Characterization of Celastrol to Inhibit Hsp90 and Cdc37 Interaction

Abstract

The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotidefree Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect. Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.Heat shock protein 90 (Hsp90) 2 is a highly abundant and essential molecular chaperone in eukaryotic cells, accounting for as much as 1-2% of the cytosolic protein even under nonstressed conditions (1). Hsp90 protects cells not only through correcting the misfolded proteins under stress conditions, but also plays a key role under normal conditions in regulating the stability, maturation, and activation of a wide range of client substrates, including kinases, hormone receptors, and transcription factors (2). There is strong evidence that Hsp90 plays an important role in disease states, particularly in cancer. Hsp90 is expressed 2-10-fold higher in cancer cells compared with their normal counterparts, implying its crucial role in tumor cell growth or survival (3). The largest subset of Hsp90 clients is the protein kinase, many of which are mutated and/or overexpressed signaling proteins in cancers (4 -6). Furthermore, cancer cells are significantly more sensitive to Hsp90 inhibition than non-transformed cells (7). Therefore, Hsp90 has emerged as a promising target for cancer treatment.The crystal structure reveals that Hsp90 consists of three highly conserved domains: an N-terminal ATP-binding domain (25 kDa), a middle domain (35 kDa), and a C-terminal dimerization domain (12 kDa) (8 -10). Hsp90 exists as a homodimer (11). The N-terminal domain contains a specific ATP-binding pocket, which has been well characterized (9, 12). The middle domain is highly charged, and its major role is to distinguish various types of client proteins and adjust the molecular chaperone for proper substrate activation (13). The C-terminal domain strengthens the weak association between the two N-terminal domains of the Hsp90 dimer (10). A second ATP-binding site is located in the C terminus, which does not exhibit ATPase activity (14).Hsp90 chaperone function depends on the conformational changes driven by its ATPase activity (15). Numerous Hsp90 inhibitors, ranging from...

Inhibitory action of Celastrol on hypoxia-mediated angiogenesis and metastasis via the HIF-1α pathway

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Quinone methide tripterine, celastrol, induces apoptosis in human myeloma cells via NF-κB pathway

“…Recently, celastrol has been found to be a novel Hsp90 inhibitor (35,36). However, no study has found that the celastrol-induced apoptosis of myeloma cells was induced by Hsp90 inhibition.…”

Celastrol enhances AAV1-mediated gene expression in mice adipose tissues

“…Celastrol has been reported to possess antioxidative activities and interfere with the interaction between HSP90 and CDC37. 33,34 Also, downmodulation of HSP90 potentially increase AAV transduction through the interaction with FKBP52 to increase AAV second-strand synthesis. 35 However, the HSP90 protein levels were not significantly different in 3T3-L1 preadipocytes with and without celastrol as determined by western blot shown in Supplementary Figure 3a.…”