Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70

The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.     

Identification of mammalian protein quality control factors by high-throughput cellular imaging

Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress.     

Protein quality control at the Golgi

The majority of the proteome in eukaryotic cells is targeted to organelles. To maintain protein homeostasis (proteostasis), distinct protein quality control (PQC) machineries operate on organelles, where they detect misfolded proteins, orphaned and mis-localized proteins and selectively target these proteins into different ubiquitin-dependent or -independent degradation pathways. Thereby, PQC prevents proteotoxic effects that would disrupt organelle integrity and cause cellular damage that leads to diseases. Here, we will discuss emerging mechanisms for PQC machineries at the Golgi apparatus, the central station for the sorting and the modification of proteins that traffic to the endo-lysosomal system, or along the secretory pathway to the PM and to the extracellular space. We will focus on Golgi PQC pathways that (1) retrieve misfolded and orphaned proteins from the Golgi back to the endoplasmic reticulum, (2) extract these proteins from Golgi membranes for proteasomal degradation, (3) or selectively target these proteins to lysosomes for degradation.     

Regulation of protein turnover by heat shock proteins

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin–proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.     

Exploring proteasome complexes by proteomic approaches

The ubiquitin proteasome system (UPS) represents a major pathway for intracellular protein degradation. Proteasome dependent protein quality control participates in cell cycle, immune response and apoptosis. Therefore, the UPS is in focus of therapeutic investigations and the development of pharmaceutical agents. Detailed analyses on proteasome structure and function are the foundation for drug development and clinical studies. Proteomic approaches contributed significantly to our current knowledge in proteasome research. In particular, 2-DE has been essential in facilitating the development of current models on molecular composition and assembly of proteasome complexes. Furthermore, developments in MS enabled identification of UPS proteins and their PTMs at high accuracy and high-throughput. First results on global characterization of the UPS are also available. Although the UPS has been intensively investigated within the last two decades, its functional significance and contribution to the regulation of cell and tissue phenotypes remain to be explored. This review recapitulates a variety of applied proteomic approaches in proteasome exploration, and presents an overview of current technologies and their potential in driving further investigations.     

Inhibition of retroviral Gag assembly by non-silencing miRNAs promotes autophagic viral degradation

We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-mediated Gag assembly at the plasma membrane. Consequently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demonstrate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endosomes/lysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autophagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus (MLV) assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by nonspecific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possibility that similar regulations can occur in other biological contexts.     

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome.     

Regulation of p97 in the ubiquitin-proteasome system by the UBX protein-family

The AAA protein p97 is a central component in the ubiquitin-proteasome system, in which it is thought to act as a molecular chaperone, guiding protein substrates to the 26S proteasome for degradation. This function is dependent on association with cofactors that are specific to the different biological pathways p97 participates in. The UBX-protein family (ubiquitin regulatory X) constitutes the largest known group of p97 cofactors. We propose that the regulation of p97 by UBX-proteins utilizes conserved structural features of this family. Firstly, they act as scaffolding subunits in p97-containing multiprotein complexes, by providing additional interaction motifs. Secondly, they provide regulation of multiprotein complex assembly and we suggest two possible models for p97 substrate recruitment in the UPS pathway. Lastly, they impose constraints on p97 and its interaction with substrates and further cofactors. These features allow the regulation, within the UPS, of the competitive interactions on p97, a regulation that is crucial to allow the diverse functionality of p97.     

Inhibition of the extracellular signal-regulated kinase signaling pathway is correlated with proteasome inhibitor suppression of coxsackievirus replication

The ubiquitin/proteasome system (UPS), a major intracellular protein degradation pathway, plays a critical role in coxsackieviral replication. To elucidate the mechanisms by which the UPS regulates viral replication, we studied the influence of proteasome inhibition on signaling through the extracellular signal-regulated kinase (ERK) pathway, a pathway which has been previously demonstrated to be necessary for coxsackieviral replication and contribute to virus-mediated pathogenesis. We found that proteasome inhibition reduced coxsackievirus-induced ERK phosphorylation in a dose-dependent manner, which is correlated with an induction of the mitogen-activated protein kinase phosphatase-1 (MKP-1). Blockade of MKP induction by short-interfering RNA attenuated the loss of ERK phosphorylation, and subsequently restored viral replication. Our results suggest that inhibition of the ERK signaling pathway contributes, as least in part, to proteasome inhibitor-mediated reduction of coxsackievirus replication, demonstrating a converging function of major intracellular signaling and protein degradation pathways in the regulation of viral replication.     

Exposed hydrophobicity is a key determinant of nuclear quality control degradation

Protein quality control (PQC) degradation protects the cell by preventing the toxic accumulation of misfolded proteins. In eukaryotes, PQC degradation is primarily achieved by ubiquitin ligases that attach ubiquitin to misfolded proteins for proteasome degradation. To function effectively, PQC ubiquitin ligases must distinguish misfolded proteins from their normal counterparts by recognizing an attribute of structural abnormality commonly shared among misfolded proteins. However, the nature of the structurally abnormal feature recognized by most PQC ubiquitin ligases is unknown. Here we demonstrate that the yeast nuclear PQC ubiquitin ligase San1 recognizes exposed hydrophobicity in its substrates. San1 recognition is triggered by exposure of as few as five contiguous hydrophobic residues, which defines the minimum window of hydrophobicity required for San1 targeting. We also find that the exposed hydrophobicity recognized by San1 can cause aggregation and cellular toxicity, underscoring the fundamental protective role for San1-mediated PQC degradation of misfolded nuclear proteins.     

Regulation of nicotinic receptor expression by the ubiquitin-proteasome system

Control of ligand-gated ion channel (LGIC) expression is essential for the formation, maintenance and plasticity of synapses. Treatment of mouse myotubes with proteasome inhibitors increased the number of surface nicotinic acetylcholine receptors (AChRs), indicating LGIC expression is regulated by the ubiquitin-proteasome system (UPS). Elevated surface expression resulted from increased AChR delivery to the plasma membrane and not from decreased turnover from the surface. The rise in AChR trafficking was the direct result of increased assembly of subunits in the endoplasmic reticulum (ER). Because proteasome inhibitors also blocked ER-associated degradation (ERAD) of unassembled AChR subunits, the data indicate that the additional AChRs were assembled from subunits normally targeted for ERAD. Our data show that AChR surface expression is regulated by the UPS through ERAD, whose activity determines oligomeric receptor assembly efficiency.     

Molecular mechanisms of spatial protein quality control

Evidence is now accumulating that damaged proteins are not randomly distributed but often concentrated in microscopically visible and functionally distinct inclusion bodies. How misfolded proteins are organized into these compartments, however, is still unknown. We have recently begun to investigate stress-inducible protein quality control (PQC) bodies in yeast cells. Surprisingly, we found that protein misfolding and aggregation were not sufficient to trigger body formation under mild heat stress conditions. Rather, compartment assembly also required the concerted action of molecular chaperones, protein-sorting factors and protein-sequestration factors, thus defining a minimal machinery for spatial PQC. Expression of this machinery was limited to times of acute stress through rapid changes in mRNA abundance and a proteasomal feedback mechanism. These findings demonstrate that yeast cells can control the amount of soluble misfolded proteins through regulated phase transitions in the cytoplasm, thus allowing them to rapidly adapt to changing environmental conditions.     

Carbon and nitrogen metabolism regulated by the ubiquitin-proteasome system

The ubiquitin-proteasome system (UPS) is a unique protein degradation mechanism conserved in the eukaryotic cell. In addition to the control of protein quality, UPS regulates diverse cellular signal transduction via the fine-tuning of target protein degradation. Protein ubiquitylation and subsequent degradation by the 26S proteasome are involved in almost all aspects of plant growth and development and response to biotic and abiotic stresses. Recent studies reveal that the UPS plays an essential role in adaptation to carbon and nitrogen availability in plants. Here we highlight ubiquitin ligase ATL31 and the homologue ATL6 target 14-3-3 proteins for ubiquitylation to be degraded, which control signaling for carbon and nitrogen metabolisms and C/N balance response. We also give an overview of the UPS function involved in carbon and nitrogen metabolisms.     

Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis

Ubiquitin functions as a signal for sorting cargo at multiple steps of the endocytic pathway and controls the activity of trans-acting components of the endocytic machinery (reviewed in refs 1, and 2). By contrast to proteasome degradation, which generally requires a polyubiquitin chain that is at least four subunits long, internalization and sorting of endocytic cargo at the late endosome are mediated by mono-ubiquitination. Here, we demonstrate that ubiquitin-interacting motifs (UIMs) found in epsins and Vps27p (ref. 9) from Saccharomyces cerevisiae are required for ubiquitin binding and protein transport. Epsin UIMs are important for the internalization of receptors into vesicles at the plasma membrane. Vps27p UIMs are necessary to sort biosynthetic and endocytic cargo into vesicles that bud into the lumen of a late endosomal compartment, the multivesicular body. We propose that mono-ubiquitin regulates internalization and endosomal sorting by interacting with modular ubiquitin-binding domains in core components of the protein transport machinery. UIM domains are found in a broad spectrum of proteins, consistent with the idea that mono-ubiquitin can function as a regulatory signal to control diverse biological activities.     

Molecular mechanisms of spatial protein quality control

Evidence is now accumulating that damaged proteins are not randomly distributed but often concentrated in microscopically visible and functionally distinct inclusion bodies. How misfolded proteins are organized into these compartments, however, is still unknown. We have recently begun to investigate stress-inducible protein quality control (PQC) bodies in yeast cells. Surprisingly, we found that protein misfolding and aggregation were not sufficient to trigger body formation under mild heat stress conditions. Rather, compartment assembly also required the concerted action of molecular chaperones, protein-sorting factors and protein-sequestration factors, thus defining a minimal machinery for spatial PQC. Expression of this machinery was limited to times of acute stress through rapid changes in mRNA abundance and a proteasomal feedback mechanism. These findings demonstrate that yeast cells can control the amount of soluble misfolded proteins through regulated phase transitions in the cytoplasm, thus allowing them to rapidly adapt to changing environmental conditions.     

Substrate Recognition in Nuclear Protein Quality Control Degradation Is Governed by Exposed Hydrophobicity That Correlates with Aggregation and Insolubility

Misfolded proteins present an escalating deleterious challenge to cells over the course of their lifetime. One mechanism the cell possesses to prevent misfolded protein accumulation is their destruction by protein quality control (PQC) degradation systems. In eukaryotes, PQC degradation typically proceeds via multiple ubiquitin-protein ligases that act throughout the cell to ubiquitinate misfolded proteins for proteasome degradation. What the exact feature of misfolding that each PQC ubiquitin-protein ligase recognizes in their substrates remains an open question. Our previous studies of the budding yeast nuclear ubiquitin-protein ligase San1 indicated that it recognizes exposed hydrophobicity within its substrates, with the threshold of hydrophobicity equivalent to that of 5 contiguous hydrophobic residues. Here, we uncover an additional parameter: the nature of the exposed hydrophobicity that confers San1-mediated degradation correlates with significant protein insolubility. San1 particularly targets exposed hydrophobicity that leads to insolubility and aggregation above a certain threshold. Our studies presented here provide additional insight into the details of misfolded nuclear protein recognition and demonstrate that there is selectivity for the type of exposed hydrophobicity.     

Stress-induced polyubiquitination of proteasomal ubiquitin receptors targets the proteolytic complex for autophagic degradation

Ubiquitin (Ub) is a small protein (8 kDa) found in all eukaryotic cells, which is conjugated covalently to numerous proteins, tagging them for recognition by a downstream effector. One of the best characterized functions of Ub is targeting proteins for either selective degradation by the proteasome, or for bulk degradation by the autophagy-lysosome system. The executing arm of the UPS is the 26S proteasome, a large multicatalytic complex. While much is known about the synthesis and assembly of the proteasome's subunits, the mechanism(s) underlying its removal has remained obscure, similar to that of many other components of the ubiquitin-proteasome system. Our recent study identified autophagy as the degrading mechanism for the mammalian proteasome, mostly under stress conditions. Amino acid starvation induces specific ubiquitination of certain 19S proteasomal subunits that is essential for its binding to SQSTM1/p62, the protein that shuttles the ubiquitinated proteasome to the autophagic machinery. SQSTM1 delivers ubiquitinated substrates for proteasomal degradation via interaction of its PB1 domain with the 19S proteasomal subunit PSMD4/Rpn10, in situations where the proteasome serves as a “predator." In contrast, we found that the UBA domain of SQSTM1 is essential for its interaction with the ubiquitinated proteasome and its delivery to the autophagosome, rendering the proteasome a “prey.”     

Disease-associated prion protein oligomers inhibit the 26S proteasome

The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic beta subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the beta subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative beta sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein.