Effects of cancer-associated point mutations on the structure, function, and stability of succinate dehydrogenase A

After the discovery of mutations in the gene encoding the Krebs cycle enzyme succinate dehydrogenase A (SDHA) in paragangliomas and other tumors, genetic SDHA variants have been reported to be important biomarkers. However, the molecular mechanisms in SDHA mutation-induced carcinogenesis remain unclear. Using overlapping PCR, protein expression and purification, immunoblotting and -precipitation, enzyme activity measurements, protein aggregation and degradation assays, and a homology model, here we demonstrate that the properties of cancer-associated SDHA variants arising from point mutations are closely related to their structure and stability. Furthermore, SDHA mutations resulted in a temperature/urea concentration-dependent increase in aggregation of the purified SDHA protein. Compared with wild-type SDHA, the degradation of the SDHA variants was increased in 293T cells after 17-(allylamino)geldanamycin (17-AAG)-induced inhibition of heat shock protein 90 (HSP90). The homology model further revealed that changes in conformational and SDHA protein stability appear to be associated with these point mutations. Taken together, our findings provide information important for understanding the molecular mechanisms of SDHA mutations in tumors.

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HSP (HSP90)