Characterization of the novel human prolyl 4-hydroxylases and asparaginyl hydroxylase that modify the hypoxia-inducible factor
1University of Oulu, Faculty of Medicine, Department of Medical Biochemistry and Molecular Biology
2University of Oulu, Biocenter Oulu
|Online Access:||PDF Full Text (PDF, 1.3 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514275756
|Publish Date:|| 2004-12-03
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Pharmacology and Toxicology, on December 3rd, 2004, at 10 a.m.
Doctor Eric Huang
Doctor Eric Metzen
HIF prolyl 4-hydroxylases (HIF-P4Hs) and HIF asparaginyl hydroxylase (FIH) are novel members of the 2-oxoglutarate dioxygenase family that play key roles in the regulation of the hypoxia-inducible transcription factor (HIF). They hydroxylate specific proline and asparagine residues in HIF-α, leading to its proteasomal degradation and inhibition of its transcriptional activity, respectively. These enzymes are inhibited in hypoxia, and as a consequence HIF-α becomes stabilized, forms a dimer with HIF-β, attains its maximal transcriptional activity and induces expression of many genes that are important for cell survival under hypoxic conditions.
The three HIF-P4Hs and FIH were expressed here as recombinant proteins in insect cells and purified to near homogeneity. All these enzymes were found to require long peptide substrates. The three HIF-P4Hs and FIH acted differently on the various potential hydroxylation sites in the HIF-α isoforms. The HIF-P4Hs acted well on sequences with cores distinctly different from the core motif -Leu-X-X-Leu-Ala-Pro-, suggested based on sequence analysis studies, the alanine being the only relatively strict requirement in addition to the proline itself. Acidic residues around the hydroxylation site also played a distinct role. These results together with those of others provide evidence that there is no conserved core motif for the hydroxylation by HIF-P4Hs.
The Km values of the HIF-P4Hs for O2 were slightly above its atmospheric concentration, while the Km of FIH was about one-third of these values but still 2.5 times that of the type I collagen P4H. The HIF-P4Hs are thus effective oxygen sensors, as even small decreases in the amount of O2 affect their activities, while a more severe decrease is required to inhibit FIH activity. Small molecule inhibitors of the collagen P4Hs also inhibited the HIF-P4Hs and FIH but with distinctly different Ki values, indicating that it should be possible to develop specific inhibitors for the HIF-P4Hs and FIH.
The HIF-P4Hs were found to bind the iron cosubstrate more tightly than FIH and the collagen P4Hs, and the chelator desferrioxamine was an ineffective inhibitor of the HIF-P4Hs in vitro. Several metals were effective competitive inhibitors of FIH but they were ineffective inhibitors of the HIF-P4Hs. The well-known stabilization of HIF-1α by cobalt and nickel is thus not due to a simple competitive inhibition of the HIF-P4Hs, and is probably at least in part due to HIF-P4H-independent mechanisms. The effective inhibition of FIH by these metals nevertheless indicates that the stabilized HIF-1α is transcriptionally fully active.
Acta Universitatis Ouluensis. D, Medica
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