Nancy N. Fang

Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins

Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with numerous aggregation diseases. Several protein quality control mechanisms degrade non-native proteins by the ubiquitin–proteasome system. Here, we use quantitative mass spectrometry to demonstrate that heat-shock triggers a large increase in the level of ubiquitylation associated with misfolding of cytosolic proteins. We discover that the Hul5 HECT ubiquitin ligase participates in this heat-shock stress response. Hul5 is required to maintain cell fitness after heat-shock and to degrade short-lived misfolded proteins. In addition, localization of Hul5 in the cytoplasm is important for its quality control function. We identify potential Hul5 substrates in heat-shock and physiological conditions to reveal that Hul5 is required for ubiquitylation of low-solubility cytosolic proteins including the Pin3 prion-like protein. These findings indicate that Hul5 is involved in a cytosolic protein quality control pathway that targets misfolded proteins for degradation.

Rsp5/Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress

The heat-shock response is a complex cellular program that induces major changes in protein translation, folding and degradation to alleviate toxicity caused by protein misfolding. Although heat shock has been widely used to study proteostasis, it remained unclear how misfolded proteins are targeted for proteolysis in these conditions. We found that Rsp5 and its mammalian homologue Nedd4 are important E3 ligases responsible for the increased ubiquitylation induced by heat stress. We determined that Rsp5 ubiquitylates mainly cytosolic misfolded proteins upon heat shock for proteasome degradation. We found that ubiquitylation of heat-induced substrates requires the Hsp40 co-chaperone Ydj1 that is further associated with Rsp5 upon heat shock. In addition, ubiquitylation is also promoted by PY Rsp5-binding motifs found primarily in the structured regions of stress-induced substrates, which can act as heat-induced degrons. Our results support a bipartite recognition mechanism combining direct and chaperone-dependent ubiquitylation of misfolded cytosolic proteins by Rsp5.

Polarization of the Endoplasmic Reticulum by ER-Septin Tethering

Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.

The Yeast Ubr1 Ubiquitin Ligase Participates in a Prominent Pathway That Targets Cytosolic Thermosensitive Mutants for Degradation

Mutations causing protein misfolding and proteolysis are associated with many genetic diseases. The degradation of these aberrant proteins typically is mediated by protein-quality control pathways that recognize misfolded domains. Several E3 ubiquitin ligases have been shown to target cytosolic misfolded proteins to the proteasome. In this study, we characterized a panel of more than 20 cytosolic thermosensitive mutants from six essential genes in Saccharomyces cerevisiae. These wild-type proteins are stable at restrictive temperature. In contrast, we found that a large portion of the mutants is degraded at nonpermissive temperature in a proteasome-dependent manner. Approximately one-third of the assessed unstable mutants are targeted by the Ubr1 ubiquitin ligase. In two cases, efficient degradation of the thermosensitive mutants is abrogated in the absence of Ubr1 alone, whereas in a third case it is reliant on the dual deletion of Ubr1 and the nuclear E3 ligase San1. We found that the impairment of the degradation of these quality control substrates at the restrictive temperature is associated with the suppression of thermosensitive phenotype. This study confirms that Ubr1 plays an important role in the degradation of cytosolic misfolded proteins and indicates that degradation mediated by protein quality control is a major cause for the conditional lethality of mutated essential genes.

Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions.

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.

Hul5 ubiquitin ligase: Good riddance to bad proteins

Failure to eliminate abnormal proteins in the cell is associated with numerous aggregation diseases. Misfolded proteins are normally detected by protein quality control and either refolded or eliminated. The ubiquitin-proteasome system is a major pathway that degrades these unwanted proteins. Ubiquitin ligases are central to these degradation pathways as they recognize aberrant proteins and covalently attach a polyubiquitin chain to target them to the proteasome. We discovered that the Hul5 ubiquitin ligase is a major player in a novel protein quality control pathway that targets cytosolic misfolded proteins. Hul5 is required for the maintenance of cell fitness and the increased ubiquitination of low solubility proteins after heat-shock in yeast cells. We identified several low-solubility substrates of Hul5, including the prion-like protein Pin3. It is now apparent that in the cytoplasm, misfolded proteins can be targeted by multiple degradation pathways. In this review, we discuss how the Hul5 protein quality control pathway may specifically target low solubility cytosolic proteins in the cell.

A Role for the Budding Yeast Separase, Esp1, in Ty1 Element Retrotransposition

Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast esp1-1 separase mutation. We identified 161 genes that when mutated aggravate esp1-1 growth and 44 genes that upon increased dosage are detrimental to esp1-1 viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the esp1-1 genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the SUF16 tRNA gene are both reduced in an esp1-1 strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition – one to remove cohesin and the second to target Ty1-IN to chromatin.

Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA splicing factors during adipogenesis

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope–tagged 14-3-3ζ molecule. After inducing adipogenesis, TAP–14-3-3ζ complexes were purified, followed by MS analysis to determine the 14-3-3ζ interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis (i.e. Ptrf/Cavin1) within the 14-3-3ζ interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3ζ interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of Pparg and the adipogenesis-associated gene Lpin1. In summary, we have identified novel adipogenic factors within the 14-3-3ζ interactome. Further characterization of additional proteins within the 14-3-3ζ interactome may help identify novel targets to block obesity-associated expansion of adipose tissues.

Systems-Wide Analysis of Protein Ubiquitylation: We Finally Have the Tiger by the Tail

Ubiquitylation is a posttranslational modification in which ubiquitin, a conserved polypeptide, is conjugated to targeted proteins. The human genome encodes for over a thousand proteins that can either mediate ubiquitylation, remove the modification, or bind to modified proteins. Hence, the ubiquitin system is expected to affect a large portion of the proteome. Since its discovery, our understanding of the ubiquitin system has come a long way. Great efforts made by several prominent members of the scientific community have paid off, with leaps and bounds being made in areas such as the discovery of ubiquitylated proteins and ubiquitylation sites. Boosting the success of classical proteomic approaches, the recent introduction of a new method (by which ubiquitylated peptides are captured by antibodies) enabled the identification of a large number of ubiquitylation sites. In this chapter, we will review the different proteomic strategies that have been established in order to uncover which proteins are ubiquitylated in the cell, and then further discuss the novel biological insights revealed by these systems-wide studies.