Nitric oxide donor, (+/-)-S-nitroso-N-acetylpenicillamine, stabilizes transactive hypoxia-inducible factor-1alpha by inhibiting von Hippel-Lindau recruitment and asparagine hydroxylation.
We have confirmed that the NO donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) stabilizes the transactive form of hypoxia-inducible factor-1alpha (HIF-1alpha), leading to the induction of HIF-1alpha target genes such as vascular endothelial growth factor and carbonic anhydrase 9. Activation of HIF-1alpha should require inhibition of the dual system that keeps it inactive. One is ubiquitination, which is triggered by hydroxylation of HIF-1alpha-proline and the subsequent binding of E3 ubiquitin ligase, the von Hippel Lindau (VHL) protein. The other is hydroxylation of HIF-1alpha-asparagine, which reduces the affinity of HIF-1alpha 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-1alpha peptides by measuring the activities of the corresponding enzymes, HIF-1alpha-specific proline hydroxylase 2 (PHD2) and the HIF-1alpha-specific asparagine hydroxylase, designated factor inhibiting HIF-1alpha (FIH-1), respectively. We found that the SNAP did not prevent PHD2 from hydroxylating the proline of HIF-1alpha. Instead, it blocked the interaction between VHL and the proline-hydroxylated HIF-1alpha, but only when the reducing agents Fe(II) and vitamin C were limiting. The fact that the absence of cysteine 520 of HIF-1alpha abolishes its responsiveness to SNAP suggests that this residue mediates the inhibition by SNAP of the interaction between VHL and HIF-1alpha, presumably by S-nitrosylation of HIF-1alpha. 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-1alpha with alanine failed to reverse the inhibitory effects of SNAP on asparagine hydroxylation, implying that FIH-1, not its substrate HIF-1alpha, is inhibited by SNAP.
Park
Young-Kwon
YK
Department of Life Science, University of Seoul, Seoul, Korea.
Ahn
Dae-Ro
DR
Oh
Myoungsuk
M
Lee
Taekyoung
T
Yang
Eun Gyeong
EG
Son
Miwon
M
Park
Hyunsung
H
D001216:Asparagine / Q000037:antagonists & inhibitors*; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000037:antagonists & inhibitors*; D001616:beta-Galactosidase / Q000378:metabolism
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D015534:Trans-Activators; D006639:Histidine; D001216:Asparagine; D026423:S-Nitroso-N-Acetylpenicillamine; D008156:Luciferases; D011393:Procollagen-Proline Dioxygenase; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D005982:Glutathione Transferase; D001616:beta-Galactosidase