Nitric oxide donor, (+/-)-S-nitroso-N-acetylpenicillamine, stabilizes transactive hypoxia-inducible factor-1 alpha by inhibiting von Hippel-Lindau recruitment and asparagine hydroxylation

Abstract

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