Redox regulation of the stability of the SUMO protease SENP3 via interactions with CHIP and Hsp90

The molecular chaperone heat shock protein 90 (Hsp90) and the co-chaperone/ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) control the turnover of client proteins. How this system decides to stabilize or degrade the client proteins under particular physiological or pathological conditions is unclear. We report here a novel client protein, the SUMO2/3 protease SENP3, that is sophisticatedly regulated by CHIP and Hsp90. SENP3 is maintained at a low basal level under non-stress condition due to Hsp90-independent CHIP-mediated ubiquitination. Upon mild oxidative stress, SENP3 undergoes thiol modification, which recruits Hsp90. Hsp90/SENP3 association protects SENP3 from CHIP-mediated ubiquitination and subsequent degradation, but this effect of Hsp90 requires the presence of CHIP. Our data demonstrate for the first time that CHIP and Hsp90 interplay with a client alternately under non-stress and stress conditions, and the choice between stabilization and degradation is made by the redox state of the client. In addition, enhanced SENP3/Hsp90 association is found in cancer. These findings provide new mechanistic insight into how cells regulate the SUMO protease in response to oxidative stress.     

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.     

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 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.     

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.     

Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor

Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.     

Chaperone-dependent regulation of endothelial nitric-oxide synthase intracellular trafficking by the co-chaperone/ubiquitin ligase CHIP

Endothelial nitric-oxide synthase (eNOS), the enzyme responsible for production of endothelial NO, is under tight and complex regulation. Proper cellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. In addition, the molecular chaperone Hsp90 interacts with eNOS and positively regulates eNOS activity. Hsp90 is modulated by physical interaction with its co-chaperones. CHIP (carboxyl terminus of Hsp70-interacting protein) is such a co-chaperone that remodels the Hsp90 heterocomplex and causes protein degradation of some Hsp90 substrates through the ubiquitin-protein isopeptide ligase activity of CHIP. Here we show that CHIP incorporated into the eNOS.Hsp90 complex and specifically decreased soluble eNOS levels in transiently transfected COS cells. Surprisingly, in contrast to the effects of the Hsp90 inhibitor geldanamycin, which induces eNOS ubiquitylation and its subsequent protein degradation, CHIP did not target eNOS for ubiquitylation and proteasome-dependent degradation. Instead, CHIP partitioned soluble eNOS into an insoluble and inactive cellular compartment, presumably through its co-chaperone activity. This effect seems to be due to displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation. Consistent with observations from overexpression studies, eNOS localization to the membrane and activity were increased in mouse lung endothelial cells lacking CHIP. Taken together, these results demonstrate a novel co-chaperone-dependent mechanism through which eNOS trafficking is regulated and suggest a potentially generalized role for CHIP in protein trafficking through the Golgi compartment.     

Ubiquitin-proteasome degradation of serum- and glucocorticoid-regulated kinase-1 (SGK-1) is mediated by the chaperone-dependent E3 ligase CHIP

SGK-1 (serum- and glucocorticoid-regulated kinase-1) is a stress-induced serine/threonine kinase that is phosphorylated and activated downstream of PI3K (phosphoinositide 3-kinase). SGK-1 plays a critical role in insulin signalling, cation transport and cell survival. SGK-1 mRNA expression is transiently induced following cellular stress, and SGK-1 protein levels are tightly regulated by rapid proteasomal degradation. In the present study we report that SGK-1 forms a complex with the stress-associated E3 ligase CHIP [C-terminus of Hsc (heat-shock cognate protein) 70-interacting protein]; CHIP is required for both the ubiquitin modification and rapid proteasomal degradation of SGK-1. We also show that CHIP co-localizes with SGK-1 at or near the endoplasmic reticulum. CHIP-mediated regulation of SGK-1 steady-state levels alters SGK-1 kinase activity. These data suggest a model that integrates CHIP function with regulation of the PI3K/SGK-1 pathway in the stress response.     

Geldanamycins trigger a novel Ron degradative pathway, hampering oncogenic signaling

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron(M1254T) receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron(M1254T), are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved.     

The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways

Alpha-synuclein is a major component of Lewy bodies, the pathological hallmark of Parkinson disease, dementia with Lewy bodies, and related disorders. Misfolding and aggregation of alpha-synuclein is thought to be a critical cofactor in the pathogenesis of certain neurodegenerative diseases. In the current study, we investigate the role of the carboxyl terminus of Hsp70-interacting protein (CHIP) in alpha-synuclein aggregation. We demonstrate that CHIP is a component of Lewy bodies in the human brain, where it colocalizes with alpha-synuclein and Hsp70. In a cell culture model, endogenous CHIP colocalizes with alpha-synuclein and Hsp70 in intracellular inclusions, and overexpression of CHIP inhibits alpha-synuclein inclusion formation and reduces alpha-synuclein protein levels. We demonstrate that CHIP can mediate alpha-synuclein degradation by two discrete mechanisms that can be dissected using deletion mutants; the tetratricopeptide repeat domain is critical for proteasomal degradation, whereas the U-box domain is sufficient to direct alpha-synuclein toward the lysosomal degradation pathway. Furthermore, alpha-synuclein, synphilin-1, and Hsp70 all coimmunoprecipitate with CHIP, raising the possibility of a direct alpha-synuclein-CHIP interaction. The fact that the tetratricopeptide repeat domain is required for the effects of CHIP on alpha-synuclein inclusion morphology, number of inclusions, and proteasomal degradation as well as the direct interaction of CHIP with Hsp70 implicates a cooperation of CHIP and Hsp70 in these processes. Taken together, these data suggest that CHIP acts a molecular switch between proteasomal and lysosomal degradation pathways.     

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.     

Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase

The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.     

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.     

CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

CIB1 is a homolog of calmodulin that regulates cell adhesion, migration, and differentiation. It has been considered as an oncogene in many tumor cells; however, its role in lung adenocarcinoma (LAC) has not been studied. In this study, the expression levels of CIB1 in LAC tissues and adjacent normal tissues were examined by immunohistochemistry, and the relationship between CIB1 expression and patient clinicopathological characteristics was analyzed. The effects of CIB1 on epithelial–mesenchymal transition (EMT), migration, and metastasis of LAC cells were determined in vitro and vivo. Proteins interacting with CIB1 were identified using electrospray mass spectrometry (LS-MS), and CHIP was selected in the following assays. Carboxyl-terminus of Hsp70-interacting protein (CHIP) is a ubiquitin E3 ligase. We show that CHIP can degrade CIB1 via promoting polyubiquitination of CIB1 and its subsequent proteasomal degradation. Besides, lysine residue 10 and 65 of CIB1 is the ubiquitinated site of CIB1. Furthermore, CHIP-mediated CIB1 downregulation is critical for the suppression of metastasis and migration of LAC. These results indicated that CHIP-mediated CIB1 ubiquitination could regulate epithelial–mesenchymal and tumor metastasis in LAC.Subject terms: Metastasis, Oncogenes, Non-small-cell lung cancer     

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.     

CHIP targets toxic alpha-Synuclein oligomers for degradation

alpha-Synuclein (alphaSyn) can self-associate, forming oligomers, fibrils, and Lewy bodies, the pathological hallmark of Parkinson disease. Current dogma suggests that oligomeric alphaSyn intermediates may represent the most toxic alphaSyn species. Here, we studied the effect of a potent molecular chaperone, CHIP (carboxyl terminus of Hsp70-interacting protein), on alphaSyn oligomerization using a novel bimolecular fluorescence complementation assay. CHIP is a multidomain chaperone, utilizing both a tetratricopeptide/Hsp70 binding domain and a U-box/ubiquitin ligase domain to differentially impact the fate of misfolded proteins. In the current study, we found that co-expression of CHIP selectively reduced alphaSyn oligomerization and toxicity in a tetratricopeptide domain-dependent, U-box-independent manner by specifically degrading toxic alphaSyn oligomers. We conclude that CHIP preferentially recognizes and mediates degradation of toxic, oligomeric forms of alphaSyn. Further elucidation of the mechanisms of CHIP-induced degradation of oligomeric alphaSyn may contribute to the successful development of drug therapies that target oligomeric alphaSyn by mimicking or enhancing the powerful effects of CHIP.     

Heat shock protein 90 regulates the stability of MEKK3 in HEK293 cells

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the conformational maturation and function of certain signaling proteins. Hsp90 inhibitors cause the inactivation, destabilization and eventual degradation of Hsp90 client proteins through occupying the ATP/ADP binding pocket of Hsp90. In the present study, we found that Hsp90 interacted with MEKK3 in HEK293 cells. Hsp90 inhibitors reduced the level of endogenous MEKK3 in time- and dose-dependent manners, and this decrease was reversed by Hsp90 overexpression. In addition, Hsp90 RNAi destabilized MEKK3. A selective inhibitor of Hsp90, geldanamycin (GA), shortened MEKK3 half-life, and induced ubiquitination and proteasomal degradation of MEKK3. These results strongly suggested that Hsp90 could work as the molecular chaperone of MEKK3.     

Role of oxidative stress in geldanamycin-induced cytotoxicity and disruption of Hsp90 signaling complex

Heat shock protein 90 (Hsp90) is a chaperone protein regulating PC-12 cell survival by binding and stabilizing Akt, Raf-1, and Cdc37. Hsp90 inhibitor geldanamycin (GA) cytotoxicity has been attributed to the disruption of Hsp90 binding, and the contribution of oxidative stress generated by its quinone group has not been studied in this context. Reactive oxygen species (ROS) and cell survival were assessed in PC-12 cells exposed to GA or menadione (MEN), and Akt, Raf-1, and Cdc37 expression and binding to Hsp90 were determined. GA disrupted Hsp90 binding and increased ROS production starting at 1 h, and cell death occurred at 6 h, inhibited by N-acetylcysteine (NAC) without preventing dissociation of proteins. At 24 h, NAC prevented cytotoxicity and Hsp90 complex disruption. However, MnTBAP antioxidant treatment failed to inhibit GA cytotoxicity, suggesting that NAC acts by restoring glutathione. In contrast, 24 h MEN treatment induced cytotoxicity without disrupting Hsp90 binding. GA and MEN decreased Hsp90-binding protein expression, and proteasomal inhibition prevented MEN-, but not GA-induced degradation. In conclusion, whereas MEN cytotoxicity is mediated by ROS and proteasomal degradation, GA-induced cytotoxicity requires ROS but induces Hsp90 complex dissociation and proteasome-independent protein degradation. These differences between MEN- and GA-induced cytotoxicity may allow more specific targeting of cancer cells.