Received for publication January 15, 2008.
Revised April 18, 2008.
Accepted for publication April 21, 2008.

THE NITRIC OXIDE DONOR, SNAP, STABILIZES TRANS-ACTIVE HYPOXIA-INDUCIBLE FACTOR-1 BY INHIBITING VHL RECRUITMENT AND ASPARAGINE HYDROXYLATION

Abstract

We have confirmed that the NO donor, SNAP, stabilizes the trans-active form of Hypoxia-Inducible Factor-1 (HIF-1), leading to the induction of HIF-1 target genes such as VEGF and CA9. 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, CBP/p300. We examined the effects of NO donor, SNAP on proline and asparagines 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 asparagines 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. 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 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.

Key words: Nitric oxide, Transcriptional coactivators, Immunocytochemistry, Mass Spectroscopy, Regulation of gene expression, Radical intermediates, Oxidative stress, Transcription targets