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