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.
Highlights
Maintenance of the collective proteome is achieved through the careful balance of protein synthesis and degradation [1, 2]
All unstable reporter proteins accumulated upon treatment with the highest concentration of epoxomicin compared with loading controls (Fig. 1A)
We quantitatively monitored changes in abundance of ub-modified peptides upon proteasome inhibition or activation of the unfolded protein response (UPR) because we have previously demonstrated that a subset of regulatory 40S ribosomal ubiquitylation (RRub) events is stimulated by cell stressors that activate the UPR [25]
Summary
Reagents—Epoxomicin (EMD Millipore, Billerica, MA) and MG132 (Enzo, Farmingdale, NY) were resuspended in DMSO and used at 1 M or 10 M, respectively unless otherwise noted. Resuspended peptides were incubated with ␣-diGly antibody (Cell Signaling Technologies) preconjugated to Protein-A (Thermo) beads at 4 °C for 2 h with rotating. Each collected fraction was mixed 1:1 with urea lysis buffer (8 M urea, 75 mM NaCl, 50 mM Tris pH 8.2, Roche complete protease inhibitor, 1 mM NaF, 1 mM -glycerophosphate, 1 mM Na orthovanadate, 1 mM PMSF, 5 mM N-ethylmaleimide (NEM)) prior to Lys-C and trypsin digestion, peptide clean-up, diGLY immuno-affinity enrichment and subsequent LCMS/MS analysis. SILAC-based quantitative data for ub-modified peptide enrichment experiments with subcellular fractionation was performed with three technical replicates for each sample. Each experimental data set with SILAC quantification was done with a control untreated sample in both the heavy and light population to determine the population-level standard deviation. Peptides with a SILAC ratio outside this standard deviation for all treatment samples were considered as altered in abundance
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
