CHARACTERIZATION OF A NOVEL VARIANT (L163R) OF THE VON HIPPEL LINDAU PROTEIN (pVHL)

MATHO, CECILIA; MERLINO, ALICIA; SANSO G; COITIÑO, ELENA LAURA; PENNISI PATRICIA A

Montevideo

Congreso; XXIII Reunion Anula de la Sociedad Latinoameicana de Endocrinología Pediátrica; 2012

SLEP-Sociedad Latinoamericana de Endocrinología Pediátrica

Background: von Hippel-Lindau (VHL) is a hereditary syndrome caused by VHL gene mutations. VHL protein (pVHL) forms a multiprotein complex that polyubiquitylates and determines the proteasomic degradation of HIF1α, a transcription factor involved in the regulation of genes implicated in angiogenesis, apoptosis and cell proliferation. Aim: To analyze in silico the structural characteristics and in vitro the functional properties of the novel variant L163R. Methods: Bioinformatic and molecular modeling tools were used to predict and compare the structure and properties under normoxia/hypoxia conditions of L163R and native pVHL, evaluating the MM-PBSA energy for complex formation. RCC786-0-VHL-/- cells,  both parental or stably transfected with pBabe-puro-HA-VHL-L163R (obtained by site-directed mutagenesis) were used for proliferation assays. Results:  In silico, the complex formed by L163R was more unstable than the one formed by the native protein. In vitro, the proliferation rates of RCC786-0-VHLL163R and RCC786-0-VHL-/- were similar, which was suggestive of the expression of an inactive protein. Conclusion: L163R genetic variant might decrease the stability or even prevent the formation of the multiprotein complex, suggesting a possible pathogenic role for this variant. We have combined molecular modeling with in vitro experiments for functional characterization, to better understand pathogenic mechanism of L163R variant.