title: Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction.
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.
authors.affiliation: From the ‡Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Gendron
authors.affiliation: From the ‡Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California.
authors.forename: Kristofor
authors.initials: K
authors.lastname: Webb
authors.affiliation: From the ‡Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California.
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.affiliation: From the ‡Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California.
authors.forename: Lisa
authors.initials: L
authors.lastname: Rising
authors.affiliation: From the ‡Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California.
authors.forename: Nathan
authors.initials: N
authors.lastname: Zuzow
authors.affiliation: From the ‡Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California e1bennett@ucsd.edu.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Bennett
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors; D002853:Chromatography, Liquid; D045325:HCT116 Cells; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D060066:Protein Interaction Maps; D020543:Proteome / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014452:Ubiquitins / Q000378:metabolism*; D000074405:Valosin Containing Protein
chemical_list: D018797:Cell Cycle Proteins; D061988:Proteasome Inhibitors; D020543:Proteome; D014452:Ubiquitins; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein