Mircea Ivan

Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel-Lindau protein

von Hippel–Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel–Lindau protein (pVHL). Here we show that pVHL, through its β-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an α-domain-dependent manner. This is the first function to be ascribed to the pVHL β-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.

Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor

The product of the von Hippel–Lindau gene, pVHL, targets the α subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF) for polyubiquitination in the presence of oxygen. The binding of pVHL to HIF is governed by the enzymatic hydroxylation of conserved prolyl residues within peptidic motifs present in the HIFα family members. By using a biochemical purification strategy, we have identified a human homolog of Caenorhabditis elegans Egl9 as a HIF prolyl hydroxylase. In addition, we studied the activity of a structurally diverse collection of low molecular weight inhibitors of procollagen prolyl 4-hydroxylase as potential inhibitors of the HIF hydroxylase. A model compound of this series stabilized HIF in a variety of cells, leading to the increased production of its downstream target, vascular endothelial growth factor.

The von Hippel–Lindau tumor suppressor protein

The von Hippel-Lindau tumor suppressor protein (pVHL) has been shown to bind directly to the alpha subunits of the heterodimeric transcription factor HIF (hypoxia inducible factor). pVHL directs the polyubiquitination and, hence, destruction of HIF in the presence of oxygen. Loss of pVHL function leads to deregulation of HIF target genes, which play critical roles in angiogenesis.

Analysis of von Hippel–Lindau Hereditary Cancer Syndrome: Implications of Oxygen Sensing

Publisher Summary This chapter focuses on oxygen sensing during the analysis of von Hippel–Lindau (VHL) hereditary cancer syndrome and the implications of oxygen sensing. VHL disease is a hereditary cancer syndrome caused by germline mutations that inactivate the VHL tumor suppressor gene that resides on chromosome 3p25. The cardinal features of this disorder are blood vessel tumors (hemangioblastomas) of the central nervous system (CNS) and retina, clear cell renal carcinomas, and pheochromocytomas. Tumor development in VHL disease is linked to somatic inactivation of the remaining wild-type VHL allele, thus depriving a susceptible cell of the wild-type VHL gene product (pVHL).The chapter discusses the expression and purification of GST–VBC complex and VBC complex, production of pure VBC complex and GST–VBC for pull down, far western blot analysis with VBC complex, i n vitro ubiquitination assay, and prolyl hydroxylation protocols