VHL mutations linked to type 2C von Hippel-Lindau disease cause extensive structural perturbations in pVHL.

pVHL (von Hippel-Lindau tumor suppressor protein) is the substrate recognition subunit of the CBC(VHL) ubiquitin ligase complex promoting the degradation of hypoxia-inducible factor subunits, HIF-1/2alpha. Mutational inactivation of pVHL causes the hereditary von Hippel-Lindau tumor syndrome, which predisposes affected individuals to hemangioblastomas, renal cell carcinomas, and pheochromocytomas. Whereas the development of hemangioblastomas and renal cell carcinomas has been attributed to impaired HIF-1/2alpha down-regulation by pVHL mutant proteins, the molecular defects underlying the development of pheochromocytomas are still unknown. Here, we present a detailed biochemical analysis of pVHL mutant proteins linked to type 2C (pheochromocytoma only) von Hippel-Lindau disease. Type 2C-associated mutations caused extensive structural perturbations of pVHL, as revealed by the reduced stability, increased proteolytic susceptibility, and dramatically altered NMR spectrum of recombinant, mutant pVHL-ElonginC-ElonginB complexes in vitro. In human cell lines, type 2C-linked mutations destabilized the CBC(VHL) ubiquitin ligase complex and resulted in reduced cellular pVHL levels. Together, our data reveal unexpectedly strong structural defects of type 2C-associated pVHL mutant proteins that are likely to affect both HIF-1/2alpha-related and -unrelated pVHL functions in the pathogenesis of pheochromocytomas.