von Hippel-Lindau β-domain-luciferase fusion protein as a bioluminescent hydroxyproline sensor for a hypoxia-inducible factor prolyl hydroxylase assay.
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.
Hong
Sungchae
S
College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea.
Yum
Soohwan
S
Ha
Nam-Chul
NC
Jung
Yunjin
Y
D000600:Amino Acids, Dicarboxylic / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D049308:Dioxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006909:Hydroxyproline / Q000032:analysis*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007656:Ketoglutaric Acids / Q000737:chemistry; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000600:Amino Acids, Dicarboxylic; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049308:Dioxygenases; D008156:Luciferases; D011393:Procollagen-Proline Dioxygenase; C447780:EGLN2 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D005982:Glutathione Transferase; D006909:Hydroxyproline; C040947:oxalylglycine