Abstract
As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site. Using a model substrate, we demonstrate that ISG15 can modify ubiquitin, which is immobilized on its substrate, to form ISG15-ubiquitin mixed chains. Furthermore, our results indicate that ISG15-ubiquitin mixed chains do not serve as degradation signals for a ubiquitin fusion degradation substrate. Accordingly, an ISG15-ubiquitin fusion protein, which mimics an ISG15-ubiquitin mixed chain, negatively regulates cellular turnover of ubiquitylated proteins. In addition, ISG15-ubiquitin mixed chains, which are detectable on endogenously ubiquitylated proteins, dampen cellular turnover of these proteins. Thus, our studies unveil an unanticipated interplay between two protein modification systems and highlight its role in coordinating protein homeostasis.
Highlights
IntroductionNEDD8 modifies ubiquitin at Lys 48 and these NEDD8-ubiquitin heterologous chains are recognized essentially like ubiquitin chains[21]
The SUMO chains recruit the ubiquitin ligase RING finger protein 4 (RNF4), by which the ubiquitin moieties are added to the SUMO chain and result in proteasomal degradation of PML
Relative to what we have learned about ubiquitin and the ubiquitin-like modifiers SUMOs and NEDD8, little is known about protein modification by ISG15
Summary
NEDD8 modifies ubiquitin at Lys 48 and these NEDD8-ubiquitin heterologous chains are recognized essentially like ubiquitin chains[21] Another ubiquitin-like protein, FAT10, has been reported as a degradation signal when it is fused to the N-terminus of long-lived proteins[24,25]. ISG15 is the first identified ubiquitin-like modifier, which can covalently conjugate to cellular proteins[26,27]. As a ubiquitin-like modifier, the mature form of ISG15 contains two ubiquitin-like domains and a C-terminus ending with the amino acid sequence ‘Leu Arg Leu Arg Gly Gly’ (LRLRGG). Similar levels of polyubiquitylated proteins were detected in ISGylation deficient cells as in wild-type cells[52] These findings suggest that the apparent antagonistic relationship between ISGylation and ubiquitylation might be restricted to specific cell conditions. We report the formation of ISG15-ubiquitin mixed chains and describe their role in regulating the turnover of ubiquitylated proteins
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