Assays to Study Hypoxia-Inducible Factor Prolyl Hydroxylase Domain 2 (PHD2), a Key Human Oxygen Sensing Protein.

Abstract

Molecular oxygen is essential for all multicellular life forms. In humans, the hypoxia-inducible factor (HIF) prolyl hydroxylase domain-containing enzymes (PHDs) serve as important oxygen sensors by regulating the activity of HIF, the master regulator that mediates cellular oxygen homeostasis, in an oxygen-dependent manner. In normoxia, PHDs catalyze the prolyl hydroxylation of HIF, which leads to its degradation and prevents cellular hypoxic response to be triggered. PHDs are current inhibition targets for the potential treatments of a number of diseases. In this chapter, we discuss in vitro and cell-based methods to study the modulation of PHD2, the most important human PHD isoform in normoxia and mild hypoxia. These include the production and purification of recombinant PHD2, the use of mass spectrometry to follow PHD2-catalyzed reactions and the studies of HIF stabilization in cells by immunoblotting.