Sumbayev Vadim V, Budde Andreja, Zhou Jie, Brüne Bernhard
University of Kaiserslautern, Faculty of Biology, Department of Cell Biology, Erwin Schrödinger-Strasse, 67663 Kaiserslautern, Germany.
FEBS Lett. 2003 Jan 30;535(1-3):106-12. doi: 10.1016/s0014-5793(02)03887-5.
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.
缺氧诱导因子-1α（HIF-1α）是感知氧分压降低的主要调节因子。HIF-1α稳定性调节引发复杂的生物学反应，使细胞能够做出适当反应以应对氧供应减少的病理生理状况。最近，一氧化氮作为一种信使出现，具有在常氧条件下稳定HIF-1α并反式激活HIF-1的能力。鉴于活性氮物种可进行翻译后蛋白质修饰，尤其是S-亚硝基化，我们探究HIF-1α是否是S-亚硝基化的靶点。体外，供体NO+的NO供体如GSNO和SNAP可引发纯化的HIF-1α大量S-亚硝基化。在人HIF-1α中发现的所有15个游离巯基均会发生S-亚硝基化。精胺-亚硝酰基供体（一种NO自由基供体化合物）不会发生巯基修饰。然而，在黄嘌呤/黄嘌呤氧化酶产生的O(2)(-)存在下，精胺-亚硝酰基供体恢复了S-亚硝基化，很可能是通过形成类似N(2)O(3)的物种。在体外，添加谷胱甘肽或抗坏血酸可减弱HIF-1α的S-亚硝基化。在RCC4和HEK293细胞中，GSNO或SNAP可重现HIF-1α的S-亚硝基化，但效力显著降低，仅能修饰三到四个巯基。重要的是，RCC4细胞中通过诱导型一氧化氮合酶内源性生成的NO引发了HIF-1α的S-亚硝基化，该过程对用N-甲基-L-精氨酸抑制诱导型一氧化氮合酶活性敏感。NO稳定的HIF-1α对添加N-乙酰半胱氨酸敏感，N-乙酰半胱氨酸使HIF-1α不稳定，且与S-亚硝基化的HIF-1α消失密切相关。总之，HIF-1α是外源性和内源性产生的NO进行S-亚硝基化的靶点。
