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In vitro analysis of proteasome-associated USP14 activity for substrate degradation and deubiquitylation

Title
In vitro analysis of proteasome-associated USP14 activity for substrate degradation and deubiquitylation
Authors
Muniyappan, SrinivasanLee, Byung-Hoon
DGIST Authors
Muniyappan, Srinivasan; Lee, Byung-Hoon
Issue Date
2019-02
Citation
Methods in Enzymology, 619, 249-268
Type
Article
Article Type
Review; Book Chapter
Author Keywords
Deubiquitinating enzymeIn vitro deubiquitination assayProteasomeUbiquitin adductsUbiquitin conjugateUSP14
Keywords
UBIQUITINIMPAIRMENTINHIBITOR
ISSN
0076-6879
Abstract
The ubiquitin-proteasome pathway plays an essential role in maintaining protein homeostasis and regulates almost every aspect of cellular processes in eukaryotes. Emerging evidence indicates that the proteasome does not work as a simple unidirectional molecular machinery for substrate proteolysis. In fact, proteasome activity should be tightly regulated, and the proteasome itself can be dynamically engaged in the degradation cycle. Proteasome-mediated degradation can occur through multistep mechanisms such as ubiquitin-dependent substrate recognition, deubiquitination, and ATP-driven unfolding and translocation of the substrate into 20S chamber for proteolytic cleavage. Deubiquitination is particularly interesting because this reaction may impose a critical checkpoint for substrate turnover on the proteasome. Notably, there are three major deubiquitinating enzymes (DUBs) on human proteasomes: USP14, UCH37, and RPN11. USP14 can spare the substrate from degradation prior to the proteasome's commitment step, suggesting that USP14 inhibition may stimulate proteasomal degradation of undesirable proteins under certain proteotoxic conditions. Furthermore, USP14 deubiquitinates multichain conjugates, the first among ~ 100 DUBs found to have this striking specificity. In this chapter, we describe in vitro methods to test proteasome-associated USP14 activity for substrate degradation and deubiquitylation. © 2019 Elsevier Inc.
URI
http://hdl.handle.net/20.500.11750/9781
DOI
10.1016/bs.mie.2018.12.028
Publisher
Academic Press
Related Researcher
  • Author Lee, Byung-Hoon Lab of Protein Homeostasis and Drug Discovery
  • Research Interests Ubiquitin-proteasome system; Protein homeostasis; Small-molecule chemical screening and drug discovery in human disease
Files:
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Collection:
Department of New BiologyLab of Protein Homeostasis and Drug Discovery1. Journal Articles


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