Identification of Residues on Hsp70 and Hsp90 Ubiquitinated by the Cochaperone CHIP

Molecular chaperones Hsp70 and Hsp90 are in part responsible for maintaining the viability of cells by facilitating the folding and maturation process of many essential client proteins. The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) has been shown in vitro and in vivo to associate with Hsp70 and Hsp90 and ubiquitinate them, thus targeting them to the proteasome for degradation. Here, we study one facet of this CHIP-mediated turnover by determining the lysine residues on human Hsp70 and Hsp90 ubiquitinated by CHIP. We performed in vitro ubiquitination reactions of the chaperones using purified components and analyzed the samples by tandem mass spectrometry to identify modified lysine residues. Six such ubiquitination sites were identified on Hsp70 (K325, K451, K524, K526, K559, and K561) and 13 ubiquitinated lysine residues were found on Hsp90 (K107, K204, K219, K275, K284, K347, K399, K477, K481, K538, K550, K607, and K623). We mapped the ubiquitination sites on homology models of almost full-length human Hsp70 and Hsp90, which were found to cluster in certain regions of the structures. Furthermore, we determined that CHIP forms polyubiquitin chains on Hsp70 and Hsp90 linked via K6, K11, K48, and K63. These findings clarify the mode of ubiquitination of Hsp70 and Hsp90 by CHIP, which ultimately leads to their degradation.     

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.     

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.     

Ubiquitylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome

BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus of Hsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70- and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70.BAG-1.CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitin-like domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome.     

Chaperone functions of the E3 ubiquitin ligase CHIP

The carboxyl terminus of the Hsc70-interacting protein (CHIP) is an Hsp70 co-chaperone as well as an E3 ubiquitin ligase that protects cells from proteotoxic stress. The abilities of CHIP to interact with Hsp70 and function as a ubiquitin ligase place CHIP at a pivotal position in the protein quality control system, where its entrance into Hsp70-substrate complexes partitions nonnative proteins toward degradation. However, the manner by which Hsp70 substrates are selected for ubiquitination by CHIP is not well understood. We discovered that CHIP possesses an intrinsic chaperone activity that enables it to selectively recognize and bind nonnative proteins. Interestingly, the chaperone function of CHIP is temperature-sensitive and is dramatically enhanced by heat stress. The ability of CHIP to recognize nonnative protein structure may aid in selection of slow folding or misfolded polypeptides for ubiquitination.     

Parkin mediates the degradation-independent ubiquitination of Hsp70

Mutations in the parkin gene cause autosomal recessive, juvenile-onset parkinsonism. Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. Disease-associated mutations cause a loss-of-function of parkin which may compromise the poly-ubiquitination and proteasomal degradation of specific protein substrates, potentially leading to their deleterious accumulation. Here, we identify the molecular chaperones, Hsp70 and Hsc70, as substrates for parkin. Parkin mediates the ubiquitination of Hsp70 both in vitro and in cultured cells. Parkin interacts with Hsp70 via its second RING finger domain and mutations in/near this domain compromise Hsp70 ubiquitination. Ubiquitination of Hsp70 fails to alter its steady-state levels or turnover, nor does it promote its proteasomal degradation. Consistent with this observation, Hsp70 levels remain unaltered in brains from parkin-deficient autosomal recessive, juvenile-onset parkinsonism subjects, whereas alternatively, Hsp70 levels are elevated in the detergent-insoluble fraction of sporadic Parkinson's disease/dementia with Lewy bodies brains. Parkin mediates the multiple mono-ubiquitination of Hsp70/Hsc70 consistent with a degradation-independent role for this ubiquitin modification. Our observations support a novel functional relationship between parkin and Hsc/Hsp70 and support the notion that parkin is a multi-purpose E3 ubiquitin ligase capable of modifying proteins either via attachment of alternatively linked poly-ubiquitin chains or through multiple mono-ubiquitination to achieve alternate biological outcomes.     

Molecular mechanism of the negative regulation of Smad1/5 protein by carboxyl terminus of Hsc70-interacting protein (CHIP)

The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner.     

The cochaperone HspBP1 inhibits the CHIP ubiquitin ligase and stimulates the maturation of the cystic fibrosis transmembrane conductance regulator

The CHIP ubiquitin ligase turns molecular chaperones into protein degradation factors. CHIP associates with the chaperones Hsc70 and Hsp90 during the regulation of signaling pathways and during protein quality control, and directs chaperone-bound clients to the proteasome for degradation. Obviously, this destructive activity should be carefully controlled. Here, we identify the cochaperone HspBP1 as an inhibitor of CHIP. HspBP1 attenuates the ubiquitin ligase activity of CHIP when complexed with Hsc70. As a consequence, HspBP1 interferes with the CHIP-induced degradation of immature forms of the cystic fibrosis transmembrane conductance regulator (CFTR) and stimulates CFTR maturation. Our data reveal a novel regulatory mechanism that determines folding and degradation activities of molecular chaperones.     

Hsp70 and Hsp90 oppositely regulate TGF-β signaling through CHIP/Stub1

Transforming growth factor-β (TGF-β) signaling plays an important role in regulation of a wide variety of cellular processes. Canonical TGF-β signaling is mediated by Smads which were further regulated by several factors. We previously reported that E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein, also named Stub1) controlled the sensitivity of TGF-β signaling by modulating the basal level of Smad3 through ubiquitin-mediated degradation. Here, we present evidence that Hsp70 and Hsp90 regulate the complex formation of Smad3/CHIP. Furthermore, we observed that over-expressed Hsp70 or inhibition of Hsp90 by geldanamycin (GA) leads to facilitated CHIP-induced ubiquitination and degradation of Smad3, which finally enhances TGF-β signaling. In contrast, over-expressed Hsp90 antagonizes CHIP mediated Smad3 ubiquitination and degradation and desensitizes cells in response to TGF-β signaling. Taken together, our data reveal an opposite role of Hsp70 and Hsp90 in regulating TGF-β signaling by implicating CHIP-mediated Smad3 ubiquitination and degradation. This study provides a new insight into understanding the regulation of the TGF-β signaling by chaperones.     

CHIP: a link between the chaperone and proteasome systems

CHIP, carboxy terminus of Hsc70 interacting protein, is a cytoplasmic protein whose amino acid sequence is highly conserved across species. It is most highly expressed in cardiac and skeletal muscle and brain. The primary amino acid sequence is characterized by 3 domains, a tetratricopeptide repeat (TPR) domain at its amino terminus, a U-box domain at its carboxy terminus, and an intervening charged domain. CHIP interacts with the molecular chaperones Hsc70-Hsp70 and Hsp90 through its TPR domain, whereas its U-box domain contains its E3 ubiquitin ligase activity. Its interaction with these molecular chaperones results in client substrate ubiquitylation and degradation by the proteasome. Thus, CHIP acts to tilt the folding-refolding machinery toward the degradative pathway, and it serves as a link between the two. Because protein degradation is required for healthy cellular function, CHIP's ability to degrade proteins that are the signature of disease, eg, ErbB2 in breast and ovarian cancers, could prove to be a point of therapeutic intervention.     

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca²⁺/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca²⁺/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca²⁺/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca²⁺/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca²⁺-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.     

Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.     

CHIP: a quality-control E3 ligase collaborating with molecular chaperones

It is notable that both the chaperone and ubiquitin-proteasome systems are required for removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. Carboxyl-terminus of Hsc70 interacting protein (CHIP), originally identified as a co-chaperone of Hsc70, has both a tetratricopeptide repeat (TPR) motif and a U-box domain. The TPR motif associates with Hsc70 and Hsp90, while the U-box domain executes a ubiquitin ligase activity. Thus, CHIP is an ideal molecule acting as a protein quality-control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. Accumulating evidence from in vitro studies indicates that this is apparently the case. Here, we present and discuss several unresolved but critical issues related to the molecular mechanism and in vivo roles of CHIP.     

CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70

Over 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heat-shock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SOD1-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and polyubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1(G93A) transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome.     

CHIP functions an E3 ubiquitin ligase of Runx1

Runx1 is a key factor in the generation and maintenance of hematopoietic stem cells. Improper expression and mutations in Runx1 are frequently implicated in human leukemia. Here, we report that CHIP, the carboxyl terminus of Hsc70-interacting protein, also named Stub1, physically interacts with Runx1 through the TPR and Charged domains in the nucleus. Over-expression of CHIP directly induced Runx1 ubiquitination and degradation through the ubiquitin-proteasome pathway. Interestingly, we found that CHIP-mediated degradation of Runx1 is independent of the molecular chaperone Hsp70/90. Taken together, we propose that CHIP serves as an E3 ubiquitin ligase that regulates Runx1 protein stability via an ubiquitination and degradation mechanism that is independent of Hsp70/90.     

Co-chaperone CHIP associates with expanded polyglutamine protein and promotes their degradation by proteasomes

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasomes are not known. Here, we demonstrate that CHIP (C terminus of Hsp70-interacting protein) co-immunoprecipitates with the polyglutamine-expanded huntingtin or ataxin-3 and associates with their aggregates. Transient overexpression of CHIP increases the ubiquitination and the rate of degradation of polyglutamine-expanded huntingtin or ataxin-3. Finally, we show that overexpression of CHIP suppresses the aggregation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is overexpressed along with Hsc70.     

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.     

Regulation of proto-oncogenic dbl by chaperone-controlled, ubiquitin-mediated degradation

The dbl proto-oncogene product is a prototype of a growing family of guanine nucleotide exchange factors (GEFs) that stimulate the activation of small GTP-binding proteins from the Rho family. Mutations that result in the loss of proto-Dbl's amino terminus produce a variant with constitutive GEF activity and high oncogenic potential. Here, we show that proto-Dbl is a short-lived protein that is kept at low levels in cells by efficient ubiquitination and degradation. The cellular fate of proto-Dbl is regulated by interactions with the chaperones Hsc70 and Hsp90 and the protein-ubiquitin ligase CHIP, and these interactions are mediated by the spectrin domain of proto-Dbl. We show that CHIP is the E3 ligase responsible for ubiquitination and proteasomal degradation of proto-Dbl, while Hsp90 functions to stabilize the protein. Onco-Dbl, lacking the spectrin homology domain, cannot bind these regulators and therefore accumulates in cells at high levels, leading to persistent stimulation of its downstream signaling pathways.     

Ubiquitination and degradation of mutant p53

While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. In this study, we show that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degradation of both mutant and wild-type p53, our data suggest that the ability of Mdm2 to function as a ubiquitin ligase is less important in the degradation of mutant p53, which is heavily ubiquitinated in an Mdm2-independent manner. Our initial attempts to identify ubiquitin ligases that are responsible for the ubiquitination of mutant p53 have suggested a role for the chaperone-associated ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein), although other unidentified ubiquitin ligases also appear to contribute. The contribution of Mdm2 to the degradation of mutant p53 may reflect the ability of Mdm2 to deliver the ubiquitinated mutant p53 to the proteasome.