The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1.

The molecular co-chaperone BAG1 and other members of the BAG family bind to Hsp70/Hsc70 heat shock proteins through a conserved BAG domain that interacts with the ATPase domain of the chaperone. BAG1 and other accessory proteins stimulate ATP hydrolysis and regulate the ATP-driven activity of the chaperone complexes. Contacts are made through residues in helices alpha2 and alpha3 of the BAG domain and predominantly residues in the C-terminal lobe of the bi-lobed Hsc70 ATPase domain. Within the C-terminal lobe, a subdomain exists that contains all the contacts shown by mutagenesis to be required for BAG1 recognition. In this study, the subdomain, representing Hsc70 residues 229-309, was cloned and expressed as a separately folded unit. The results of in vitro binding assays demonstrate that this subdomain is sufficient for binding to BAG1. Binding analyses with surface plasmon resonance indicated that the subdomain binds to BAG1 with a 10-fold decrease in equilibrium dissociation constant (K(D) = 22 nM) relative to the intact ATPase domain. This result suggests that the stabilizing contacts for docking of BAG1 to Hsc70 are located in the C-terminal lobe of the ATPase domain. These findings provide new insights into the role of co-chaperones as nucleotide exchange factors.     

BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion

BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.     

Regulation of the cytoplasmic quality control protein degradation pathway by BAG2

The cytoplasm is protected against the perils of protein misfolding by two mechanisms: molecular chaperones (which facilitate proper folding) and the ubiquitin-proteasome system, which regulates degradation of misfolded proteins. CHIP (carboxyl terminus of Hsp70-interacting protein) is an Hsp70-associated ubiquitin ligase that participates in this process by ubiquitylating misfolded proteins associated with cytoplasmic chaperones. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. Using a proteomics approach, we have identified BAG2, a previously uncharacterized BAG domain-containing protein, as a common component of CHIP holocomplexes in vivo. Binding assays indicate that BAG2 associates with CHIP as part of a ternary complex with Hsc70, and BAG2 colocalizes with CHIP under both quiescent conditions and after heat shock. In vitro and in vivo ubiquitylation assays indicate that BAG2 is an efficient and specific inhibitor of CHIP-dependent ubiquitin ligase activity. This activity is due, in part, to inhibition of interactions between CHIP and its cognate ubiquitin-conjugating enzyme, UbcH5a, which may in turn be facilitated by ATP-dependent remodeling of the BAG2-Hsc70-CHIP heterocomplex. The association of BAG2 with CHIP provides a cochaperone-dependent regulatory mechanism for preventing unregulated ubiquitylation of misfolded proteins by CHIP.     

Gene and protein expression of Drosophila Starvin during cold stress and recovery from chill coma

In Drosophila melanogaster, the sole member of the Bcl-2-associated anthanogene (BAG)-family proteins, called Starvin (Stv), has only been recently described. BAG proteins regulate a large range of physiological processes including life/death cell balance and stress response. The role of Stv has been poorly studied in the context of abiotic stress and particularly during and after cold stress. In this study we investigated the temporal expression of Stv gene and protein in adult flies during both the cold stress (up to 9 h at 0 °C) and the subsequent recovery phase (up to 8 h at 25 °C). Because BAG proteins can regulate positively and negatively the function of Hsp70/Hsc70, we also checked whether Stv expression was related to Hsp70 and Hsc70. Stv mRNA and Stv protein both showed a similar expression pattern: no modulation during the cold period followed by a significant up-regulation during the recovery period. A coordinated response of Stv and Hsp70 mRNA was observed, but not for Hsc70. Our findings indicate that Stv expression is part of a stress-induced program in D. melanogaster. It probably acts as a co-chaperone modulating the activity of Hsp70 chaperone machinery during recovery from cold stress. Finally our results support the suggestion that Stv and human BAG3 may be functional homologs.     

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.     

The co-chaperone BAG3 interacts with the cytosolic chaperonin CCT: New hints for actin folding

It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-γ and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner. The interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT.     

Distinct isoforms of the cofactor BAG-1 differentially affect Hsc70 chaperone function

In the mammalian cytosol and nucleus the activity of the molecular chaperone Hsc70 is regulated by chaperone cofactors that modulate ATP binding and hydrolysis by Hsc70. Among such cofactors is the anti-apoptotic protein BAG-1. Remarkably, BAG-1 is expressed as multiple isoforms, which are distinguished by their amino termini. We investigated whether distinct isoforms differ with respect to their Hsc70-regulating activity. By comparing the mainly cytosolic isoforms BAG-1M and BAG-1S, opposite effects of the two isoforms were observed in chaperone-assisted folding reactions. Whereas BAG-1M was found to inhibit the Hsc70-mediated refolding of nonnative polypeptide substrates, the BAG-1S isoform stimulated Hsc70 chaperone activity. The opposite effects are not due to differences in the regulation of the ATPase activity of Hsc70 by the two isoforms. Both isoforms stimulated ATP hydrolysis by Hsc70 in an Hsp40-dependent manner through an acceleration of ADP-ATP exchange. Our results reveal that the different amino termini of the distinct BAG-1 isoforms determine the outcome of an Hsc70-mediated folding event, most likely by transiently interacting with the polypeptide substrate. Employing isoforms of a cofactor with different substrate binding properties appears to provide the means to influence the chaperone function of Hsc70 in addition to modulating its ATPase cycle.     

Heat Shock Protein Cognate 70-4 and an E3 Ubiquitin Ligase,CHIP, Mediate Plastid-Destined Precursor Degradation through the Ubiquitin-26S Proteasome System in Arabidopsis

Plastid-targeted proteins pass through the cytosol as unfolded precursors. If proteins accumulate in the cytosol, they can form nonspecific aggregates that cause severe cellular damage. Here, we demonstrate that high levels of plastid precursors are degraded through the ubiquitin-proteasome system (UPS) in Arabidopsis thaliana cells. The cytosolic heat shock protein cognate 70-4 (Hsc70-4) and E3 ligase carboxy terminus of Hsc70-interacting protein (CHIP) were highly induced in plastid protein import2 plants, which had a T-DNA insertion at Toc159 and showed an albino phenotype and a severe defect in protein import into chloroplasts. Hsc70-4 and CHIP together mediated plastid precursor degradation when import-defective chloroplast-targeted reporter proteins were transiently expressed in protoplasts. Hsc70-4 recognized specific sequence motifs in transit peptides and thereby led to precursor degradation through the UPS. CHIP, which interacted with Hsc70-4, functioned as an E3 ligase in the Hsc70-4–mediated protein degradation. The physiological role of Hsc70-4 was confirmed by analyzing Hsc70-4 RNA interfernce plants in an hsc70-1 mutant background. Plants with lower Hsc70 levels exhibited abnormal embryogenesis, resulting in defective seedlings that displayed high levels of reactive oxygen species and monoubiquitinated Lhcb4 precursors. We propose that Hsc70-4 and CHIP mediate plastid-destined precursor degradation to prevent cytosolic precursor accumulation and thereby play a critical role in embryogenesis.     

CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation

Proper folding of proteins (either newly synthesized or damaged in response to a stressful event) occurs in a highly regulated fashion. Cytosolic chaperones such as Hsc/Hsp70 are assisted by cofactors that modulate the folding machinery in a positive or negative manner. CHIP (carboxyl terminus of Hsc70-interacting protein) is such a cofactor that interacts with Hsc70 and, in general, attenuates its most well characterized functions. In addition, CHIP accelerates ubiquitin-dependent degradation of chaperone substrates. Using an in vitro ubiquitylation assay with recombinant proteins, we demonstrate that CHIP possesses intrinsic E3 ubiquitin ligase activity and promotes ubiquitylation. This activity is dependent on the carboxyl-terminal U-box. CHIP interacts functionally and physically with the stress-responsive ubiquitin-conjugating enzyme family UBCH5. Surprisingly, a major target of the ubiquitin ligase activity of CHIP is Hsc70 itself. CHIP ubiquitylates Hsc70, primarily with short, noncanonical multiubiquitin chains but has no appreciable effect on steady-state levels or half-life of this protein. This effect may have heretofore unanticipated consequences with regard to the chaperoning activities of Hsc70 or its ability to deliver substrates to the proteasome. These studies demonstrate that CHIP is a bona fide ubiquitin ligase and indicate that U-box-containing proteins may comprise a new family of E3s.     

Arginine 469 is a pivotal residue for the Hsc70-GlcNAc-binding property

The members of the 70 kDa-heat shock proteins (HSP70) family play numerous fundamental functions in the cell such as promoting the assembly of multimeric complexes or helping the correct folding of nascent proteins to take place. In numerous previous studies we demonstrated that Hsp70 and its constitutive isoform Hsc70 are endowed of a GlcNAc-binding activity. The molecular modeling of the substrate binding domain of Hsc70 and in silico docking experiments using Ser/Thr-O-GlcNAc motifs allowed to define the potential carbohydrate-recognition region and to point out the crucial position of Arg469 as an amino-acid directly interacting with the sugar moiety. We cloned a flagged Hsc70 in a pCMV.SPORT6 vector and we showed that the mutation R469A decreased the GlcNAc-binding property of the chaperone of around 70%. This is the first work reporting the localization of the GlcNAc-binding domain of a member of the HSP70 family.     

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.     

Multiple Molecules of Hsc70 and a Dimer of DjA1 Independently Bind to an Unfolded Protein

Protein folding is a prominent chaperone function of the Hsp70 system. Refolding of an unfolded protein is efficiently mediated by the Hsc70 system with either type 1 DnaJ protein, DjA1 or DjA2, and a nucleotide exchange factor. A surface plasmon resonance technique was applied to investigate substrate recognition by the Hsc70 system and demonstrated that multiple Hsc70 proteins and a dimer of DjA1 initially bind independently to an unfolded protein. The association rate of the Hsc70 was faster than that of DjA1 under folding-compatible conditions. The Hsc70 binding involved a conformational change, whereas the DjA1 binding was bivalent and substoichiometric. Consistently, we found that the bound ¹⁴C-labeled Hsc70 to the unfolded protein became more resistant to tryptic digestion. The gel filtration and cross-linking experiments revealed the predominant presence of the DjA1 dimer. Furthermore, the Hsc70 and DjA1 bound to distinct sets of peptide array sequences. All of these findings argue against the generality of the widely proposed hypothesis that the DnaJ-bound substrate is targeted and transferred to Hsp70. Instead, these results suggest the importance of the bivalent binding of DjA1 dimer that limits unfavorable transitions of substrate conformations in protein folding.     

BAG4/SODD protein contains a short BAG domain

BAG (Bcl-2-associated athanogene) proteins are molecular chaperone regulators that affect diverse cellular pathways. All members share a conserved motif, called the BAG domain (BD), which binds to Hsp70/Hsc70 family proteins and modulates their activity. We have determined the solution structure of BD from BAG4/SODD (silencer of death domains) by multidimensional nuclear magnetic resonance methods and compared it to the corresponding domain in BAG1 (Briknarová, K., Takayama, S., Brive, L., Havert, M. L., Knee, D. A., Velasco, J., Homma, S., Cabezas, E., Stuart, J., Hoyt, D. W., Satterthwait, A. C., Llinás, M., Reed, J. C., and Ely, K. R. (2001) Nat. Struct. Biol. 8, 349-352). The difference between BDs from these two BAG proteins is striking, and the structural comparison defines two subfamilies of mammalian BD-containing proteins. One subfamily includes the closely related BAG3, BAG4, and BAG5 proteins, and the other is represented by BAG1, which contains a structurally and evolutionarily distinct BD. BDs from both BAG1 and BAG4 are three-helix bundles; however, in BAG4, each helix in this bundle is three to four turns shorter than its counterpart in BAG1, which reduces the length of the domain by one-third. BAG4 BD thus represents a prototype of the minimal functional fragment that is capable of binding to Hsc70 and modulating its chaperone activity.     

EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

Background

The uptake of abortion-inducing pathogens by trophoblast giant (TG) cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70) contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this.

Methods

Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR) domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice.

Results

The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70.

Conclusions

Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.     

Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum-associated degradation

The Hsp/c70 cytosolic chaperone system facilitates competing pathways of protein folding and degradation. Here we use a reconstituted cell-free system to investigate the mechanism and extent to which Hsc70 contributes to these co- and posttranslational decisions for the membrane protein cystic fibrosis transmembrane conductance regulator (CFTR). Hsc70 binding to CFTR was destabilized by the C-terminal domain of Bag-1 (CBag), which stimulates client release by accelerating ADP-ATP exchange. Addition of CBag during CFTR translation slightly increased susceptibility of the newly synthesized protein to degradation, consistent with a profolding function for Hsc70. In contrast, posttranslational destabilization of Hsc70 binding nearly completely blocked CFTR ubiquitination, dislocation from the endoplasmic reticulum, and proteasome-mediated cleavage. This effect required molar excess of CBag relative to Hsc70 and was completely reversed by the CBag-binding subdomain of Hsc70. These results demonstrate that the profolding role of Hsc70 during cotranslational CFTR folding is counterbalanced by a dominant and essential role in posttranslational targeting to the ubiquitin-proteasome system. Moreover, the degradative outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is in turn governed by the integrated expression of regulatory cochaperones.     

The lid domain of Caenorhabditis elegans Hsc70 influences ATP turnover, cofactor binding and protein folding activity

Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date.We find that CeHsc70 is characterized by a high ATP turnover rate and limited by post-hydrolysis nucleotide exchange. This rate-limiting step is defined by the helical lid domain at the C-terminus. A certain truncation in this domain (CeHsc70-Δ545) reduces the turnover rate and renders the hydrolysis step rate-limiting. The helical lid domain also affects cofactor affinities as the lidless mutant CeHsc70-Δ512 binds more strongly to DNJ-13, forming large protein complexes in the presence of ATP. Despite preserving the ability to hydrolyze ATP and interact with its cofactors DNJ-13 and BAG-1, the truncation of the helical lid domain leads to the loss of all protein folding activity, highlighting the requirement of this domain for the functionality of the nematode's Hsc70 protein.     

Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.     

BAG-1 induces autophagy for cardiac cell survival

The Bcl-2 associated athanogene (BAG) family of proteins function as cochaperones by bridging molecules that recruit molecular chaperones to target proteins. BAG-1 provides a physical link between the heat shock proteins Hsc70/Hsp70 and the proteasome to facilitate ubiquitin-proteasome-mediated protein degradation. In addition to the proteasome, protein degradation via autophagy is responsible for maintaining cellular metabolism, organelle homeostasis and redox equilibrium. Our recent report shows that autophagy plays an important role in cardiac adaptation-induced cell survival against ischemia-reperfusion injury in association with the BAG-1 protein. BAG-1 is associated with the autophagosomal membrane protein LC3-II and it may participate in the induction of autophagy via Hsc70. Moreover, another BAG family member, BAG-3, is responsible for the induction of macroautophagy in association with HspB8. These results show the involvement of BAG family members in the induction of autophagy for the degradation of damaged or oxidized proteins to promote cell survival.