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Abstract:
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Human ISG15 is a 17 kDa ubiquitin -like protein (Ubl ) that is induced by type I interferons (interferons [alpha] and [beta] ) 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 -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 . |