Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis

The pathways that distinguish transport of folded and misfolded cargo through the exocytic (secretory) pathway of eukaryotic cells remain unknown. Using proteomics to assess global cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein interactions (the CFTR interactome), we show that Hsp90 cochaperones modulate Hsp90-dependent stability of CFTR protein folding in the endoplasmic reticulum (ER). Cell-surface rescue of the most common disease variant that is restricted to the ER, DeltaF508, can be initiated by partial siRNA silencing of the Hsp90 cochaperone ATPase regulator Aha1. We propose that failure of DeltaF508 to achieve an energetically favorable fold in response to the steady-state dynamics of the chaperone folding environment (the "chaperome") is responsible for the pathophysiology of CF. The activity of cargo-associated chaperome components may be a common mechanism regulating folding for ER exit, providing a general framework for correction of misfolding disease.     

Folding or holding?—Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease

The toxic accumulation of misfolded proteins as inclusions, fibrils, or aggregates is a hallmark of many neurodegenerative diseases. However, how molecular chaperones, such as heat shock protein 70 kDa (Hsp70) and heat shock protein 90 kDa (Hsp90), defend cells against the accumulation of misfolded proteins remains unclear. The ATP-dependent foldase function of both Hsp70 and Hsp90 actively transitions misfolded proteins back to their native conformation. By contrast, the ATP-independent holdase function of Hsp70 and Hsp90 prevents the accumulation of misfolded proteins. Foldase and holdase functions can protect against the toxicity associated with protein misfolding, yet we are only beginning to understand the mechanisms through which they modulate neurodegeneration. This review compares recent structural findings regarding the binding of Hsp90 to misfolded and intrinsically disordered proteins, such as tau, α-synuclein, and Tar DNA-binding protein 43. We propose that Hsp90 and Hsp70 interact with these proteins through an extended and dynamic interface that spans the surface of multiple domains of the chaperone proteins. This contrasts with many other Hsp90–client protein interactions for which only a single bound conformation of Hsp90 is proposed. The dynamic nature of these multidomain interactions allows for polymorphic binding of multiple conformations to vast regions of Hsp90. The holdase functions of Hsp70 and Hsp90 may thus allow neuronal cells to modulate misfolded proteins more efficiently by reducing the long-term ATP running costs of the chaperone budget. However, it remains unclear whether holdase functions protect cells by preventing aggregate formation or can increase neurotoxicity by inadvertently stabilizing deleterious oligomers.     

Protein quality control: U-box-containing E3 ubiquitin ligases join the fold

Molecular chaperones act with folding co-chaperones to suppress protein aggregation and refold stress damaged proteins. However, it is not clear how slowly folding or misfolded polypeptides are targeted for proteasomal degradation. Generally, selection of proteins for degradation is mediated by E3 ubiquitin ligases of the mechanistically distinct HECT and RING domain sub-types. Recent studies suggest that the U-box protein family represents a third class of E3 enzymes. CHIP, a U-box-containing protein, is a degradatory co-chaperone of heat-shock protein 70 (Hsp70) and Hsp90 that facilitates the polyubiquitination of chaperone substrates. These data indicate a model for protein quality control in which the interaction of Hsp70 and Hsp90 with co-chaperones that have either folding or degradatory activity helps to determine the fate of non-native cellular proteins.     

The Cdc37 protein kinase-binding domain is sufficient for protein kinase activity and cell viability

Cdc37 is a molecular chaperone required for folding of protein kinases. It functions in association with Hsp90, although little is known of its mechanism of action or where it fits into a folding pathway involving other Hsp90 cochaperones. Using a genetic approach with Saccharomyces cerevisiae, we show that CDC37 overexpression suppressed a defect in v-Src folding in yeast deleted for STI1, which recruits Hsp90 to misfolded clients. Expression of CDC37 truncation mutants that were deleted for the Hsp90-binding site stabilized v-Src and led to some folding in both sti1Delta and hsc82Delta strains. The protein kinase-binding domain of Cdc37 was sufficient for yeast cell viability and permitted efficient signaling through the yeast MAP kinase-signaling pathway. We propose a model in which Cdc37 can function independently of Hsp90, although its ability to do so is restricted by its normally low expression levels. This may be a form of regulation by which cells restrict access to Cdc37 until it has passed through a triage involving other chaperones such as Hsp70 and Hsp90.     

Human Hsp90 cochaperones: perspectives on tissue-specific expression and identification of cochaperones with similar in vivo functions

The Hsp90 molecular chaperone is required for the function of hundreds of different cellular proteins. Hsp90 and a cohort of interacting proteins called cochaperones interact with clients in an ATP-dependent cycle. Cochaperone functions include targeting clients to Hsp90, regulating Hsp90 ATPase activity, and/or promoting Hsp90 conformational changes as it progresses through the cycle. Over the last 20 years, the list of cochaperones identified in human cells has grown from the initial six identified in complex with steroid hormone receptors and protein kinases to about fifty different cochaperones found in Hsp90-client complexes. These cochaperones may be placed into three groups based on shared Hsp90 interaction domains. Available evidence indicates that cochaperones vary in client specificity, abundance, and tissue distribution. Many of the cochaperones have critical roles in regulation of cancer and neurodegeneration. A more limited set of cochaperones have cellular functions that may be limited to tissues such as muscle and testis. It is likely that a small set of cochaperones are part of the core Hsp90 machinery required for the folding of a wide range of clients. The presence of more selective cochaperones may allow greater control of Hsp90 activities across different tissues or during development.Electronic supplementary materialThe online version of this article (10.1007/s12192-020-01167-0) contains supplementary material, which is available to authorized users.     

Chaperones in control of protein disaggregation

The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat-shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates.     

Chaperone-mediated hierarchical control in targeting misfolded proteins to aggresomes

Protein misfolding is a common event in living cells. Molecular chaperones not only assist protein folding; they also facilitate the degradation of misfolded polypeptides. When the intracellular degradative capacity is exceeded, juxtanuclear aggresomes are formed to sequester misfolded proteins. Despite the well-established role of chaperones in both protein folding and degradation, how chaperones regulate the aggregation process remains controversial. Here we investigate the molecular mechanisms underlying aggresome formation in mammalian cells. Analysis of the chaperone requirements for the fate of misfolded proteins reveals an unexpected role of heat shock protein 70 (Hsp70) in promoting aggresome formation. This proaggregation function of Hsp70 relies on the interaction with the cochaperone ubiquitin ligase carboxyl terminal of Hsp70/Hsp90 interacting protein (CHIP). Disrupting Hsp70-CHIP interaction prevents the aggresome formation, whereas a dominant-negative CHIP mutant sensitizes the aggregation of misfolded protein. This accelerated aggresome formation also relies on the stress-induced cochaperone Bcl2-associated athanogene 3. Our results indicate that a hierarchy of cochaperone interaction controls different aspects of the intracellular protein triage decision, extending the function of Hsp70 from folding and degradation to aggregation.     

Distinct but overlapping functions of Hsp70, Hsp90, and an Hsp70 nucleotide exchange factor during protein biogenesis in yeast

Hsp70 and Hsp90 molecular chaperones play essential roles in protein expression and maturation, and while catalyzing protein folding they can "decide" to target mis-folded substrates for degradation. In this report, we show for the first time distinct but partially overlapping requirements for Hsp90, Hsp70, and an Hsp70 nucleotide exchange factor (NEF) at different steps during the biogenesis of a model substrate, firefly luciferase (FFLux), in yeast. By examining the inducible expression of FFLux in wild type cells and in specific yeast mutants, we find that the Fes1p NEF is required for efficient FFLux folding, whereas the Hsp70, Ssa1p, is required for both protein folding and stability, and to maintain maximal FFLux mRNA levels. In contrast, Hsp90 function was primarily necessary to express the FFLux-encoding gene from an inducible promoter. Together, these data indicate previously unknown roles for these proteins and point to the complexity with which chaperones and cochaperones function in the cell.     

Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.     

TAT-Hsp40 inhibits oxidative stress-mediated cytotoxicity via the inhibition of Hsp70 ubiquitination

Heat shock protein 40 (Hsp40) functions as a co-chaperone of mammalian Heat shock protein 70 (Hsp70) and facilitates the ATPase activity of Hsp70, and also promotes the cellular protein folding and renaturation of misfolded proteins. In an effort to assess the effects of Hsp40, we generated TAT-fused Hsp40 (TAT-Hsp40). The cells were transduced with TAT-Hsp40 and exposed to H(2)O(2). We demonstrated that the TAT-Hsp40-transduced cells were more resistant to cellular cytotoxicity and cell death. In particular, the degradation of Hsp70 was significantly reduced in TAT-Hsp40-containing cells as a consequence of reduced ubiquitin-proteasome activity after oxidative injury. These data support the notion that Hsp40 may confer resistance to oxidative stress via the prevention of proteasome activity.     

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp–substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp–substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.     

Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes

Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.     

Evolving paradigms on the interplay of mitochondrial Hsp70 chaperone system in cell survival and senescence

Abstract

The role of mitochondria within a cell has grown beyond being the prime source of cellular energy to one of the major signaling platforms. Recent evidence provides several insights into the crucial roles of mitochondrial chaperones in regulating the organellar response to external triggers. The mitochondrial Hsp70 (mtHsp70/Mortalin/Grp75) chaperone system plays a critical role in the maintenance of proteostasis balance in the organelle. Defects in mtHsp70 network result in attenuated protein transport and misfolding of polypeptides leading to mitochondrial dysfunction. The functions of Hsp70 are primarily governed by J-protein cochaperones. Although human mitochondria possess a single Hsp70, its multifunctionality is characterized by the presence of multiple specific J-proteins. Several studies have shown a potential association of Hsp70 and J-proteins with diverse pathological states that are not limited to their canonical role as chaperones. The role of mitochondrial Hsp70 and its co-chaperones in disease pathogenesis has not been critically reviewed in recent years. We evaluated some of the cellular interfaces where Hsp70 machinery associated with pathophysiological conditions, particularly in context of tumorigenesis and neurodegeneration. The mitochondrial Hsp70 machinery shows a variable localization and integrates multiple components of the cellular processes with varied phenotypic consequences. Although Hsp70 and J-proteins function synergistically in proteins folding, their precise involvement in pathological conditions is mainly idiosyncratic. This machinery is associated with a heterogeneous set of molecules during the progression of a disorder. However, the precise binding to the substrate for a specific physiological response under a disease subtype is still an undocumented area of analysis.     

Ubr1 and Ubr2 Function in a Quality Control Pathway for Degradation of Unfolded Cytosolic Proteins

Quality control systems facilitate polypeptide folding and degradation to maintain protein homeostasis. Molecular chaperones promote folding, whereas the ubiquitin/proteasome system mediates degradation. We show here that Saccharomyces cerevisiae Ubr1 and Ubr2 ubiquitin ligases promote degradation of unfolded or misfolded cytosolic polypeptides. Ubr1 also catalyzes ubiquitinylation of denatured but not native luciferase in a purified system. This activity is based on the direct interaction of denatured luciferase with Ubr1, although Hsp70 stimulates polyubiquitinylation of the denatured substrate. We also report that loss of Ubr1 and Ubr2 function suppressed the growth arrest phenotype resulting from chaperone mutation. This correlates with increased protein kinase maturation and indicates partitioning of foldable conformers toward the proteasome. Our findings, based on the efficiency of this quality control system, suggest that the cell trades growth potential to avert the potential toxicity associated with accumulation of unfolded or misfolded proteins. Ubr1 and Ubr2 therefore represent E3 components of a novel quality control pathway for proteins synthesized on cytosolic ribosomes.     

Hsp70 clears misfolded kinases that partitioned into distinct quality-control compartments

Hsp70 aids in protein folding and directs misfolded proteins to the cellular degradation machinery. We describe discrete roles of Hsp70,SSA1 as an important quality-control machinery that switches functions to ameliorate the cellular environment. SSA1 facilitates folding/maturation of newly synthesized protein kinases by aiding their phosphorylation process and also stimulates ubiquitylation and degradation of kinases in regular protein turnover or during stress when kinases are denatured or improperly folded. Significantly, while kinases accumulate as insoluble inclusions upon SSA1 inhibition, they form soluble inclusions upon Hsp90 inhibition or stress foci during heat stress. This suggests formation of inclusion-specific quality-control compartments under various stress conditions. Up-regulation of SSA1 results in complete removal of these inclusions by the proteasome. Elevation of the cellular SSA1 level accelerates kinase turnover and protects cells from proteotoxic stress. Upon overexpression, SSA1 targets heat-denatured kinases toward degradation, which could enable them to recover their functional state under physiological conditions. Thus active participation of SSA1 in the degradation of misfolded proteins establishes an essential role of Hsp70 in deciding client fate during stress.     

From the cradle to the grave: molecular chaperones that may choose between folding and degradation

Molecular chaperones are known to facilitate cellular protein folding. They bind non-native proteins and orchestrate the folding process in conjunction with regulatory cofactors that modulate the affinity of the chaperone for its substrate. However, not every attempt to fold a protein is successful and chaperones can direct misfolded proteins to the cellular degradation machinery for destruction. Protein quality control thus appears to involve close cooperation between molecular chaperones and energy-dependent proteases. Molecular mechanisms underlying this interplay have been largely enigmatic so far. Here we present a novel concept for the regulation of the eukaryotic Hsp70 and Hsp90 chaperone systems during protein folding and protein degradation.     

Dynamic association of proteasomal machinery with the centrosome

Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with gamma-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation.     

The balance between Hsp70 and its cochaperones Hdj1 and Bag1 determines its substrate-binding activity

Heat shock protein Hsp70 presents one of the most effective cell protective systems. Its protective activity is mostly due to the fact that Hsp70 is able to restore native conformation of newly synthesized or damaged proteins. Two other proteins. Hdj and Bag 1, are involved in the process, allowing Hsp70 to perform binding-release cyclec of target proteins. The aim of this study was to investigate interactions between cochaperones Hdj 1 and Bag 1, and the major cell chaperone Hsp in vitro. The accumulation of Hsp70 and Hdj 1 in human erythroleukemia K562 cells was stimulated by heat stress (43 degrees C, 60 min). Cells were collected at certain time periods after heat stress, and amounts of cell chaperones were measured using Western blotting and ELISA assay. The level of Hsp70 chaperone activity in cell extracts was estimated using original technique. The effects of exogenous cochaperones and of their parts on this activity were also investigated. The results of the study indicate that Hsp70 chaperone activity is regulated by the level of its cochaperones, especially Hdj 1. At the same time the amount of ATP appears to be critical for functional activity of Hsp70. Hdj 1 and Bag 1 peptides, which bind to Hsp70 with high affinity, are able to significally reduce its chaperone activity. This finding confirms the possibility of using peptide approach for regulation of Hsp70 function at the cellular and organismal levels.     

Localization of heat shock proteins in cerebral cortical cultures following induction by celastrol

Hsp70, Hsp32, and Hsp27 were induced by celastrol in rat cerebral cortical cultures at dosages that did not affect cell viability. Pronounced differences were observed in the cellular localization of these heat shock proteins in cell types of cerebral cortical cultures. Celastrol-induced Hsp70 localized to the cell body and cellular processes of neurons that were identified by neuron-specific βIII-tubulin. Hsp70 was not detected in adjacent GFAP-positive glial cells that demonstrated a strong signal for Hsp27 and Hsp32 in both glial cell bodies and cellular processes. Cells in the cerebral cortex region of the brain are selectively impacted during the progression of Alzheimer’s disease which is a “protein misfolding disorder.” Heat shock proteins provide a line of defense against misfolded, aggregation-prone proteins. Celastrol is a potential agent to counter this neurodegenerative disorder as recent evidence indicates that in vivo administration of celastrol in a transgenic model of Alzheimer’s reduces an important neuropathological hallmark of this disease, namely, amyloid beta pathology that involves protein aggregation.     

Design of a fluorescence polarization assay platform for the study of human Hsp70

The 70kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here, we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regulation.