Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling.

BACKGROUND: Molecular chaperones recognize nonnative proteins and orchestrate cellular folding processes in conjunction with regulatory cofactors. However, not every attempt to fold a protein is successful, and misfolded proteins can be directed to the cellular degradation machinery for destruction. Molecular mechanisms underlying the cooperation of molecular chaperones with the degradation machinery remain largely enigmatic so far. RESULTS: By characterizing the chaperone cofactors BAG-1 and CHIP, we gained insight into the cooperation of the molecular chaperones Hsc70 and Hsp70 with the ubiquitin/proteasome system, a major system for protein degradation in eukaryotic cells. The cofactor CHIP acts as a ubiquitin ligase in the ubiquitination of chaperone substrates such as the raf-1 protein kinase and the glucocorticoid hormone receptor. During targeting of signaling molecules to the proteasome, CHIP may cooperate with BAG-1, a ubiquitin domain protein previously shown to act as a coupling factor between Hsc/Hsp70 and the proteasome. BAG-1 directly interacts with CHIP; it accepts substrates from Hsc/Hsp70 and presents associated proteins to the CHIP ubiquitin conjugation machinery. Consequently, BAG-1 promotes CHIP-induced degradation of the glucocorticoid hormone receptor in vivo. CONCLUSIONS: The ubiquitin domain protein BAG-1 and the CHIP ubiquitin ligase can cooperate to shift the activity of the Hsc/Hsp70 chaperone system from protein folding to degradation. The chaperone cofactors thus act as key regulators to influence protein quality control.     

Hsp70 Cochaperones HspBP1 and BAG-1M Differentially Regulate Steroid Hormone Receptor Function

Hsp70 binding protein 1 (HspBP1) and Bcl2-associated athanogene 1 (BAG-1), the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70) chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the androgen receptor (AR). BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.     

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.     

Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways

The mechanisms by which molecular chaperones assist quality control of cytosolic proteins are poorly understood. Analysis of the chaperone requirements for degradation of misfolded variants of a cytosolic protein, the VHL tumor suppressor, reveals that distinct chaperone pathways mediate its folding and quality control. While both folding and degradation of VHL require Hsp70, the chaperonin TRiC is essential for folding but is dispensable for degradation. Conversely, the chaperone Hsp90 neither participates in VHL folding nor is required to maintain misfolded VHL solubility but is essential for its degradation. The cochaperone HOP/Sti1p also participates in VHL quality control and may direct the triage decision by bridging the Hsp70-Hsp90 interaction. Our finding that a distinct chaperone complex is uniquely required for quality control provides evidence for active and specific chaperone participation in triage decisions and suggests that a hierarchy of chaperone interactions can control the alternate fates of a cytosolic protein.     

Hsp33 Controls Elongation Factor-Tu Stability and Allows Escherichia coli Growth in the Absence of the Major DnaK and Trigger Factor Chaperones

Intracellular de novo protein folding is assisted by cellular networks of molecular chaperones. In Escherichia coli, cooperation between the chaperones trigger factor (TF) and DnaK is central to this process. Accordingly, the simultaneous deletion of both chaperone-encoding genes leads to severe growth and protein folding defects. Herein, we took advantage of such defective phenotypes to further elucidate the interactions of chaperone networks in vivo. We show that disruption of the TF/DnaK chaperone pathway is efficiently rescued by overexpression of the redox-regulated chaperone Hsp33. Consistent with this observation, the deletion of hslO, the Hsp33 structural gene, is no longer tolerated in the absence of the TF/DnaK pathway. However, in contrast with other chaperones like GroEL or SecB, suppression by Hsp33 was not attributed to its potential overlapping general chaperone function(s). Instead, we show that overexpressed Hsp33 specifically binds to elongation factor-Tu (EF-Tu) and targets it for degradation by the protease Lon. This synergistic action of Hsp33 and Lon was responsible for the rescue of bacterial growth in the absence of TF and DnaK, by presumably restoring the coupling between translation and the downstream folding capacity of the cell. In support of this hypothesis, we show that overexpression of the stress-responsive toxin HipA, which inhibits EF-Tu, also rescues bacterial growth and protein folding in the absence of TF and DnaK. The relevance for such a convergence of networks of chaperones and proteases acting directly on EF-Tu to modulate the intracellular rate of protein synthesis in response to protein aggregation is discussed.     

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.     

Folding and stability of the ligand-binding domain of the glucocorticoid receptor

A complex pathway involving many molecular chaperones has been proposed for the folding, assembly, and maintenance of a high-affinity ligand-binding form of steroid receptors in vivo, including the glucocorticoid receptor. To better understand this intricate folding and assembly process, we studied the folding of the ligand-binding domain of the glucocorticoid receptor in vitro. We found that this domain can be refolded into a compact, highly structured state in vitro in the absence of chaperones. However, the presence of zwitterionic detergent is required to maintain the domain in a soluble form. In this state, the protein is dimeric and has considerable helical structure as shown by far-UV circular dichroism. Further investigation of the properties of this in vitro refolded state show that it is stable and resistant to denaturation by heat or low concentrations of chemical denaturants. A detailed analysis of the unfolding equilibria using three different structural probes demonstrated that this state unfolds via a highly populated dimeric intermediate state. Together, these data clearly show that the ligand-binding domain of the glucocorticoid receptor does not require chaperones for folding per se. However, this in vitro refolded state binds the ligand dexamethasone only weakly (K(d) = 45 microM) compared to the in vivo assembled receptor (K(d) = 3.4 nM). We suggest that the role of Hsp90 and associated chaperones is to bind to, and stabilize, a specific conformational state of the receptor which binds ligand with high affinity.     

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.     

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.     

A toxic imbalance of Hsp70s in Saccharomyces cerevisiae is caused by competition for cofactors

Molecular chaperones are responsible for managing protein folding from translation through degradation. These crucial machines ensure that protein homeostasis is optimally maintained for cell health. However, ‘too much of a good thing’ can be deadly, and the excess of chaperones can be toxic under certain cellular conditions. For example, overexpression of Ssa1, a yeast Hsp70, is toxic to cells in folding‐challenged states such as [PSI+]. We discovered that overexpression of the nucleotide exchange factor Sse1 can partially alleviate this toxicity. We further argue that the basis of the toxicity is related to the availability of Hsp70 cofactors, such as Hsp40 J‐proteins and nucleotide exchange factors. Ultimately, our work informs future studies about functional chaperone balance and cautions against therapeutic chaperone modifications without a thorough examination of cofactor relationships.     

The evolutionary and ecological role of heat shock proteins

Most heat shock proteins (Hsp) function as molecular chaperones that help organisms to cope with stress of both an internal and external nature. Here, we review the recent evidence of the relationship between stress resistance and inducible Hsp expression, including a characterization of factors that induce the heat shock response and a discussion of the associated costs. We report on studies of stress resistance including mild stress, effects of high larval densities, inbreeding and age on Hsp expression, as well as on natural variation in the expression of Hsps. The relationship between Hsps and life history traits is discussed with special emphasis on the ecological and evolutionary relevance of Hsps. It is known that up‐regulation of the Hsps is a common cellular response to increased levels of non‐native proteins that facilitates correct protein folding/refolding or degradation of non‐functional proteins. However, we also suggest that the expression level of Hsp in each species and population is a balance between benefits and costs, i.e. a negative impact on growth, development rate and fertility as a result of overexpression of Hsps. To date, investigations have focused primarily on the Hsp70 family. There is evidence that representatives of this Hsp family and other molecular chaperones play significant roles in relation to stress resistance. Future studies including genomic and proteonomic analyses will increase our understanding of molecular chaperones in stress research.     

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.     

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.     

OEP61 is a chaperone receptor at the plastid outer envelope

Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 (heat-shock protein 90) delivers precursors via its receptor Toc64, which transfers precursors to the core translocase in the outer envelope. In the present paper, we identify an uncharacterized protein in Arabidopsis thaliana OEP61 which shares common features with Toc64, and potentially provides an alternative route to the chloroplasts. Sequence analysis indicates that OEP61 possesses a clamp-type TPR (tetratricopeptide repeat) domain capable of binding molecular chaperones, and a C-terminal TMD (transmembrane domain). Phylogenetic comparisons show sequence similarities between the TPR domain of OEP61 and those of the Toc64 family. Expression of mRNA and protein was detected in all plant tissues, and localization at the chloroplast outer envelope was demonstrated by a combination of microscopy and in vitro import assays. Binding assays show that OEP61 interacts specifically with Hsp70 (heat-shock protein 70) via its TPR clamp domain. Furthermore, OEP61 selectively recognizes chloroplast precursors via their targeting sequences, and a soluble form of OEP61 inhibits chloroplast targeting. We therefore propose that OEP61 is a novel chaperone receptor at the chloroplast outer envelope, mediating Hsp70-dependent protein targeting to chloroplasts.     

Steered molecular dynamics simulation of the binding of the bovine auxilin J domain to the Hsc70 nucleotide-binding domain

The Hsp70 and Hsp40 chaperone machine plays critical roles in protein folding, membrane translocation, and protein degradation by binding and releasing protein substrates in a process that utilizes ATP. The activities of the Hsp70 family of chaperones are recruited and stimulated by the J domains of Hsp40 chaperones. However, structural information on the Hsp40–Hsp70 complex is lacking, and the molecular details of this interaction are yet to be elucidated. Here we used steered molecular dynamics (SMD) simulations to investigate the molecular interactions that occur during the dissociation of the auxilin J domain from the Hsc70 nucleotide-binding domain (NBD). The changes in energy observed during the SMD simulation suggest that electrostatic interactions are the dominant type of interaction. Additionally, we found that Hsp70 mainly interacts with auxilin through the surface residues Tyr866, Arg867, and Lys868 of helix II, His874, Asp876, Lys877, Thr879, and Gln881 of the HPD loop, and Phe891, Asn895, Asp896, and Asn903 of helix III. The conservative residues Tyr866, Arg867, Lys868, His874, Asp876, Lys877, and Phe891 were also found in a previous study to be indispensable to the catalytic activity of the DnaJ J domain and the binding of it with the NBD of DnaK. The in silico identification of the importance of auxilin residues Asn895, Asp896, and Asn903 agrees with previous mutagenesis and NMR data suggesting that helix III of the J domain of the T antigen interacts with Hsp70. Furthermore, our data indicate that Thr879 and Gln881 from the HPD loop are also important as they mediate the interaction between the bovine auxilin J domain and Hsc70.     

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.