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.     

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.     

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.     

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.     

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.     

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.     

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.     

Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.     

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.     

Nuclear protein quality control in yeast: The latest INQuiries

The nucleus is a highly organized organelle with an intricate substructure of chromatin, RNAs, and proteins. This environment represents a challenge for maintaining protein quality control, since non-native proteins may interact inappropriately with other macromolecules and thus interfere with their function. Maintaining a healthy nuclear proteome becomes imperative during times of stress, such as upon DNA damage, heat shock, or starvation, when the proteome must be remodeled to effect cell survival. This is accomplished with the help of nuclear-specific chaperones, degradation pathways, and specialized structures known as protein quality control (PQC) sites that sequester proteins to help rapidly remodel the nuclear proteome. In this review, we focus on the current knowledge of PQC sites in Saccharomyces cerevisiae, particularly on a specialized nuclear PQC site called the intranuclear quality control site, a poorly understood nuclear inclusion that coordinates dynamic proteome triage decisions in yeast.     

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.     

The metazoan protein disaggregase and amyloid depolymerase system

A baffling aspect of metazoan proteostasis is the lack of an Hsp104 ortholog that rapidly disaggregates and reactivates misfolded polypeptides trapped in stress induced disordered aggregates, preamyloid oligomers, or amyloid fibrils. By contrast, in bacteria, protozoa, chromista, fungi, and plants, Hsp104 orthologs are highly conserved and confer huge selective advantages in stress tolerance. Moreover, in fungi, the amyloid remodeling activity of Hsp104 has enabled deployment of prions for various beneficial modalities. Thus, a longstanding conundrum has remained unanswered: how do metazoan cells renature aggregated proteins or resolve amyloid fibrils without Hsp104? Here, we highlight recent advances that unveil the metazoan protein-disaggregase machinery, comprising Hsp110, Hsp70, and Hsp40, which synergize to dissolve disordered aggregates, but are unable to rapidly solubilize stable amyloid fibrils. However, Hsp110, Hsp70, and Hsp40 exploit the slow monomer exchange dynamics of amyloid, and can slowly depolymerize amyloid fibrils from their ends in a manner that is stimulated by small heat shock proteins. Upregulation of this system could have key therapeutic applications in various protein-misfolding disorders. Intriguingly, yeast Hsp104 can interface with metazoan Hsp110, Hsp70, and Hsp40 to rapidly eliminate disease associated amyloid. Thus, metazoan proteostasis is receptive to augmentation with exogenous disaggregases, which opens a number of therapeutic opportunities.     

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.     

Contribution of small heat shock proteins to muscle development and function

Investigations undertaken over the past years have led scientists to introduce the concept of protein quality control (PQC) systems, which are responsible for polypeptide processing. The PQC system monitors proteostasis and involves activity of different chaperones such as small heat shock proteins (sHSPs). These proteins act during normal conditions as housekeeping proteins regulating cellular processes, and during stress conditions. They also mediate the removal of toxic misfolded polypeptides and thereby prevent development of pathogenic states. It is postulated that sHSPs are involved in muscle development. They could act via modulation of myogenesis or by maintenance of the structural integrity of signaling complexes. Moreover, mutations in genes coding for sHSPs lead to pathological states affecting muscular tissue functioning.     

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.     

Metazoan Hsp70 machines use Hsp110 to power protein disaggregation

Accumulation of aggregation‐prone misfolded proteins disrupts normal cellular function and promotes ageing and disease. Bacteria, fungi and plants counteract this by solubilizing and refolding aggregated proteins via a powerful cytosolic ATP‐dependent bichaperone system, comprising the AAA+ disaggregase Hsp100 and the Hsp70‐Hsp40 system. Metazoa, however, lack Hsp100 disaggregases. We show that instead the Hsp110 member of the Hsp70 superfamily remodels the human Hsp70‐Hsp40 system to efficiently disaggregate and refold aggregates of heat and chemically denatured proteins in vitro and in cell extracts. This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP‐driven chaperoning is not required. Knock‐down of nematode Caenorhabditis elegans Hsp110, but not an unrelated nucleotide exchange factor, compromises dissolution of heat‐induced protein aggregates and severely shortens lifespan after heat shock. We conclude that in metazoa, Hsp70‐Hsp40 powered by Hsp110 nucleotide exchange represents the crucial disaggregation machinery that reestablishes protein homeostasis to counteract protein unfolding stress.     

Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting

Ubiquitin accumulation in amyloid plaques is a pathological marker observed in the vast majority of neurodegenerative diseases, yet ubiquitin function in these inclusions is controversial. It has been suggested that ubiquitylated proteins are directed to inclusion bodies under stress conditions, when both chaperone-mediated refolding and proteasomal degradation are compromised or overwhelmed. Alternatively, ubiquitin and chaperones may be recruited to preformed inclusions to promote their elimination. We address this issue using a yeast model system, based on expression of several mildly misfolded degradation substrates in cells with altered chaperone content. We find that the heat shock protein 70 (Hsp70) chaperone pair Ssa1/Ssa2 and the Hsp40 cochaperone Sis1 are essential for degradation. Substrate ubiquitylation is strictly dependent on Sis1, whereas Ssa1 and Ssa2 are dispensable. Remarkably, in Ssa1/Ssa2-depleted cells, ubiquitylated substrates are sequestered into detergent-insoluble, Hsp42-positive inclusion bodies. Unexpectedly, sequestration is abolished by preventing substrate ubiquitylation. We conclude that Hsp40 is required for the targeting of misfolded proteins to the ubiquitylation machinery, whereas the decision to degrade or sequester ubiquitylated proteins is mediated by the Hsp70s. Accordingly, diminished Hsp70 levels, as observed in aging or certain pathological conditions, might be sufficient to trigger ubiquitin-dependent sequestration of partially misfolded proteins into inclusion bodies.     

A biosensor of protein foldedness identifies increased “holdase” activity of chaperones in the nucleus following increased cytosolic protein aggregation

Chaperones and other quality control machinery guard proteins from inappropriate aggregation, which is a hallmark of neurodegenerative diseases. However, how the systems that regulate the “foldedness” of the proteome remain buffered under stress conditions and in different cellular compartments remains incompletely understood. In this study, we applied a FRET-based strategy to explore how well quality control machinery protects against the misfolding and aggregation of “bait” biosensor proteins, made from the prokaryotic ribonuclease barnase, in the nucleus and cytosol of human embryonic kidney 293T cells. We found that those barnase biosensors were prone to misfolding, were less engaged by quality control machinery, and more prone to inappropriate aggregation in the nucleus as compared with the cytosol, and that these effects could be regulated by chaperone Hsp70-related machinery. Furthermore, aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to outcompete and thus prevented the engagement of quality control machinery with the biosensor in the cytosol. This effect correlated with reduced levels of DNAJB1 and HSPA1A chaperones in the cell outside those sequestered to the aggregates, particularly in the nucleus. Unexpectedly, we found Httex1 aggregation also increased the apparent engagement of the barnase biosensor with quality control machinery in the nucleus suggesting an independent implementation of “holdase” activity of chaperones other than DNAJB1 and HSPA1A. Collectively, these results suggest that proteostasis stress can trigger a rebalancing of chaperone abundance in different subcellular compartments through a dynamic network involving different chaperone–client interactions.     

Polyphenolic flavonoid (Myricetin) upregulated proteasomal degradation mechanisms: Eliminates neurodegenerative proteins aggregation

Major neurodegenerative disorders are characterized by the formation of misfolded proteins aggregates inside or outside the neuronal cells. Previous studies suggest that aberrant proteins aggregates play a critical role in protein homeostasis imbalance and failure of protein quality control (PQC) mechanism, leading to disease conditions. However, we still do not understand the precise mechanisms of PQC failure and cellular dysfunctions associated with neurodegenerative diseases caused by the accumulation of protein aggregates. Here, we show that Myricetin, a flavonoid, can eliminate various abnormal proteins from the cellular environment via modulating endogenous levels of Hsp70 chaperone and quality control (QC)-E3 ubiquitin ligase E6-AP. We have observed that Myricetin treatment suppresses the aggregation of different aberrant proteins. Myricetin also enhances the elimination of various toxic neurodegenerative diseases associated proteins from the cells, which could be reversed by the addition of putative proteasome inhibitor (MG132). Remarkably, Myricetin can also stabilize E6-AP and reduce the misfolded proteins inclusions, which further alleviates cytotoxicity. Taken together these findings suggested that new mechanistic and therapeutic insights based on small molecules mediated regulation of disturbed protein quality control mechanism, which may result in the maintenance of the state of proteostasis.