HIF-1α protein as a target for S-nitrosation

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a master regulator to sense decreased oxygen partial pressure. HIF-1alpha 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-1alpha 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-1alpha is a target for S-nitrosation. In vitro NO+ donating NO donors such as GSNO and SNAP provoked massive S-nitrosation of purified HIF-1alpha. All 15 free thiol groups found in human HIF-1alpha 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 N2O3-like species. In vitro, S-nitrosation of HIF-1alpha was attenuated by the addition of GSH or ascorbate. In RCC4 and HEK293 cells GSNO or SNAP reproduced S-nitrosation of HIF-1alpha, 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-1alpha that was sensitive to inhibition of inducible NO synthase activity with N-monomethyl-L-arginine. NO-stabilized HIF-1alpha was susceptible to the addition of N-acetyl-cysteine that destabilized HIF-1alpha in close correlation to the disappearance of S-nitrosated HIF-1alpha. In conclusion, HIF-1alpha is a target for S-nitrosation by exogenously and endogenously produced NO. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.