System-wide Analysis Reveals Intrinsically Disordered Proteins Are Prone to Ubiquitylation after Misfolding Stress

Damaged and misfolded proteins that are no longer functional in the cell need to be eliminated. Failure to do so might lead to their accumulation and aggregation, a hallmark of many neurodegenerative diseases. Protein quality control pathways play a major role in the degradation of these proteins, which is mediated mainly by the ubiquitin proteasome system. Despite significant focus on identifying ubiquitin ligases involved in these pathways, along with their substrates, a systems-level understanding of these pathways has been lacking. For instance, as misfolded proteins are rapidly ubiquitylated, unconjugated ubiquitin is rapidly depleted from the cell upon misfolding stress; yet it is unknown whether certain targets compete more efficiently to be ubiquitylated. Using a system-wide approach, we applied statistical and computational methods to identify characteristics enriched among proteins that are further ubiquitylated after heat shock. We discovered that distinct populations of structured and, surprisingly, intrinsically disordered proteins are prone to ubiquitylation. Proteomic analysis revealed that abundant and highly structured proteins constitute the bulk of proteins in the low-solubility fraction after heat shock, but only a portion is ubiquitylated. In contrast, ubiquitylated, intrinsically disordered proteins are enriched in the low-solubility fraction after heat shock. These proteins have a very low abundance in the cell, are rarely encoded by essential genes, and are enriched in binding motifs. In additional experiments, we confirmed that several of the identified intrinsically disordered proteins were ubiquitylated after heat shock and demonstrated for two of them that their disordered regions are important for ubiquitylation after heat shock. We propose that intrinsically disordered regions may be recognized by the protein quality control machinery and thereby facilitate the ubiquitylation of proteins after heat shock. Damaged and misfolded proteins that are no longer functional in the cell need to be eliminated. Failure to do so might lead to their accumulation and aggregation, a hallmark of many neurodegenerative diseases. Protein quality control pathways play a major role in the degradation of these proteins, which is mediated mainly by the ubiquitin proteasome system. Despite significant focus on identifying ubiquitin ligases involved in these pathways, along with their substrates, a systems-level understanding of these pathways has been lacking. For instance, as misfolded proteins are rapidly ubiquitylated, unconjugated ubiquitin is rapidly depleted from the cell upon misfolding stress; yet it is unknown whether certain targets compete more efficiently to be ubiquitylated. Using a system-wide approach, we applied statistical and computational methods to identify characteristics enriched among proteins that are further ubiquitylated after heat shock. We discovered that distinct populations of structured and, surprisingly, intrinsically disordered proteins are prone to ubiquitylation. Proteomic analysis revealed that abundant and highly structured proteins constitute the bulk of proteins in the low-solubility fraction after heat shock, but only a portion is ubiquitylated. In contrast, ubiquitylated, intrinsically disordered proteins are enriched in the low-solubility fraction after heat shock. These proteins have a very low abundance in the cell, are rarely encoded by essential genes, and are enriched in binding motifs. In additional experiments, we confirmed that several of the identified intrinsically disordered proteins were ubiquitylated after heat shock and demonstrated for two of them that their disordered regions are important for ubiquitylation after heat shock. We propose that intrinsically disordered regions may be recognized by the protein quality control machinery and thereby facilitate the ubiquitylation of proteins after heat shock. Cells face the constant threat of protein misfolding and aggregation, and thus protein quality control pathways are important in selectively targeting damaged and misfolded proteins for degradation (1Balch W.E. Morimoto R.I. Dillin A. Kelly J.W. Adapting proteostasis for disease intervention.Science. 2008; 319: 916-919Crossref PubMed Scopus (1747) Google Scholar, 2Hartl F.U. Bracher A. Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis.Nature. 2011; 475: 324-332Crossref PubMed Scopus (2189) Google Scholar). The ubiquitin proteasome system serves as a major mediator of this pathway by conjugating the small protein ubiquitin onto substrates through the E1-E2-E3 (ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase, respectively) cascade for their recognition and degradation by the proteasome (3Pickart C.M. Eddins M.J. Ubiquitin: structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1021) Google Scholar, 4Finley D. Recognition and processing of ubiquitin-protein conjugates by the proteasome.Ann. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1275) Google Scholar). It is known that the activity of the ubiquitin-proteasome system is associated with many neurodegenerative diseases. For instance, ubiquitin is found enriched in protein inclusions associated with these diseases (5Berke S.J. Paulson H.L. Protein aggregation and the ubiquitin proteasome pathway: gaining the UPPer hand on neurodegeneration.Curr. Opin. Genet. Dev. 2003; 13: 253-261Crossref PubMed Scopus (152) Google Scholar). Furthermore, proteasome activity has been shown to decrease with age in a large variety of organisms (6Carrard G. Bulteau A.-L. Petropoulos I. Friguet B. Impairment of proteasome structure and function in aging.Int. J. Biochem. Cell Biol. 2002; 34: 1461-1474Crossref PubMed Scopus (244) Google Scholar), leading to increased proteotoxicity in the cell. Because of the importance of maintaining protein homeostasis, numerous ubiquitin ligases in different cellular compartments function in protein quality control pathways to target misfolded or damaged proteins for degradation via the proteasome. For instance, the conserved Hrd1 ubiquitin ligase is involved in the endoplasmic-reticulum-associated degradation pathway that targets endoplasmic reticulum proteins for retro-translocation to the cytoplasm and proteasome degradation (7Vembar S.S. Brodsky J.L. One step at a time: endoplasmic reticulum-associated degradation.Nat. Rev. Mol. Cell Biol. 2008; 9: 944-957Crossref PubMed Scopus (1019) Google Scholar). A major question is what features are recognized by ubiquitin ligases that allow them to selectively target terminally misfolded proteins for degradation, given that the folding rates and physicochemical properties vary largely from protein to protein. Several E3 ubiquitin ligases involved in cytosolic protein quality control target their substrates via their interactions with chaperone proteins. For instance, the CHIP ubiquitin ligase can directly bind to Hsp70 and Hsp90 proteins (8Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins.Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar), which may hand over client proteins that are not successfully folded. Understanding which features are recognized by these degradation quality-control pathways might help us understand how certain misfolded proteins evade this system, leading to their accumulation and aggregation in the cell. Many studies investigating degradation protein quality control have employed model substrates (e.g. mutated proteins that misfold) to reveal which components are involved in a given quality control machinery. However, these approaches do not typically reveal the whole spectrum of substrates for these pathways. Thus, alternative system-wide approaches are also needed to provide a bigger picture. Heat shock (HS) 1The abbreviations used are:HSHeat shockFDRfalse discovery rateGlyGlytwo remnant glycine residues of ubiquitinHSheat shockIMACimmobilized metal affinity chromatography. 1The abbreviations used are:HSHeat shockFDRfalse discovery rateGlyGlytwo remnant glycine residues of ubiquitinHSheat shockIMACimmobilized metal affinity chromatography. induces general misfolding at the proteome level by increasing thermal energy and was shown to cause an increase in ubiquitylation levels in the cell over 25 years ago (9Hough R. Pratt G. Rechsteiner M. Ubiquitin-lysozyme conjugates.J. Biol. Chem. 1986; 261: 2400-2408Abstract Full Text PDF PubMed Google Scholar, 10Parag H.A. Raboy B. Kulka R.G. Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.EMBO J. 1987; 6: 55-61Crossref PubMed Scopus (171) Google Scholar). However, the exact mechanism and pathways that target misfolded proteins have remained uncharacterized for a long time. We recently showed that the Hul5 ubiquitin ligase plays a major role in this heat stress response that mainly affects cytosolic proteins (11Fang N.N. Ng A.H.M. Measday V. Hul5 ubiquitin ligase plays a major role in the ubiquitylation and of cytosolic misfolded proteins.Nat. Cell Biol. 2011; 13: PubMed Scopus Google Scholar). of Hul5 the ubiquitylation in the cytoplasm of several misfolded targets after as as low-solubility proteins in E3 ubiquitin ligases are involved in this pathway N.N. Hul5 ubiquitin to 6: PubMed Scopus Google Scholar). as ubiquitin only of the unconjugated ubiquitin is rapidly depleted stress Rechsteiner M. of in protein degradation in to Cell Biol. 1987; PubMed Scopus Google Scholar, J. A ubiquitin activity to Cell Biol. PubMed Scopus Google Scholar). the of this how the cell triage ubiquitin among an of misfolded In to systems-level we to identify characteristics enriched among proteins ubiquitylated after a of statistical and computational and we additional and to We discovered an of intrinsically disordered proteins for ubiquitylation after misfolding Heat shock discovery two remnant glycine residues of ubiquitin heat shock metal affinity chromatography. Heat shock discovery two remnant glycine residues of ubiquitin heat shock metal affinity chromatography. protein were from the C. R. G. S.S. J. M. the of Scopus Google Scholar). and were the R. R. a for analysis and of and Scholar). were a on and and exact on were used for statistical but were for to is an in the protein were with L.J. and functional analysis of in proteins from the of Mol. Biol. 2004; PubMed Scopus Google the structure was B. P. M. C. 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M.J. of ubiquitylated proteins proteasome substrates and the by the 6: Full Text Full Text PDF PubMed Scopus Google or in which the ubiquitin was in or an were in or For the by their were in a a by a and at the and For and were in a and and to the to were at 25 and at their was at for to in a of and metal affinity was as (11Fang N.N. Ng A.H.M. Measday V. Hul5 ubiquitin ligase plays a major role in the ubiquitylation and of cytosolic misfolded proteins.Nat. Cell Biol. 2011; 13: PubMed Scopus Google Scholar). of the are in the were with and and were used for analysis in a were a to an were with a with a at from to and to the in were from for the as were were to were the the with protein and the or for the were were by proteins with at two with an of at discovery of on analysis was We the on the in the control so that of the proteins were enriched in the control M. 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