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.     

Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates

Protein quality control (PQC) degradation systems protect the cell from the toxic accumulation of misfolded proteins. Because any protein can become misfolded, these systems must be able to distinguish abnormal proteins from normal ones, yet be capable of recognizing the wide variety of distinctly shaped misfolded proteins they are likely to encounter. How individual PQC degradation systems accomplish this remains an open question. Here we show that the yeast nuclear PQC ubiquitin ligase San1 directly recognizes its misfolded substrates via intrinsically disordered N- and C-terminal domains. These disordered domains are punctuated with small segments of order and high sequence conservation that serve as substrate-recognition sites San1 uses to target its different substrates. We propose that these substrate-recognition sites, interspersed among flexible, disordered regions, provide San1 an inherent plasticity which allows it to bind its many, differently shaped misfolded substrates.     

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.     

Selective destruction of abnormal proteins by ubiquitin-mediated protein quality control degradation

Misfolded proteins are continuously produced in the cell and present an escalating detriment to cellular physiology if not managed effectively. As such, all organisms have evolved mechanisms to address misfolded proteins. One primary way eukaryotic cells handle the complication of misfolded proteins is by destroying them through the ubiquitin-proteasome system. To do this, eukaryotes possess specialized ubiquitin-protein ligases that have the capacity to recognize misfolded proteins over normally folded proteins. The strategies used by these Protein Quality Control (PQC) ligases to target the wide variety of misfolded proteins in the cell will likely be different than those used by ubiquitin-protein ligases that function in regulated degradation to target normally folded proteins. In this review, we highlight what is known about how misfolded proteins are recognized by PQC ubiquitin-protein ligases.     

The Protein Quality Control Machinery Regulates Its Misassembled Proteasome Subunits

Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with various aggregation diseases. In eukaryotes, the ubiquitin proteasome system (UPS) plays a vital role in protein quality control (PQC), by selectively targeting misfolded proteins for degradation. While the assembly of the proteasome can be naturally impaired by many factors, the regulatory pathways that mediate the sorting and elimination of misassembled proteasomal subunits are poorly understood. Here, we reveal how the dysfunctional proteasome is controlled by the PQC machinery. We found that among the multilayered quality control mechanisms, UPS mediated degradation of its own misassembled subunits is the favored pathway. We also demonstrated that the Hsp42 chaperone mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments. Thus, we show that proteasome homeostasis is controlled through probing the level of proteasome assembly, and the interplay between UPS mediated degradation or their sorting into distinct cellular compartments.     

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.     

Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases

Motoneuron diseases, like spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS), are associated with proteins that because of gene mutation or peculiar structures, acquire aberrant (misfolded) conformations toxic to cells. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Here, using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrate that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome, rather than by autophagy and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner.     

The Diverse Functions of Small Heat Shock Proteins in the Proteostasis Network

The protein quality control (PQC) system maintains protein homeostasis by counteracting the accumulation of misfolded protein conformers. Substrate degradation and refolding activities executed by ATP-dependent proteases and chaperones constitute major strategies of the proteostasis network. Small heat shock proteins represent ATP-independent chaperones that bind to misfolded proteins, preventing their uncontrolled aggregation. sHsps share the conserved α-crystallin domain (ACD) and gain functional specificity through variable and largely disordered N- and C-terminal extensions (NTE, CTE). They form large, polydisperse oligomers through multiple, weak interactions between NTE/CTEs and ACD dimers. Sequence variations of sHsps and the large variability of sHsp oligomers enable sHsps to fulfill diverse tasks in the PQC network. sHsp oligomers represent inactive yet dynamic resting states that are rapidly deoligomerized and activated upon stress conditions, releasing substrate binding sites in NTEs and ACDs Bound substrates are usually isolated in large sHsp/substrate complexes. This sequestration activity of sHsps represents a third strategy of the proteostasis network. Substrate sequestration reduces the burden for other PQC components during immediate and persistent stress conditions. Sequestered substrates can be released and directed towards refolding pathways by ATP-dependent Hsp70/Hsp100 chaperones or sorted for degradation by autophagic pathways. sHsps can also maintain the dynamic state of phase-separated stress granules (SGs), which store mRNA and translation factors, by reducing the accumulation of misfolded proteins inside SGs and preventing unfolding of SG components. This ensures SG disassembly and regain of translational capacity during recovery periods.     

An overview of the role of molecular chaperones in protein homeostasis

Cells require a protein quality control (PQC) system to obtain a correct balance between folding and the degradation of incorrectly folded or misfolded proteins. This system maintains protein homeostasis and is essential for life. Key components of the PQC are molecular chaperones, which compose a ubiquitous class of proteins that mediate protein quality control by aiding in both the correct folding of proteins and the elimination of proteins that are misfolded due to cellular stress or mutation. Recent studies showed that protein homeostasis has an important role in nutrition and aging, increasing the relevance of the heat shock response to human health. This review summarizes our current knowledge of the molecular chaperone system and its role in protein homeostasis.     

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.     

Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases

Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER alpha1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N-glycans. Here we demonstrate that overexpression of Golgi alpha1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human alpha1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi alpha1,2-mannosidases can also contribute to NHK degradation.     

Deubiquitinases USP20/33 promote the biogenesis of tail-anchored membrane proteins

Numerous proteins that have hydrophobic transmembrane domains (TMDs) traverse the cytosol and posttranslationally insert into cellular membranes. It is unclear how these hydrophobic membrane proteins evade recognition by the cytosolic protein quality control (PQC), which typically recognizes exposed hydrophobicity in misfolded proteins and marks them for proteasomal degradation by adding ubiquitin chains. Here, we find that tail-anchored (TA) proteins, a vital class of membrane proteins, are recognized by cytosolic PQC and are ubiquitinated as soon as they are synthesized in cells. Surprisingly, the ubiquitinated TA proteins are not routed for proteasomal degradation but instead are handed over to the targeting factor, TRC40, and delivered to the ER for insertion. The ER-associated deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins after their insertion into the ER. Thus, our data suggest that deubiquitinases rescue posttranslationally targeted membrane proteins that are inappropriately ubiquitinated by PQC in the cytosol.     

The Type II Hsp40 Sis1 Cooperates with Hsp70 and the E3 Ligase Ubr1 to Promote Degradation of Terminally Misfolded Cytosolic Protein

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.     

Amino-terminal arginylation as a degradation signal for selective autophagy

The ubiquitin-proteasome system and the autophagy lysosome system are the two major protein degradation machineries in eukaryotic cells. These two systems coordinate the removal of unwanted intracellular materials, but the mechanism by which they achieve this synchronization is largely unknown. The ubiquitination of substrates serves as a universal degradation signal for both systems. Our study revealed that the amino-terminal Arg, a canonical N-degron in the ubiquitin-proteasome system, also acts as a degradation signal in autophagy. We showed that many ER residents, such as BiP, contain evolutionally conserved arginylation permissive pro-N-degrons, and that certain inducers like dsDNA or proteasome inhibitors cause their translocation into the cytoplasm where they bind misfolded proteins and undergo amino-terminal arginylation by arginyl transferase 1 (ATE1). The amino-terminal Arg of BiP binds p62, which triggers p62 oligomerization and enhances p62-LC3 interaction, thereby stimulating autophagic delivery and degradation of misfolded proteins, promoting cell survival. This study reveals a novel ubiquitin-independent mechanism for the selective autophagy pathway, and provides an insight into how these two major protein degradation pathways communicate in cells to dispose the unwanted proteins. [BMB Reports 2015; 48(9): 487-488]     

Hul5 ubiquitin ligase

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.     

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.     

N‐glycan processing selects ERAD‐resistant misfolded proteins for ER‐to‐lysosome‐associated degradation

Efficient degradation of by‐products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER‐associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER‐phagy receptors for ER‐to‐lysosome‐associated degradation (ERLAD). Demannosylation of N‐linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro‐collagen that cycles of de‐/re‐glucosylation of selected N‐glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD‐resistant misfolded proteins for FAM134B‐driven lysosomal delivery. In summary, we show that mannose and glucose processing of N‐glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.     

Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase （IKK）

Autophagic and proteasomal proteolysis are two major pathways for degradation of cellular constituents. Current models suggest that autophagy is responsible for the nonselective bulk degradation of long-lived proteins and organelles while the proteasome specifically degrades short-lived proteins including misfolded proteins caused by the absence of Hsp90 function. Here, we show that the IκB kinase （IKK）, an essential activator of NF-κB, is selectively degraded by autophagy when Hsp90 is inhibited by geldanamycin （GA）, a specific Hsp90 inhibitor showing highly effective anti-tumor activity. We find that in this case inactivation of ubiquitination or proteasome fails to block IKK degradation. However, inhibition of autophagy by an autophagy inhibitor or knockout of Atg5, a key component of the autophagy pathway, significantly rescues IKK from GA-induced degradation. These findings provide the first evidence that an Hsp90 client may be degraded by a mechanism different from the proteasome pathway and establish a molecular link among Hsp90, NF-κB and autophagy     

Type 2 transglutaminase is involved in the autophagy-dependent clearance of ubiquitinated proteins

Eukaryotic cells are equipped with an efficient quality control system to selectively eliminate misfolded and damaged proteins, and organelles. Abnormal polypeptides that escape from proteasome-dependent degradation and aggregate in the cytosol can be transported via microtubules to inclusion bodies called 'aggresomes', where misfolded proteins are confined and degraded by autophagy. Here, we show that Type 2 transglutaminase (TG2) knockout mice display impaired autophagy and accumulate ubiquitinated protein aggregates upon starvation. Furthermore, p62-dependent peroxisome degradation is also impaired in the absence of TG2. We also demonstrate that, under cellular stressful conditions, TG2 physically interacts with p62 and they are localized in cytosolic protein aggregates, which are then recruited into autophagosomes, where TG2 is degraded. Interestingly, the enzyme's crosslinking activity is activated during autophagy and its inhibition leads to the accumulation of ubiquitinated proteins. Taken together, these data indicate that the TG2 transamidating activity has an important role in the assembly of protein aggregates, as well as in the clearance of damaged organelles by macroautophagy.