Analysis of von Hippel-Lindau tumor suppressor as a mediator of cellular oxygen sensing.

Publisher Summary This chapter presents a study that analyzes the von Hippel–Lindau (VHL) tumor suppressor as a mediator of cellular oxygen sensing. The chapter focuses on the regulation of HIF-α stability. Three isoforms of the HIF-α subunit have been identified (HIF-1α, HIF-2α, and HIF-3α). All are subject to VHL-dependent proteolysis following the hydroxylation of specific prolyl residues. For HIF-1α and HIF-2α, prolyl hydroxylation occurs at two sites (P402 and P564 in human HIF-1α) that reside within a central oxygen-dependent degradation domain (ODDD). HIF-3α has been less completely studied but at least one site of prolyl hydroxylation has been defined. Hydroxylation at these sites regulates interaction with the VHL ubiquitin E3 ligase. The interaction appears similar for all three isoforms, and the assays described here can be used for HIF-1α, HIF-2α, or HIF-3α. This chapter describes methods for assaying interactions between HIF- α and VHL E3, methods for assaying HIF-α PHD activity by VHL capture, and methods for assaying VHL-dependent ubiquitylation of HIF-α.

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