Von Hippel–Lindau β-domain–luciferase fusion protein as a bioluminescent hydroxyproline sensor for a hypoxia-inducible factor prolyl hydroxylase assay

Abstract

Hypoxia-inducible factor prolyl hydroxylases (HPHs) are responsible for hydroxylation of proline residues in hypoxia-inducible factor-α (HIF-α), resulting in von Hippel–Lindau (VHL)-mediated proteasome degradation of the hydroxylated proteins. Pharmacological inhibition of the enzyme leads to stabilization of HIF-α proteins and consequent activation of HIF, which provides therapeutic benefit for a variety of tissues undergoing ischemic stress. In an effort to develop a new assay for measuring HPH activity, we designed a fusion protein, VHL β-domain–luciferase. Recombinant fusion protein with a glutathione S-transferase (GST) tag was purified from Escherichia coli. GST–VHL β-domain–luciferase with C-terminal deletion (GVbL–CD) was obtained as a major product and found to have luciferase activity. In a GVbL–CD capture assay using HIF peptide-bound beads, at least a 13-fold increase in luciferase activity was elicited for HIF peptide with hydroxyproline compared with unhydroxylated HIF peptide. HPH inhibitory activities of known HPH inhibitors or HIF-1α inducers were assessed using this assay, whose results were in good agreement with those obtained from conventional methods. The competitive effect of 2-ketoglutarate on dimethyloxalylglycine-mediated HPH inhibition was assessed very well in the new assay. Taken together, the VHL β-domain protein with luciferase activity is of use for HPH activity assay.