title: HIF-1 alpha protein as a target for S-nitrosation.
abstract: Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a master regulator to sense decreased oxygen partial pressure. HIF-1 alpha stability regulation initiates a complex biological response that allows cells to act appropriately to meet patho-physiological situations of decreased oxygen availability. Recently, nitric oxide emerged as a messenger with the ability to stabilize HIF-1 alpha and to transactivate HIF-1 under normoxia. Considering that reactive nitrogen species are recognized for post-translation protein modifications, among others S-nitrosation, we asked whether HIF-1 alpha is a target for S-nitrosation. In vitro NO+ donating NO donors such as GSNO and SNAP provoked massive S-nitrosation of purified HIF-1 alpha. All 15 free thiol groups found in human HIF-1 alpha are subjected to S-nitrosation. Thiol modification is not shared by spermine-NONOate, a NO radical donating compound. However, spermine-NONOate in the presence of O(2)(-), generated by xanthine/xanthine oxidase, regained S-nitrosation, most likely via formation of a N(2)O(3)-like species. In vitro, S-nitrosation of HIF-1 alpha was attenuated by the addition of GSH or ascorbate. In RCC4 and HEK293 cells GSNO or SNAP reproduced S-nitrosation of HIF-1 alpha, however with a significantly reduced potency that amounted to modification of three to four thiols, only. Importantly, endogenous formation of NO in RCC4 cells via inducible NO synthase elicited S-nitrosation of HIF-1 alpha that was sensitive to inhibition of inducible NO synthase activity with N-monomethyl-L-arginine. NO-stabilized HIF-1 alpha was susceptible to the addition of N-acetyl-cysteine that destabilized HIF-1 alpha in close correlation to the disappearance of S-nitrosated HIF-1 alpha. In conclusion, HIF-1 alpha is a target for S-nitrosation by exogenously and endogenously produced NO.
authors.affiliation: University of Kaiserslautern, Faculty of Biology, Department of Cell Biology, Erwin Schrödinger-Strasse, 67663 Kaiserslautern, Germany.
authors.forename: Vadim V
authors.initials: VV
authors.lastname: Sumbayev
authors.forename: Andreja
authors.initials: A
authors.lastname: Budde
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brüne
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000737:chemistry / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D015538:Nitrosation / Q000187:drug effects; D010084:Oxidation-Reduction / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D026423:S-Nitroso-N-Acetylpenicillamine / Q000737:chemistry / Q000494:pharmacology; D026422:S-Nitrosoglutathione / Q000737:chemistry / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020030:Nitric Oxide Donors; D011500:Protein Synthesis Inhibitors; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D026423:S-Nitroso-N-Acetylpenicillamine; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D000111:Acetylcysteine