Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis

BackgroundP53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells.PurposeTo explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation.Study design and methodsDifferent mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53.ResultsThe triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation.ConclusionOur study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.     

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.     

The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation

The cellular level of the tumor suppressor p53 is tightly regulated through induced degradation via the ubiquitin/proteasome system. The ubiquitin ligase Mdm2 plays a pivotal role in stimulating p53 turnover. However, recently additional ubiquitin ligases have been identified that participate in the degradation of the tumor suppressor. Apparently, multiple degradation pathways are employed to ensure proper destruction of p53. Here we show that the chaperone-associated ubiquitin ligase CHIP is able to induce the proteasomal degradation of p53. CHIP-induced degradation was observed for mutant p53, which was previously shown to associate with the chaperones Hsc70 and Hsp90, and for the wild-type form of the tumor suppressor. Our data reveal that mutant and wild-type p53 transiently associate with molecular chaperones and can be diverted onto a degradation pathway through this association.     

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.     

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.     

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.     

Hsp70 acts as a fine-switch that controls E3 ligase CHIP-mediated TAp63 and ΔNp63 ubiquitination and degradation

The major clinical problem in human cancer is metastasis. Metastases are the cause of 90% of human cancer deaths. TAp63 is a critical suppressor of tumorigenesis and metastasis. ΔNp63 acts as a dominant-negative inhibitor to block the function of p53 and TAp63. Although several ubiquitin E3 ligases have been reported to regulate p63 stability, the mechanism of p63 regulation remains partially understood. Herein, we show that CHIP, an E3 ligase with a U-box domain, physically interacts with p63 and promotes p63 degradation. Notably, Hsp70 depletion by siRNA stabilizes TAp63 in H1299 cells and destabilizes ΔNp63 in SCC9 cells. Loss of Hsp70 results in a reduction in the TAp63-CHIP interaction in H1299 cells and an increase in the interaction between ΔNp63 and CHIP in SCC9 cells. Our results reveal that Hsp70 acts as a molecular switch to control CHIP-mediated ubiquitination and degradation of p63 isoforms. Furthermore, regulation of p63 by the Hsp70-CHIP axis contributes to the migration and invasion of tumor cells. Hence, our findings demonstrate that Hsp70 is a crucial regulator of CHIP-mediated ubiquitination and degradation of p63 isoforms and identify a new pathway for maintaining TAp63 or ΔNp63 stability in cancers.     

Co-chaperone CHIP Stabilizes Aggregate-prone Malin, a Ubiquitin Ligase Mutated in Lafora Disease

Lafora disease (LD) is an autosomal recessive neurodegenerative disorder caused by mutation in either the dual specificity phosphatase laforin or ubiquitin ligase malin. A pathological hallmark of LD is the accumulation of cytoplasmic polyglucosan inclusions commonly known as Lafora bodies in both neuronal and non-neuronal tissues. How mutations in these two proteins cause disease pathogenesis is not well understood. Malin interacts with laforin and recruits to aggresomes upon proteasome inhibition and was shown to degrade misfolded proteins. Here we report that malin is spontaneously misfolded and tends to be aggregated, degraded by proteasomes, and forms not only aggresomes but also other cytoplasmic and nuclear aggregates in all transfected cells upon proteasomal inhibition. Malin also interacts with Hsp70. Several disease-causing mutants of malin are comparatively more unstable than wild type and form aggregates in most transfected cells even without the inhibition of proteasome function. These cytoplasmic and nuclear aggregates are immunoreactive to ubiquitin and 20 S proteasome. Interestingly, progressive proteasomal dysfunction and cell death is also most frequently observed in the mutant malin-overexpressed cells compared with the wild-type counterpart. Finally, we demonstrate that the co-chaperone carboxyl terminus of the Hsc70-interacting protein (CHIP) stabilizes malin by modulating the activity of Hsp70. All together, our results suggest that malin is unstable, and the aggregate-prone protein and co-chaperone CHIP can modulate its stability.     

Down-regulation of the mixed-lineage dual leucine zipper-bearing kinase by heat shock protein 70 and its co-chaperone CHIP

Dual leucine zipper-bearing kinase (DLK) is a mixed-lineage kinase family member that acts as an upstream activator of the c-Jun N-terminal kinases. As opposed to other components of this pathway, very little is currently known regarding the mechanisms by which DLK is regulated in mammalian cells. Here we identify the stress-inducible heat shock protein 70 (Hsp70) as a negative regulator of DLK expression and activity. Support for this notion derives from data showing that Hsp70 induces the proteasomal degradation of DLK when both proteins are co-expressed in COS-7 cells. Hsp70-mediated degradation occurs with expression of wild-type DLK, which functions as a constitutively activated protein in these cells but not kinase-defective DLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiquitin ligase, seems to be indispensable for this process since Hsp70 failed to induce DLK degradation in COS-7 cells expressing a CHIP mutant unable to catalyze ubiquitination or in immortalized fibroblasts derived from CHIP knock-out mice. Consistent with these data, we have found that endogenous DLK becomes sensitive to CHIP-dependent proteasomal degradation when it is activated by okadaic acid and that down-regulation of Hsp70 levels with an Hsp70 antisense attenuates this sensitivity. Therefore, our studies suggest that Hsp70 contributes to the regulation of activated DLK by promoting its CHIP-dependent proteasomal degradation.     

CHIP and BAP1 Act in Concert to Regulate INO80 Ubiquitination and Stability for DNA Replication

The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its half-life. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.     

CHIP-dependent p53 regulation occurs specifically during cellular senescence

p53 regulates several biological processes, including senescence. Its protein stability is regulated by ubiquitination and proteasomal degradation, mainly mediated by Mdm2. However, other E3 ligases have been identified, such as the chaperone-associated ligase CHIP, although their precise function regarding p53 degradation remains elusive. Interestingly, CHIP deficiency has been recently shown to result in accelerated aging in mice, although the molecular basis of this phenotype was not completely understood. In this study, we explore the role of CHIP in regulating p53 in senescence. We demonstrate that in senescent human fibroblasts, CHIP is up-regulated concomitant with a significant down-regulation of p53. Moreover, CHIP partially translocates to the nucleus and acquires higher ubiquitination levels in senescent cells. Notably, CHIP overexpression in young cells, to levels similar to those recorded during senescence, leads to p53 degradation to below its basal levels. In addition, whereas CHIP silencing has no effect on p53 stability in young cells, a considerable p53 accumulation occurs in their senescent counterparts. Finally, we have observed an attenuation of the CHIP-associated molecular folding-refolding machinery during senescence, and supportively, inhibition of Hsp90 activity leads to rapid p53 degradation only in senescent cells. Taking these results together, we conclude that CHIP-dependent p53 regulation occurs specifically during senescence.     

CHIP chaperones wild type p53 tumor suppressor protein

Wild type p53 exists in a constant state of equilibrium between wild type and mutant conformation and undergoes conformational changes at elevated temperature. We have demonstrated that the co-chaperone CHIP (carboxyl terminus of Hsp70-interacting protein), which suppressed aggregation of several misfolded substrates and induced the proteasomal degradation of both wild type and mutant p53, physically interacts with the amino terminus of WT53 and prevented it from irreversible thermal inactivation. CHIP preferentially binds to the p53 mutant phenotype and restored the DNA binding activity of heat-denatured p53 in an ATP-independent manner. In cells under elevated temperatures that contained a higher level of p53 mutant phenotype, CHIP restored the native-like conformation of p53 in the presence of geldanamycin, whereas CHIP-small interfering RNA considerably increased the mutant form. Further, under elevated temperatures, the levels of CHIP and p53 were higher in nucleus, and chromatin immunoprecipitation shows the presence of p53 and CHIP together upon the DNA binding site in the p21 and p53 promoters. We propose that CHIP might be a direct chaperone of wild type p53 that helps p53 in maintaining wild type conformation under physiological condition as well as help resurrect p53 mutant phenotype into a folded native state under stress condition.     

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.     

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.     

Proteasome inhibitors suppress the protein expression of mutant p53

Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53.     

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.