DOCSLIB.ORG

Anahita Dastur's Dissertation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Anahita Dastur's Dissertation Copyright by Anahita R Dastur 2007 The Dissertation Committee for Anahita R Dastur Certifies that this is the approved version of the following dissertation: Characterization of Herc5: the major ligase for ISG15, an antiviral ubiquitin-like protein Committee: Jon Huibregtse, Supervisor Robert Krug Makkuni Jayaram Tanya Paull John Sisson Characterization of Herc5: the major ligase for ISG15, an antiviral ubiquitin-like protein by Anahita R Dastur, B.Sc., M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August 2007 Acknowledgements I am grateful to my adviser, Dr. Jon Huibregtse, who is not only an excellent scientist but also a wonderful person. In the five years in his lab, I enjoyed complete scientific freedom, but he was always around for guidance and advice. I would also like to extend my gratitude to the members of my committee, Drs. Robert Krug, Makkuni Jayaram, Tanya Paull and John Sisson, for their interest in my work and useful suggestions. I was lucky to have really nice lab members – Nancy, Sylvie, Younghoon, Hyung Cheol, Larissa, Tracy and William. They made each day in the lab a day to look forward to. My friends – Anirvan, Raju, Priya, Younghoon, Nivedita and Niloufer - have been good sounding boards as well as a great support system. I would like to thank Dr. Umesh Varshney, whose passion for science inspired me to pursue a career in this field. My parents have always been proud of my achievements and what I am today is because of their hard work and unconditional love, so I cannot thank them enough. Finally, I am eternally grateful to my husband, Vishal – I could not have done this without his patience, support and love. iv Characterization of Herc5: the major ligase for ISG15, an antiviral ubiquitin-like protein Publication No._____________ Anahita R Dastur, Ph.D. The University of Texas at Austin, 2007 Supervisor: Jon Huibregtse Human ISG15 is a 17 kDa ubiquitin-like protein (Ubl) that is induced by type I interferons (interferons α and β) and plays a role in antiviral responses. ISG15 is conjugated via its C-terminus to more than 150 cellular proteins, and like ubiquitin, an E1-E2-E3 enzymatic cascade is required for conjugation. Ube1L and UbcH8 were previously identified as the E1 and E2 enzymes for this pathway. My experiments identified Herc5, a HECT domain E3, as the major ligase for ISG15. Like ISG15, Ube1L, and UbcH8, expression of Herc5 is transcriptionally induced by type I interferons. siRNAs against Herc5 abrogated ISG15 conjugation to the vast majority of target proteins in interferon-treated cells. Wild type Herc5, but not the catalytically inactive C994A mutant, supported conjugation of ISG15 in non-interferon-treated cells co- v transfected with Ube1L, UbcH8 and ISG15. IQGAP1, a scaffold protein, was identified as another essential component of the ISG15 system. IQGAP1 was discovered to interact with Herc5, and this interaction was mediated by the C-terminal domain of IQGAP1 and the N-terminal RCC1-like repeats of Herc5. IQGAP1 was required for auto-conjugation of ISG15 to Herc5, and I propose a model where IQGAP1 functions, at least in part, by relieving an auto-inhibitory conformation of Herc5. Thus, I have identified two factors that are critical for ISG15 conjugation and my discoveries have increased our understanding of the ISG15 pathway. Identification and characterization of the conjugation apparatus will aid in establishing an in vitro biochemical system for ISG15 conjugation, which in turn, will be important to decipher the biological function of ISG15 modification. vi Table of Contents List of Tables ......................................................................................................... ix List of Figures......................................................................................................... x Chapter 1: Introduction 1.1 Prelude ......................................................................................................1 1.2 A brief overview of the ubiquitin-proteasome system..............................3 1.3 HECT E3s:................................................................................................7 1.4 Ubiquitin-like proteins............................................................................17 1.5 Innate immune response .........................................................................29 Chapter 2: Materials and Methods Chapter 3: Herc5, an interferon-induced hect E3 enzyme, is required for conjugation of ISG15 in human cells 3.1 Introduction.............................................................................................50 3.2 Results.....................................................................................................53 3.3 Discussion...............................................................................................74 vii Chapter 4: Analysis of Herc5 mutants and Herc5-Herc6 chimeras 4.1 Introduction.............................................................................................78 4.2 Results.....................................................................................................83 4.3 Discussion.............................................................................................100 Chapter 5: IQGAP1 is required for ISG15 conjugation 5.1 Introduction...........................................................................................103 5.2 Results...................................................................................................107 5.3 Discussion.............................................................................................125 Chapter 6: Future directions ............................................................................130 Bibliography ........................................................................................................135 Vita …………………………………………………………………………….150 viii List of Tables Table 1: Sequences of siRNAs used for this research………………………47 Table 2: List of Plasmids used in this study………………………………...48 ix List of Figures Figure 1.1 The E6AP HECT – UbcH7 complex forms a U-shaped structure……………………………………………………………..9 Figure 1.2 Comparison of the crystal structures of three HECT domains……12 Figure 1.3 Structure of ISG15 resembles that of ubiquitin…………………...24 Figure 1.4 Type I IFN induction, signaling and action……………………….34 Figure 3.1 Herc5 and Herc6 siRNAs are effective at the mRNA as well as protein levels……………………………………………………....54 Figure 3.2 siRNAs against Herc5 lead to a decrease in ISG15 conjugates…...56 Figure 3.3 Co-transfection of Herc5-Herc6 siRNAs did not lead to a further decrease in ISG15 conjugates……………………………………..57 Figure 3.4 siRNAs against Herc5 block ISG15 conjugation of p56 and MxA……………………………………………………………….58 Figure 3.5 Herc5 and Herc6 expression is induced by interferon-β………….60 Figure 3.6 Basal level of Herc5 is responsible for ISG15 conjugation in non- interferon-treated cells transfected with E1, E2 and ISG15………62 Figure 3.7 Herc5 boosts ISG15 conjugation in non-interferon-treated cells…63 Figure 3.8 Herc5 boosts ISG15 conjugation of Moesin and p56 in non- interferon-treated cells…………………………………………….65 Figure 3.9 UbcH8, but not UbcH7, functions with Herc5 in vivo……………66 x Figure 3.10 siRNAs against TRIM25 are effective at the protein level……….68 Figure 3.11 siRNAs against TRIM25 do not affect overall ISG15 conjugation………………………………………………………..68 Figure 3.12 TRIM25 does not boost ISG15 conjugates in non-interferon-treated cells………………………………………………………………..69 Figure 3.13 Herc5 is auto-conjugated to ISG15 in vivo………………………..71 Figure 3.14 In vitro assay for ISG15 conjugation……………………………...73 Figure 4.1 A schematic showing the substrate binding box (SBB)…………...81 Figure 4.2 A schematic comparing Her4, Herc5 and Herc6………………….82 Figure 4.3 The ∆F mutant, similar to -4F mutants of ubiquitin ligases, is defective in auto-conjugation ...…………………………………..85 Figure 4.4 Schematic showing the RLD mutants and the SBB mutant………86 Figure 4.5 Auto-conjugation and substrate conjugation activities of the RLD- mutants…………………………………………………………….87 Figure 4.6 Auto-conjugation and substrate conjugation activities of ∆SBB mutant……………………………………………………………..89 Figure 4.7 Herc6 is conjugated to ISG15 in the presence of Ube1L and UbcH8……………………………………………………………..91 Figure 4.8 Herc6-ISG15 conjugation is a combination of auto-conjugation and Herc5-dependent conjugation……………………………………..92 Figure 4.9 The ability to function with ISG15 is limited to Herc5 and xi Herc6……………………………………………………………....94 Figure 4.10 Schematic showing Herc5-Herc6 chimeras……………………….96 Figure 4.11 Substrate conjugation and auto-conjugation activities of Herc5- Herc6 chimeras……………………………………………………97 Figure 4.12 Auto-conjugation and substrate conjugation activities of the C5-6 chimera………………………………………………………….....99 Figure 5.1 A schematic showing the domain architecture of IQGAP1….…..106 Figure 5.2 IQGAP1 is conjugated to ISG15 in vivo………………………...108 Figure 5.3 siRNAs against IQGAP1 effectively reduce the levels of IQGAP1 protein……………………………………………………………110 Figure 5.4 A siRNAs against IQGAP1 reduce ISG15 conjugation in IFN-treated cells……………………………………………………………....112 Figure 5.4 B IQGAP1 is required for ISG15 conjugation in cells transfected with E1, E2 and ISG15………………………………………………..113 Figure 5.5 IQGAP1 is required for conjugation of ISG15 to TrxR1 and Moesin…………………………………………………………....115
Load more

Recommended publications
  • 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.
    [Show full text]
  • Differential Expression of Multiple Disease-Related Protein Groups
    brain sciences Article Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells 1,2, 1, 1 1 Tsung-Ming Hu y, Hsiang-Sheng Chung y, Lieh-Yung Ping , Shih-Hsin Hsu , Hsin-Yao Tsai 1, Shaw-Ji Chen 3,4 and Min-Chih Cheng 1,* 1 Department of Psychiatry, Yuli Branch, Taipei Veterans General Hospital, Hualien 98142, Taiwan; [email protected] (T.-M.H.); [email protected] (H.-S.C.); [email protected] (L.-Y.P.); fi[email protected] (S.-H.H.); [email protected] (H.-Y.T.) 2 Department of Future Studies and LOHAS Industry, Fo Guang University, Jiaosi, Yilan County 26247, Taiwan 3 Department of Psychiatry, Mackay Medical College, New Taipei City 25245, Taiwan; [email protected] 4 Department of Psychiatry, Taitung Mackay Memorial Hospital, Taitung County 95064, Taiwan * Correspondence: [email protected]; Tel.: +886-3888-3141 (ext. 475) These authors contributed equally to this work. y Received: 10 July 2020; Accepted: 8 August 2020; Published: 12 August 2020 Abstract: Valproic acid (VPA) is a multifunctional medication used for the treatment of epilepsy, mania associated with bipolar disorder, and migraine. The pharmacological effects of VPA involve a variety of neurotransmitter and cell signaling systems, but the molecular mechanisms underlying its clinical efficacy is to date largely unknown. In this study, we used the isobaric tags for relative and absolute quantitation shotgun proteomic analysis to screen differentially expressed proteins in VPA-treated SH-SY5Y cells. We identified changes in the expression levels of multiple proteins involved in Alzheimer’s disease, Parkinson’s disease, chromatin remodeling, controlling gene expression via the vitamin D receptor, ribosome biogenesis, ubiquitin-mediated proteolysis, and the mitochondrial oxidative phosphorylation and electron transport chain.
    [Show full text]
  • The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-To-ER Retrograde Transport
    Supplementary Materials The proteasomal deubiquitinating enzyme PSMD14 regulates macroautophagy by controlling Golgi-to-ER retrograde transport Bustamante HA., et al. Figure S1. siRNA sequences directed against human PSMD14 used for Validation Stage. Figure S2. Primer pairs sequences used for RT-qPCR. Figure S3. The PSMD14 DUB inhibitor CZM increases the Golgi apparatus area. Immunofluorescence microscopy analysis of the Golgi area in parental H4 cells treated for 4 h either with the vehicle (DMSO; Control) or CZM. The Golgi marker GM130 was used to determine the region of interest in each condition. Statistical significance was determined by Student's t-test. Bars represent the mean ± SEM (n =43 cells). ***P <0.001. Figure S4. CZM causes the accumulation of KDELR1-GFP at the Golgi apparatus. HeLa cells expressing KDELR1-GFP were either left untreated or treated with CZM for 30, 60 or 90 min. Cells were fixed and representative confocal images were acquired. Figure S5. Effect of CZM on proteasome activity. Parental H4 cells were treated either with the vehicle (DMSO; Control), CZM or MG132, for 90 min. Protein extracts were used to measure in vitro the Chymotrypsin-like peptidase activity of the proteasome. The enzymatic activity was quantified according to the cleavage of the fluorogenic substrate Suc-LLVY-AMC to AMC, and normalized to that of control cells. The statistical significance was determined by One-Way ANOVA, followed by Tukey’s test. Bars represent the mean ± SD of biological replicates (n=3). **P <0.01; n.s., not significant. Figure S6. Effect of CZM and MG132 on basal macroautophagy. (A) Immunofluorescence microscopy analysis of the subcellular localization of LC3 in parental H4 cells treated with either with the vehicle (DMSO; Control), CZM for 4 h or MG132 for 6 h.
    [Show full text]
  • Systemic Analysis of Heat Shock Response Induced by Heat Shock and a Proteasome Inhibitor MG132
    Systemic Analysis of Heat Shock Response Induced by Heat Shock and a Proteasome Inhibitor MG132 Hee-Jung Kim1, Hye Joon Joo2, Yung Hee Kim1, Soyeon Ahn4, Jun Chang1, Kyu-Baek Hwang3, Dong-Hee Lee2, Kong-Joo Lee1* 1 The Center for Cell Signaling & Drug Discovery Research, College of Pharmacy, Ewha Womans University, Seoul, Korea, 2 Department of Life Science, Division of Life & Pharmaceutical Sciences, Department of Bioinspired Science, Ewha Womans University, Seoul, Korea, 3 School of Computer Science and Engineering, Soongsil University, Seoul, Korea, 4 Department of Research and Education, Seoul National University Bundang Hospital, Seongnam, Korea Abstract The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF- 1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly- ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line.
    [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]
  • RIG-I Regulates Myeloid Differentiation by Promoting TRIM25-Mediated Isgylation
    RIG-I regulates myeloid differentiation by promoting TRIM25-mediated ISGylation Song-Fang Wua,b,1, Li Xiaa,1, Xiao-Dong Shia, Yu-Jun Daia, Wei-Na Zhanga, Jun-Mei Zhaoa, Wu Zhanga, Xiang-Qin Wenga, Jing Lua, Huang-Ying Leb, Sheng-ce Taob, Jiang Zhua, Zhu Chena,b,2, Yue-Ying Wanga,2, and Saijuan Chena,b,2 aState Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, National Research Center for Translational Medicine at Shanghai, Rui Jin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; and bKey Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China Contributed by Zhu Chen, April 21, 2020 (sent for review October 24, 2019; reviewed by Christine Chomienne and Chi Wai Eric So) Retinoic acid-inducible gene I (RIG-I) is up-regulated during granu- a process similar to ubiquitination (7). ISGylation is substantially locytic differentiation of acute promyelocytic leukemia (APL) cells involved in immune regulation, DNA repair, tumorigenesis, and induced by all-trans retinoic acid (ATRA). It has been reported that hematopoiesis (7–9). It has been reported that ISGylation also RIG-I recognizes virus-specific 5′-ppp-double-stranded RNA (dsRNA) plays an important and conservative role for normal tissue dif- and activates the type I interferons signaling pathways in innate ferentiation, especially in placental and fetal development, with immunity. However, the functions of RIG-I in hematopoiesis re- ISG15 and other ISGylation-associated genes being specifically main unclear, especially regarding its possible interaction with and highly expressed in the placenta of a wide range of species endogenous RNAs and the associated pathways that could con- from mouse to human (10–15).
    [Show full text]
  • Chemosensitivity Is Controlled by P63 Modification with Ubiquitin-Like Protein ISG15
    Chemosensitivity is controlled by p63 modification with ubiquitin-like protein ISG15 Young Joo Jeon, … , Yong Keun Jung, Chin Ha Chung J Clin Invest. 2012;122(7):2622-2636. https://doi.org/10.1172/JCI61762. Research Article Oncology Identification of the cellular mechanisms that mediate cancer cell chemosensitivity is important for developing new cancer treatment strategies. Several chemotherapeutic drugs increase levels of the posttranslational modifier ISG15, which suggests that ISGylation could suppress oncogenesis. However, how ISGylation of specific target proteins controls tumorigenesis is unknown. Here, we identified proteins that are ISGylated in response to chemotherapy. Treatment of a human mammary epithelial cell line with doxorubicin resulted in ISGylation of the p53 family protein p63. An alternative splice variant of p63, ΔNp63α, suppressed the transactivity of other p53 family members, and its expression was abnormally elevated in various human epithelial tumors, suggestive of an oncogenic role for this variant. We showed that ISGylation played an essential role in the downregulation of ΔNp63α. Anticancer drugs, including doxorubicin, induced ΔNp63α ISGylation and caspase-2 activation, leading to cleavage of ISGylated ΔNp63α in the nucleus and subsequent release of its inhibitory domain to the cytoplasm. ISGylation ablated the ability of ΔNp63α to promote anchorage- independent cell growth and tumor formation in vivo as well to suppress the transactivities of proapoptotic p53 family members. These findings establish ISG15
    [Show full text]
  • UBE1L Is a Retinoid Target That Triggers PML RAR Degradation and Apoptosis in Acute Promyelocytic Leukemia
    UBE1L is a retinoid target that triggers PML͞RAR␣ degradation and apoptosis in acute promyelocytic leukemia Sutisak Kitareewan*†, Ian Pitha-Rowe*, David Sekula*, Christopher H. Lowrey*‡§, Michael J. Nemeth*, Todd R. Golub¶ʈ, Sarah J. Freemantle*, and Ethan Dmitrovsky*‡§ Departments of *Pharmacology and Toxicology and ‡Medicine, and §Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, NH 03755; ¶Dana Farber Cancer Institute, Boston, MA 02115; and ʈWhitehead Institute for Biomedical Research, Cambridge, MA 02139 Communicated by William T. Wickner, Dartmouth Medical School, Hanover, NH, January 7, 2002 (received for review October 2, 2001) All-trans-retinoic acid (RA) treatment induces remissions in acute not occur, further implicating UBE1L in retinoid response. promyelocytic leukemia (APL) cases expressing the t(15;17) product, Expression of E1 (that has homology to UBE1L) was not promyelocytic leukemia (PML)͞RA receptor ␣ (RAR␣). Microarray increased by RA treatment. PML͞RAR␣ cotransfected with analyses previously revealed induction of UBE1L (ubiquitin-activating UBE1L (but not E1) led to PML͞RAR␣ degradation without enzyme E1-like) after RA treatment of NB4 APL cells. We report here RA treatment. This finding indicated specificity of UBE1L for that this occurs within3hinRA-sensitive but not RA-resistant APL triggering PML͞RAR␣ degradation. Results were extended by cells, implicating UBE1L as a direct retinoid target. A 1.3-kb fragment using a UBE1L reporter assay to explore how RA regulated of the UBE1L promoter was capable of mediating transcriptional UBE1L. Retroviral transduction of UBE1L caused apoptosis ͞ ␣ response to RA in a retinoid receptor-selective manner. PML RAR ,a only in cells that expressed PML͞RAR␣. Findings that will be repressor of RA target genes, abolished this UBE1L promoter activity.
    [Show full text]
  • ISG15 Modification of the Eif4e Cognate 4EHP Enhances Cap Structure-Binding Activity of 4EHP
    Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION The binding of eukaryotic translation initiation factor ISG15 modification of the 4F (eIF4F) to the mRNA 5Ј cap structure is the rate-lim- eIF4E cognate 4EHP enhances iting step of cap structure-dependent translation initia- tion in eukaryotes (Gingras et al. 1999). eIF4F contains cap structure-binding activity cap-binding protein eIF4E, scaffold protein eIF4G, and of 4EHP RNA helicase eIF4A. The interaction between eIF4E and eIF4G results in a conformational change of both pro- Fumihiko Okumura, Weiguo Zou, teins and enhances the association between eIF4E and 1 the RNA cap structure (Gross et al. 2003). There are and Dong-Er Zhang three eIF4E-family members in mammals termed Department of Molecular and Experimental Medicine, The eIF4E-1 (eIF4E), eIF4E-2 (4EHP and 4E-LP), and eIF4E-3 Scripps Research Institute, La Jolla, California 92037, USA (Rom et al. 1998; Joshi et al. 2004). Like prototypical eIF4E, 4EHP is expressed ubiquitously; however, expres- The expression of the ubiquitin-like molecule ISG15 and sion of eIF4E-3 is detected only in heart, skeletal muscle, protein modification by ISG15 (ISGylation) are strongly lung, and spleen (Joshi et al. 2004). Similar to eIF4E, both Ј activated by interferon, genotoxic stress, and pathogen 4EHP and eIF4E-3 bind to the RNA 5 cap structure (Rom et al. 1998; Joshi et al. 2004). Furthermore, both eIF4E infection, suggesting that ISG15 plays an important role -؅ and eIF4E-3 are able to bind to eIF4G to facilitate trans in innate immune responses.
    [Show full text]
  • UBE1L Represses PML/RARA by Targeting the PML Domain for Isg15ylation
    905 UBE1L represses PML/RARA by targeting the PML domain for ISG15ylation Sumit J. Shah,1 Steven Blumen,1 Ian Pitha-Rowe,1 PML/RARA degradation through different domains and Sutisak Kitareewan,1 Sarah J. Freemantle,1 distinct mechanisms. Taken together, these findings Qing Feng,1 and Ethan Dmitrovsky1,2,3 advance prior work by establishing two pathways con- verge on the same oncogenic protein to cause its degra- Departments of 1Pharmacology and Toxicology and 2Medicine dation and thereby promote antineoplastic effects. The 3 and Norris Cotton Cancer Center, Dartmouth Medical School, molecular pharmacologic implications of these findings Hanover, New Hampshire and Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire are discussed. [Mol Cancer Ther 2008;7(4):905–14] Abstract Introduction Acute promyelocytic leukemia (APL) is characterized by Acute promyelocytic leukemia (APL) is a distinct subtype expression of promyelocytic leukemia (PML)/retinoic acid of acute myeloid leukemia (FAB, M3; ref. 1). APL is (RA) receptor A (RARA) protein and all-trans-RA-mediated characterized by accumulation of abnormal promyelocytes clinical remissions. RA treatment can confer PML/RARA expressing the oncogenic product of the balanced t(15;17) degradation, overcoming dominant-negative effects of rearrangement between the promyelocytic leukemia (PML) this oncogenic protein. The present study uncovered and retinoic acid (RA) receptor a (RARa) genes (2, 3). independent retinoid degradation mechanisms, targeting PML/RARa is a dominant-negative transcriptional factor different domains of PML/RARA. RA treatment is known (3). All-trans-RA treatment is clinically successful in APL to repress PML/RARA and augment ubiquitin-activating by triggering leukemic cell differentiation (4).
    [Show full text]
  • ISG15-Dependent Activation of the Sensor MDA5 Is Antagonized by the SARS-Cov-2 Papain-Like Protease to Evade Host Innate Immunity
    ARTICLES https://doi.org/10.1038/s41564-021-00884-1 ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity GuanQun Liu 1,2,4, Jung-Hyun Lee 1,2,4, Zachary M. Parker2, Dhiraj Acharya1,2, Jessica J. Chiang3, Michiel van Gent 1,2, William Riedl1,2, Meredith E. Davis-Gardner3, Effi Wies3, Cindy Chiang1,2 and Michaela U. Gack 1,2 ✉ Activation of the RIG-I-like receptors, retinoic-acid inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), establishes an antiviral state by upregulating interferon (IFN)-stimulated genes (ISGs). Among these is ISG15, the mechanistic roles of which in innate immunity still remain enigmatic. In the present study, we report that ISG15 conjuga- tion is essential for antiviral IFN responses mediated by the viral RNA sensor MDA5. ISGylation of the caspase activation and recruitment domains of MDA5 promotes its oligomerization and thereby triggers activation of innate immunity against a range of viruses, including coronaviruses, flaviviruses and picornaviruses. The ISG15-dependent activation of MDA5 is antagonized through direct de-ISGylation mediated by the papain-like protease of SARS-CoV-2, a recently emerged coronavirus that has caused the COVID-19 pandemic. Our work demonstrates a crucial role for ISG15 in the MDA5-mediated antiviral response, and also identifies a key immune evasion mechanism of SARS-CoV-2, which may be targeted for the development of new antivirals and vaccines to combat COVID-19. iral perturbation of host immune homoeostasis is moni- Coronaviridae family that contains several other human patho- tored by the innate immune system, which relies on recep- gens.
    [Show full text]
  • Cellular Immunology Regulation of T Cell Differentiation and Function By
    Cellular Immunology 340 (2019) 103922 Contents lists available at ScienceDirect Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm Regulation of T cell differentiation and function by ubiquitin-specific proteases T ⁎ Aiting Wanga,b, Fangming Zhua,b,c, Rui Liangb, Dan Lib, Bin Lib, a Key Laboratory of Molecular Virology and Immunology, CAS Center for Excellence in Molecular Cell Science, Unit of Molecular Immunology, Institut Pasteur of Shanghai, University of Chinese Academy of Sciences, Shanghai 200031, China b Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China c Shanghai Key Laboratory of Bio-energy Crops, School of Life Science, Shanghai University, Shanghai 200444, China ABSTRACT T cells play critical roles in immune responses to pathogens, autoimmunity, and antitumor immunity. During the past few decades, increasing numbers of studies have demonstrated the significance of protein ubiquitination in T cell-mediated immunity. Several E3 ubiquitin ligases and deubiquitinases (DUBs) have been identified as either positive or negative regulators of T cell development and function. In this review, we mainly focus on the roles of DUBs (especially ubiquitin- specific proteases (USPs)) in modulating T cell differentiation and function, as well as the molecular mechanisms. Understanding how T cell development and function is regulated by ubiquitination and deubiquitination will provide novel strategies for treating infection, autoimmune diseases, and cancer. 1. Introduction in diverse cellular processes. Extensive studies have established that Lys48-linked polyubiquitination targets proteins to undergo protea- 1.1. The ubiquitin pathway some-dependent degradation, whereas modification of target proteins by K63-linked polyubiquitination results in the regulation of cellular Ubiquitin (Ub) is a 76-amino-acid protein highly conserved in all signal transduction via proteasome-independent mechanism.
    [Show full text]