Using the Ubiquitin-modified Proteome to Monitor Protein Homeostasis Function

University of California, San Diego, United States.
Molecular & Cellular Proteomics (Impact Factor: 6.56). 05/2013; 12(12). DOI: 10.1074/mcp.R113.029744
Source: PubMed


The ubiquitin system is essential for maintenance of proper protein homeostasis function across eukaryotic species. While the general enzymatic architecture for adding and removing ubiquitin from substrates is well defined, methods for the comprehensive investigation of cellular ubiquitylation targets have just started to emerge. Recent advances in ubiquitin modified peptide enrichment have greatly increased the number of identified endogenous ubiquitylation targets as well as the sites of ubiquitin attachment within these substrates. Herein we evaluate current strategies using mass spectrometry based proteomics to characterize ubiquitin and ubiquitin like modifications. Using existing data we describe the characteristics of the ubiquitin modified proteome and discuss strategies for biological interpretation of existing and future ubiquitin-based proteomic studies.

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    • "However, both treatment with epoxomicin and the ER stressors resulted in CHOP induction (Figure 1A). To interrogate site-specific alterations to individual ub-modified proteins, we utilized a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic approach coupled with immuno-affinity enrichment of diGlycine (diGly)modified peptides (Carrano and Bennett, 2013; Kim et al., 2011). Trypsin-catalyzed cleavage of ubiquitylated proteins generates a proteotypic peptide containing the diGly remnant derived from the carboxy-terminus of ub linked to lysine residues of substrate peptides (Bustos et al., 2012). "
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    ABSTRACT: Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control. Copyright © 2015 Elsevier Inc. All rights reserved.
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    ABSTRACT: Here, we apply the COmbined FRActional DIagonal Chromatography (COFRADIC) technology to enrich for ubiquitinated peptides and identify sites of ubiquitination by mass spectrometry. Our technology bypasses the need to overexpress tagged variants of ubiquitin and the use of sequence-biased antibodies recognizing ubiquitin remnants. In brief, all protein primary amino groups are blocked by chemical acetylation after which ubiquitin chains are proteolytically and specifically removed by the catalytic core domain of the USP2 deubiquitinase (USP2cc). As USP2cc cleaves the isopeptidyl bond between the ubiquitin C-terminus and the ɛ-amino group of the ubiquitinated lysine, this enzyme re-introduces primary ɛ-amino groups in proteins. These amino groups are then chemically modified with a handle that allows specific isolation of ubiquitinated peptides during subsequent COFRADIC chromatographic runs. This method led to the identification of over 7,500 endogenous ubiquitination sites in more than 3,300 different proteins in a native human Jurkat cell lysate.
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    ABSTRACT: Maintenance of protein homeostasis is essential for cellular survival. Central to this regulation are mechanisms of protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome system. One well-studied protein quality control pathway requires ER resident, multi-subunit E3 ubiquitin ligases that function in ER-associated degradation (ERAD). Using fission yeast, our lab identified the Golgi Dsc E3 ligase as required for proteolytic activation of fungal sterol regulatory element-binding protein (SREBP) transcription factors. The Dsc E3 ligase contains 5 integral membrane subunits and structurally resembles ERAD E3 ligases. S. cerevisiae codes for homologs of Dsc E3 ligase subunits, including the Dsc1 E3 ligase homolog Tul1 that functions in Golgi protein quality control. Interestingly, S. cerevisiae lacks SREBP homologs, indicating that novel Tul1 E3 ligase substrates exist. Here, we show that the S. cerevisiae Tul1 E3 ligase consists of Tul1, Dsc2, Dsc3, and Ubx3 and define Tul1 complex architecture. Tul1 E3 ligase function required each subunit as judged by vacuolar sorting of the artificial substrate Pep12D. Genetic studies demonstrated that Tul1 E3 ligase is required in cells lacking the multivesicular body pathway and under conditions of ubiquitin depletion. To identify candidate substrates, we performed quantitative diGly proteomics using SILAC to survey ubiquitylation in wild-type and tul1∆ cells. We identified 3,116 non-redundant ubiquitylation sites, including 10 sites in candidate substrates. Quantitative proteomics found 4.5% of quantified proteins (53/1172) to be differentially expressed in tul1∆ cells. Correcting the diGly dataset for these differences increased the number of Tul1-dependent ubiquitylation sites. Together, our data demonstrate that the Tul1 E3 ligase functions in protein homeostasis under non-stress conditions and support a role in protein quality control. This quantitative diGly proteomics methodology will serve as a robust platform to screen for stress conditions that require Tul1 E3 ligase activity.
    Preview · Article · Jul 2014 · Molecular & Cellular Proteomics
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