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

Abstract

Hypoxia-inducible factor-1K K (HIF-1K K) is a master regulator to sense decreased oxygen partial pressure. HIF-1K K 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-1K K and to transactivate HIF-1 under normoxia. Considering that reactive nitrogen species are recognized for post-translation protein modi¢cations, among others S-nitrosation, we asked whether HIF-1K K is a target for S-nitrosation. In vitro NO+ donating NO donors such as GSNO and SNAP provoked massive S-nitrosation of puri¢ed HIF-1K K. All 15 free thiol groups found in human HIF-1K K are subjected to S-nitrosation. Thiol modi¢cation is not shared by spermine-NONOate, a NO radical donating compound. However, spermine-NONOate in the presence of O 3 3 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-1K K was attenuated by the addition of GSH or ascorbate. In RCC4 and HEK293 cells GSNO or SNAP reproduced S-nitrosation of HIF-1K K, however with a signi¢cantly reduced potency that amounted to modi¢ca-tion of three to four thiols, only. Importantly, endogenous formation of NO in RCC4 cells via inducible NO synthase elicited S-nitrosation of HIF-1K K that was sensitive to inhibition of inducible NO synthase activity with N-monomethyl-L-arginine. NO-stabilized HIF-1K K was susceptible to the addition of N-acetyl-cysteine that destabilized HIF-1K K in close correlation to the disappearance of S-nitrosated HIF-1K K. In conclusion, HIF-1K K is a target for S-nitrosation by exogenously and endogenously produced NO. ß

Nitric Oxide Induces Hypoxia-inducible Factor 1 Activation That Is Dependent on MAPK and Phosphatidylinositol 3-Kinase Signaling

“…However, our experimental results do not support this mechanism of action for NOC18. Another potential target is HIF-1␣ itself, since there is a report that GSNO induces nitrosylation of HIF-1␣ (44). However, our results indicate that if NOC18 induces nitrosylation of HIF-1␣, this modification does not lead to accumulation of the protein.…”

Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response

“…There are two potential mechanisms for this effect. Exposure to NO has been shown to nitrosylate thiols in the HIF1α protein leading to HIF1α stabilization under aerobic conditions 39 . Recently, the specific target has been identified to be a cysteine residue in the ODD of HIF1α, which blocks its binding to the VHl complex for subsequent degradation 27 .…”

Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia

“…This response occurred, in part, by S-nitrosylation of pVHL, as demonstrated in vitro (21). Alternate mechanisms could include S-nitrosylation of HIF-1α (20,40) and inhibition of HIF prolyl hydroxylase activity (22). However, the transgenic model used in the current study (123-aa protein domain of human HIF-1α ODD) does not contain any of the 15 cysteine residues identified as targets for Snitrosylation in HIF-1α (20,40), consistent with our finding of no increase in HIF-1α S-nitrosylation during anemia.…”