Triptolide inhibits Hsp90β atpase and chaperone activity to promote cell cycle arrest and programmed cell death through multiple regulations

Triptolide (TL) is a potent antitumor, anti-inflammatory and immunosuppressive compound extracted from Thunder God vine (Tripeterygium wilfordii Hook f.), which is traditionally used in Chinese Medicine, owing to its wide spectrum of therapeutic effects (Liu, Q 2011). In order to illuminate the molecular mechanism of TL, great efforts have been made to search for TL’s intracellular binding targets. Several intracellular targets of TL have been reported, each responsible for specific aspects of TL’s bioactivity. In this study, Hsp90β has been identified as a molecular target of TL. By using biotin-TL as a chemical probe, it is demonstrated that TL specifically and irreversibly binds to Hsp90β and exhibits a modest binding affinity. Further characterization suggests that TL most likely binds to Cys366 and Cys590 on Hsp90β with distinct functions, whilst Cys366 is the dominant binding site for TL. TL binds to Cys366 to dissociate Hsp90β-CDC37 complex and causes kinase client protein degradation via ubiquitin-dependent pathway. In addition, although TL does not bind directly to the ATP binding pocket on Hsp90β, it suppresses the chaperone activity of Hsp90β by inhibiting the ATPase activity through interaction with Cys590. At the cellular level, the shutdown of chaperone machinery by TL triggers CDK4 protein level decrease in HeLa cells. Degradation of CDK4 by TL causes down-regulation of phosphorylation level of Rb, which results a cell cycle arrest at the G1 phase. Furthermore, it is demonstrated that TL treatment triggers programmed cell death in an Hsp90β-dependent manner. Knockdown of Hsp90β increases the cytotoxicity of TL by sensitizing apoptosis induced by TL. An examination of short-term effect of TL on HeLa cells reveals that TL also up-regulates phosphorylation level of Hsp90β on both Ser226 and Ser255 in a time-dependent manner. Phosphorylation of both serines facilitates apoptosome activation inside HeLa cells, which renders these cells to programmed cell death in the early stages of drug treatment. In summary, the work carried out in this study has established that TL is a novel Hsp90β inhibitor with bi-phasic multi-functional inhibitory mechanisms. The unique regulatory mechanism of TL on Hsp90β makes it a promising lead compound for further clinical development.published_or_final_versionChemistryDoctoralDoctor of Philosoph