br Acknowledgement This work was supported by grant form EU
Acknowledgement This work was supported by grant form EU (FP7 grant agreement no.: 266033 SPonge Enzyme End Cell for Innovative AppLication-SPECIAL).
Introduction Prolyl 4-hydroxylases (P4Hs) have central roles in the synthesis of collagens and the regulation of oxygen homeostasis . The posttranslational modification, the 4 hydroxylation of a proline residue, carried out by P4Hs, is important for the stability of the collagen triple helix and the degradation of the alpha subunit of hypoxia-inducible transcription factor (HIF-α) . Vertebrate collagen prolyl-4-hydroxylases (CPHs) are alpha2beta2 tetramers with three isoenzymes differing in their catalytic alpha subunits . Another P4H family, HIF-P4Hs (HPHs), hydroxylate specific prolines in oxygen degradation domain of HIF-α and regulate von Hippel-Lindau (VHL)-dependent degradation of hydroxylated HIF-α in an oxygen-dependent manner , , . Although CPHs and HPHs catalyze prolyl 4-hydroxylation of different substrates, collagen and HIF-α, respectively, they are mechanistically identical screening libraries with a conserved active site. All P4Hs require Fe2+, 2-oxoglutarate, O2, and ascorbate to hydroxylate proline(s) in their substrates . While CPHs are regarded as attractive targets for pharmacological inhibition to control excessive collagen accumulation in fibrotic diseases and severe scarring, HPH inhibitors are believed to have beneficial effects in the treatment of diseases such as myocardial infarction, stroke, peripheral vascular disease, diabetes, and severe anemias , . Although selective modulation of activity of each enzyme holds therapeutic potential for treatment of a spectrum of pathological conditions, agents that selectively inhibit one of the P4Hs have not so far been established. As mentioned above, CPHs and HPHs require iron as a cofactor, 2-ketoglutarate and oxygen as substrates and ascorbate as an iron-reducing agent, to hydroxylate proline residues in its respective substrate, collagen and HIF-α. HPHs and CPHs have similar Km values for 2-ketoglutarate and a 2-ketoglutarate mimicking agent such as dimethyloxallyl glycine does not exhibit selective inhibition of different prolyl-4 hydroxylases . On the contrary, HPHs have much greater Km values for another substrate, dioxygen (230–250μM) than do CPHs (40μM) , suggesting that a dioxygen-mimicking agent is likely to distinguish the two enzymes. Nitric oxide (NO) was reported to inhibit HPHs, although its inhibitory mechanism remains controversial , . For catalytic hydroxylation of HIF-1α, dioxygen (O2) binds to Fe(II) in the active site of HPHs . NO is a molecular mimic for O2 binding as an Fe(II) ligand. Indeed, high resolution structures of globins and heme oxygenase complexed with O2 or with NO show very similar binding geometries and local interactions in the heme pocket , . Therefore, NO-mediated inhibition of HPHs is likely due to its competition with dioxygen, which raises the possibility of discriminative effect of NO on HPHs and CPHs. Our data provide strong evidence that NO inhibits HPH-2 directly through its competition with dioxygen binding and that NO exerts a selective inhibition of HPH-2 and HIF-1α prolyl hydroxylation without inhibiting collagen prolyl hydroxylation by CPHs, in A549 human lung adenocarcinoma cells.
Materials and methods
Discussion In this report, we provide strong evidence that nitric oxide donors inhibit HPH-2, but not CPHs, in A549 cells and that NO inhibition of HPH-2 is attenuated significantly by hyperoxia. In view of the widely different Km values of the two enzymes for dioxygen and also of a similar mode of binding of dioxygen and NO to heme-iron proteins, our data support the notion that competition of NO with dioxygen is relevant to the discriminative effect of NO against HPH-2 and CPHs. Our data further suggest that nitric oxide directly inhibits HPHs to block hydroxylation of HIF-1α and to stabilize it. This argument is supported by the suppression of HPH-2-mediated hydroxylation of the biotinylated HIF-1α peptide (WT-HIF, corresponding to HIF residues 556–574 without 520 cysteine residue) by nitric oxide donors, SNAP and GSNO in vitro as indicated by the dramatic decrease of VHL binding (Fig. 1A). Besides direct inhibition of the HPH reaction, it is plausible that NO directly inhibits VHL binding to the HIF-1α irrespective of Pro564 hydroxylation. However, this possibility can be ruled out based on the following observations: (i) Hydroxylated HIF-1α peptide binding to VHL was not affected in the presence of SNAP or GSNO (Fig. 1B), (ii) A HIF-1α peptide with alanine in the place of proline 564 failed to bind to VHL under any circumstances. These in vitro results were confirmed by cellular experiments that showed the NO donor-induced accumulation of nonhydroxylated HIF-1α (Fig. 1C and D).