Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome

Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
Molecular cell (Impact Factor: 14.02). 09/2011; 44(2):325-40. DOI: 10.1016/j.molcel.2011.08.025
Source: PubMed


Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ~19,000 diGly-modified lysine residues within ~5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.

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Available from: Edward L Huttlin, Sep 30, 2015
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    • "In fact , the abundance of more than 30% of all quantified diGly - modified peptides is reduced or unchanged upon treat - ment with a proteasome inhibitor ( Kim et al . , 2011 ) . This observa - tion suggests that regulatory ubiquitylation is pervasive and plays a substantial role in the post - translational control of protein activity . Our results suggest that a subset of 40S ubiquitylation events is regulatory in nature . Several observations support this claim . First , the abundance of site - specific ub"
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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.
    Molecular cell 06/2015; 59(1). DOI:10.1016/j.molcel.2015.04.026 · 14.02 Impact Factor
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    • "spatial proximity to the electrostatic bridge (Kim et al., 2011), suggesting that, under certain circumstances, depolymerization of the helix may also be induced. Furthermore, posttranslational modifications of residues that are located in the PB1 helix interface (Kim et al., 2011), including PKA-mediated phosphorylation of S24 (Christian et al., 2014), and modifications that affect ubiquitin binding affinity such as phosphorylation of S403 (Matsumoto et al., 2011) will also affect the polymeric assembly state. Our 2D class averages of full-length p62 suggest that, while the PB1 domain forms the stable and folded core of the helical scaffold, the C-terminal part of the protein forms a solvent-accessible stretch. "
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    ABSTRACT: The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 04/2015; 11(5). DOI:10.1016/j.celrep.2015.03.062 · 8.36 Impact Factor
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    • "One possibility is that HUWE1 assembles K63-linked ubiquitin chains on MYC that do not target the protein for degradation (Adhikary et al, 2005). Alternatively, lysines that are critical for degradation of MYC may be buried (e.g., by complex formation with MIZ1) and therefore inaccessible to HUWE1 in vivo (Kim et al, 2011). The effects of HUWE1 depletion or deletion on the function of MYC proteins have been characterized in several biological contexts: Deletion of HUWE1 enhances N-MYC levels in embryonic stem cells cultured in the absence of leukemia inhibitory factor and expands neuronal cell population in the cerebellum (D'Arca et al, 2010; Zhao et al, 2009). "
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