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Mol Cell Proteomics. 2016 Aug;15(8):2576-93. doi: 10.1074/mcp.M116.058420. Epub 2016 May 16.

Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction.

Author information

1
From the ‡Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California.
2
From the ‡Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California e1bennett@ucsd.edu.

Abstract

Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations.

PMID:
27185884
PMCID:
PMC4974337
DOI:
10.1074/mcp.M116.058420
[Indexed for MEDLINE]
Free PMC Article

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