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Nitric Oxide Donor, (±)-S-Nitroso-N-acetylpenicillamine, Stabilizes Transactive Hypoxia-Inducible Factor-1 by Inhibiting von Hippel-Lindau Recruitment and Asparagine Hydroxylation

Department of Life Science, University of Seoul, Seoul, Korea (Y.-K.P., M.O., T.L., H.P.); Life Science Division, Korea Institute of Science and Technology, Seoul, Korea (D.-R.A., E.G.Y.); and Research Labs of Dong-A Pharm. Co. Ltd., Kyunggi-do, Korea (M.S.)

We have confirmed that the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP) stabilizes the transactive form of hypoxia-inducible factor-1 (HIF-1), leading to the induction of HIF-1 target genes such as vascular endothelial growth factor and carbonic anhydrase 9. Activation of HIF-1 should require inhibition of the dual system that keeps it inactive. One is ubiquitination, which is triggered by hydroxylation of HIF-1-proline and the subsequent binding of E3 ubiquitin ligase, the von Hippel Lindau (VHL) protein. The other is hydroxylation of HIF-1-asparagine, which reduces the affinity of HIF-1 for its coactivator, cAMP responsive element binding protein/p300. We examined the effects of the NO donor SNAP on proline and asparagine hydroxylation of HIF-1 peptides by measuring the activities of the corresponding enzymes, HIF-1-specific proline hydroxylase 2 (PHD2) and the HIF-1-specific asparagine hydroxylase, designated factor inhibiting HIF-1 (FIH-1), respectively. We found that the SNAP did not prevent PHD2 from hydroxylating the proline of HIF-1. Instead, it blocked the interaction between VHL and the proline-hydroxylated HIF-1, but only when the reducing agents Fe(II) and vitamin C were limiting. The fact that the absence of cysteine 520 of HIF-1 abolishes its responsiveness to SNAP suggests that this residue mediates the inhibition by SNAP of the interaction between VHL and HIF-1, presumably by S-nitrosylation of HIF-1. Un-like PHD2, asparagine hydroxylation by FIH-1 was directly inhibited by SNAP, but again only when reducing agents were limiting. Substitution of cysteine 800 of HIF-1 with alanine failed to reverse the inhibitory effects of SNAP on asparagine hydroxylation, implying that FIH-1, not its substrate HIF-1, is inhibited by SNAP.

Address correspondence to: Dr. Hyunsung Park, 90 Cheonnong-dong, Tongdaemun-gu, Seoul, 130-743, Republic of Korea. E-mail: hspark{at}uos.ac.kr