HIF prolyl 4-hydroxylase (PHD) activity and the structure analysis: Blueprints for development of specific substrate inhibitors
Brucz, Kimberly Anne
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The maintenance of oxygen homeostasis is a primary requirement that shapes the development, growth and internal organization for all multicellular species. When the delicate balance between oxygen supply and demand is shifted, major physiological and pathological consequences arise. A conserved family of prolyl 4-hydroxylases, known as PHD enzymes, is responsible for marking the hypoxia inducible transcription factor (HIF) for degradation under normoxic conditions. Each of the PHD isoenzymes has a part in regulating the amount of functionally active HIF available to orchestrate vital responses and adaptations to changes in oxygen tension. With no prolyl 4-hydroxylase structures available and a lack of precise inhibitors, urgency exists for PHD characterization and structure determination. In this thesis, expression of milligram quantities of purified PHD enzymes and HIF substrate using cloning techniques in E.coli and chromatographic purifications are described. Conventional HIF prolyl 4-hydroxylase assays are discussed in conjunction with a proposed novel continuous assay based on oxygen consumption, the first hydroxylase assay to continuously and quantitatively measure activity using a Clark-type polarographic probe and an oxygen monitor. In addition, the generation of a homology model of the catalytically relevant PHD 1 oxygenase domain is presented. Future crystallographic experiments using X-ray diffraction can benefit from the pre-determined PHD expression and purification methods and can reference the PHD1 homology model in preliminary drug-design studies.