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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    • "(v) Redox proteomics focuses on the identification and quantification of redox-based proteome changes leading to a better understanding of protein oxidative modifications such as carbonylation , tyrosine nitration, and glutathionylation under pathological conditions (Butterfield & Dalle-Donne, 2012; Ghezzi & Bonetto, 2003; Rinalducci, Murgiano, & Zolla, 2008). (vi) Diglycine (DiGly) capture proteomics studies protein degradation facilitated by the ubiquitin–proteasome system (UPS) (Kim et al., 2011). In the UPS pathway, ubiquitin is activated and transferred to substrates via an E1–E2–E3 cascade (Ye & Rape, 2009). "
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    ABSTRACT: Neurodegenerative diseases are a major health concern worldwide. Diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, as well as many other diseases affecting the neuromuscular system, are a leading cause of disability in the aging population. Presymptomatic diagnosis of neurodegenerative disorders is challenging due to the lack of robust biomarkers. Likewise, the design of effective intervention strategies is limited because most neurodegenerative disorders are heterogeneous in nature. Reliable noninvasive biomarkers are therefore urgently needed to allow presymptomatic and accurate diagnosis, to track disease progression, to evaluate the effectiveness of new treatment regimens, and to ultimately design new therapeutic intervention strategies. Recent biological and technological advances within the field of proteomic promises to provide insight into global proteome changes in neurodegeneration, thus allowing increased understanding of molecular pathways leading to neuronal cell death and the identification of biomarkers. The combination of gel-based techniques and mass spectrometry permits large-scale identification of peptide sequences in biological samples as well as the characterization of posttranslational protein modifications. The application of comparative high-throughput proteomic analyses in animal models and human tissues will aid in the identification of both diagnostic and prognostic biomarkers and will provide a platform for a future personalized medicine approach in neurodegeneration. © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Aug 2015 · International Review of Neurobiology
    • "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.
    No preview · Article · Jun 2015 · Molecular cell
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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.
    Full-text · Article · Apr 2015 · Cell Reports
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