope in body tissues and in excreta was Received 18.5.05; accepted 18.5.05 examined. It was observed that less than one-third of the Edited by G Melino isotope was excreted in the urine, and most of it was incorporated into tissue proteins.1 Since the weight of the Abstract animals did not change during the experiment, it could be assumed that the mass and composition of body proteins also Owing to the intensive research activity on protein synthesis, did not change.
    [Show full text]
  • Hif1α Stability in Hypoxia Is Not Oxidant-Initiated but Can Be Tuned
    bioRxiv preprint doi: https://doi.org/10.1101/2020.05.05.079681; this version posted October 25, 2020. 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. Oxidants are dispensable for HIF1α stability in hypoxia Amit Kumar1, 2, 3, Manisha Vaish1, 4, Saravanan S. Karuppagounder1, 2, 3, Irina Gazaryan5, John W. Cave1, 2, 3, Anatoly A. Starkov2, 3, Elizabeth T. Anderson6, Sheng Zhang6, John T. Pinto7, Austin Rountree8, Wang Wang9, Ian R. Sweet8 and Rajiv R. Ratan1, 2, 3, * 1Burke Neurological Institute, White Plains, NY, 10605, USA 2Brain and Mind Research Institute and 3Department of Neurology, Weill Medical College of Cornell University, New York, 10016, USA 4Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, 10029, USA 5Department of Anatomy and Cell Biology, New York Medical College, Valhalla, NY, 10595, USA 6Institute for Biotechnology, Cornell University, Ithaca, NY, 14853, USA 7Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, 10595, USA 8Department of Medicine, University of Washington, Seattle, WA, 98108, USA 9Department of Pain and Anesthesiology, University of Washington, Seattle, WA, 98195, USA Correspondence should be addressed to: *Rajiv R. Ratan M.D., Ph.D. Burke Neurological Institute at Weill Cornell Medicine 785 Mamaroneck Avenue White Plains, NY, 10605, USA Email: [email protected] Phone: 914-597-2225 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.05.079681; this version posted October 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
    [Show full text]
  • Myc Targeted CDK18 Promotes ATR and Homologous Recombination to Mediate PARP Inhibitor Resistance in Glioblastoma
    Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Ning, Jian-Fang et al. “Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma.” Nature Communications 10 (2019): 2910 © 2019 The Author(s) As Published 10.1038/S41467-019-10993-5 Publisher Springer Science and Business Media LLC Version Final published version Citable link https://hdl.handle.net/1721.1/125202 Terms of Use Creative Commons Attribution 4.0 International license Detailed Terms https://creativecommons.org/licenses/by/4.0/ ARTICLE https://doi.org/10.1038/s41467-019-10993-5 OPEN Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma Jian-Fang Ning1,2, Monica Stanciu3, Melissa R. Humphrey1, Joshua Gorham4, Hiroko Wakimoto 4, Reiko Nishihara5, Jacqueline Lees3, Lee Zou6,7, Robert L. Martuza1, Hiroaki Wakimoto 1,8 & Samuel D. Rabkin 1 1234567890():,; PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA- intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc- mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recom- bination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensi- tizing GSCs.
    [Show full text]
  • Comparative Analysis of the Ubiquitin-Proteasome System in Homo Sapiens and Saccharomyces Cerevisiae
    Comparative Analysis of the Ubiquitin-proteasome system in Homo sapiens and Saccharomyces cerevisiae Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Hartmut Scheel aus Rheinbach Köln, 2005 Berichterstatter: Prof. Dr. R. Jürgen Dohmen Prof. Dr. Thomas Langer Dr. Kay Hofmann Tag der mündlichen Prüfung: 18.07.2005 Zusammenfassung I Zusammenfassung Das Ubiquitin-Proteasom System (UPS) stellt den wichtigsten Abbauweg für intrazelluläre Proteine in eukaryotischen Zellen dar. Das abzubauende Protein wird zunächst über eine Enzym-Kaskade mit einer kovalent gebundenen Ubiquitinkette markiert. Anschließend wird das konjugierte Substrat vom Proteasom erkannt und proteolytisch gespalten. Ubiquitin besitzt eine Reihe von Homologen, die ebenfalls posttranslational an Proteine gekoppelt werden können, wie z.B. SUMO und NEDD8. Die hierbei verwendeten Aktivierungs- und Konjugations-Kaskaden sind vollständig analog zu der des Ubiquitin- Systems. Es ist charakteristisch für das UPS, daß sich die Vielzahl der daran beteiligten Proteine aus nur wenigen Proteinfamilien rekrutiert, die durch gemeinsame, funktionale Homologiedomänen gekennzeichnet sind. Einige dieser funktionalen Domänen sind auch in den Modifikations-Systemen der Ubiquitin-Homologen zu finden, jedoch verfügen diese Systeme zusätzlich über spezifische Domänentypen. Homologiedomänen lassen sich als mathematische Modelle in Form von Domänen- deskriptoren (Profile) beschreiben. Diese Deskriptoren können wiederum dazu verwendet werden, mit Hilfe geeigneter Verfahren eine gegebene Proteinsequenz auf das Vorliegen von entsprechenden Homologiedomänen zu untersuchen. Da die im UPS involvierten Homologie- domänen fast ausschließlich auf dieses System und seine Analoga beschränkt sind, können domänen-spezifische Profile zur Katalogisierung der UPS-relevanten Proteine einer Spezies verwendet werden. Auf dieser Basis können dann die entsprechenden UPS-Repertoires verschiedener Spezies miteinander verglichen werden.
    [Show full text]
  • Signalling Functions of Polyubiquitin Chains and Ubiquitin-Binding Domains
    Signalling Functions of Polyubiquitin Chains and Ubiquitin- binding Domains Shengkai Zhao University College London and Cancer Research UK London Research Institute PhD Supervisor: Dr. Helle D Ulrich A thesis submitted for the degree of Doctor of Philosophy University College London September 2010 Declaration I, Shengkai Zhao, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The ubiquitin signalling system has been shown to regulate many important biological events, ranging from DNA repair to the immune response. Different polyubiquitin chains linked by various linkages have been identified in vivo, and can be recognised by proteins containing ubiquitin-binding domains that act as downstream effectors. However, functions for many of them are not well understood. I have studied the function of K63-linked and linear polyubiquitin chains on a common substrate. The other branch of my study was to investigate the role of ubiquitin binding for a novel ubiquitin-interacting protein, SPC25. K63-linked and linear polyubiquitin chains have a similar topology, but whether they convey a similar signal in vivo remains unclear. I have used the eukaryotic replication clamp PCNA, a natural substrate of K63-linked polyubiquitylation, as a model substrate to directly compare the consequences of modification by different types of polyubiquitin chains. I have shown that K63-polyubiquitylated PCNA is not subject to proteasomal degradation. In contrast, linear, non-cleavable ubiquitin chains do not promote DNA damage tolerance, but function as general degradation signals. I found that a linear tetraubiquitin chain is sufficient to afford proteasomal targeting through the Cdc48-Npl4-Ufd1 complex without further modification.
    [Show full text]
  • The Ubiquitin Gene Expression Pattern and Sensitivity to UBB and UBC Knockdown Differentiate Primary 23132/87 and Metastatic MKN45 Gastric Cancer Cells
    International Journal of Molecular Sciences Article The Ubiquitin Gene Expression Pattern and Sensitivity to UBB and UBC Knockdown Differentiate Primary 23132/87 and Metastatic MKN45 Gastric Cancer Cells Emanuele Salvatore Scarpa *, Filippo Tasini, Rita Crinelli, Chiara Ceccarini, Mauro Magnani y and Marzia Bianchi y Department of Biomolecular Sciences, University of Urbino Carlo Bo, 61029 Urbino (PU), Italy; [email protected] (F.T.); [email protected] (R.C.); [email protected] (C.C.); [email protected] (M.M.); [email protected] (M.B.) * Correspondence: [email protected]; Tel.: +39-0722-305252 These authors contributed equally to this work. y Received: 9 July 2020; Accepted: 27 July 2020; Published: 30 July 2020 Abstract: Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC, UBB and RPS27A, in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1, HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines.
    [Show full text]
  • Supplementary Data
    Supplementary Figure 1 Supplementary Figure 2 CCR-10-3244.R1 Supplementary Figure Legends Supplementary Figure 1. B-Myb is overexpressed in primary AML blasts and B-CLL cells. Baseline B-Myb mRNA levels were determined by quantitative RT-PCR, after normalization to the level of housekeeping gene, in primary B-CLL (n=10) and AML (n=5) patient samples, and in normal CD19+ (n=5) and CD34+ (n=4) cell preparations. Each sample was determined in triplicate. Horizontal bars are median, upper and lower edges of box are 75th and 25th percentiles, lines extending from box are 10th and 90th percentiles. Supplementary Figure 2. Cytotoxicity by Nutlin-3 and Chlorambucil used alone or in combination in leukemic cells. The p53wild-type EHEB and SKW6.4 cells lines, and the p53mutated BJAB cell line were exposed to Nutlin-3 or Chlorambucil used either alone or in combination. (Nutl.+Chlor.). In A, upon treatment with Nutlin-3 or Chlorambucil, used either alone (both at 10 μM) or in combination (Nutl.+Chlor.), induction of apoptosis was quantitatively evaluated by Annexin V/PI staining, while E2F1 and pRb protein levels were analyzed by Western blot. Tubulin staining is shown as loading control. The average combination index (CI) values (analyzed by the method of Chou and Talalay) for effects of Chlorambucil+Nutlin-3 on cell viability are shown. ED indicates effect dose. In B, levels of B-Myb and E2F1 mRNA were analyzed by quantitative RT- PCR. Results are expressed as fold of B-Myb and E2F1 modulation in cells treated for 24 hours as indicated, with respect to the control untreated cultures set to 1 (hatched line).
    [Show full text]