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.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 (1, 2). 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 (3, 4). 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 (5). Furthermore, proteasome activity has been shown to decrease with age in a large variety of organisms (6), 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 (7). 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 (8), 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)¹ 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 (9, 10). 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 (11). Absence of Hul5 averts the ubiquitylation in the cytoplasm of several misfolded targets after HS, as well as low-solubility proteins in unstressed cells. Other E3 ubiquitin ligases are likely involved in this pathway (12). Interestingly, as ubiquitin constitutes about only 1% of the proteome, free unconjugated ubiquitin is rapidly depleted under stress conditions (13, 14). Given the limited amount of this protein, how does the cell triage ubiquitin among an excess of misfolded proteins? In order to gain systems-level insight, we sought to identify characteristics enriched among proteins ubiquitylated after HS using a combination of statistical and computational analysis, and we conducted additional proteomics and biochemical experiments to support our hypotheses. We discovered an unexpected susceptibility of intrinsically disordered proteins for ubiquitylation after misfolding stress.