Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation
Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation
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DOI：
10.1016/j.jmb.2004.12.050
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发表时间：
2005-03-11
影响因子：
5.6
通讯作者：
Campbell, SL
中科院分区：
文献类型：
--
作者：
Heo, J;Prutzman, KC;Campbell, SL
Ras 蛋白在 GDP 结合状态和 GTP 结合状态之间循环，以调节多种细胞生长过程。在这项研究中，我们阐明了一氧化氮在氧气 (NO/O-2) 存在下调节 Ras 活性的机制。我们表明，用 NO/O-2 处理 Ras 会导致 Ras 结合的 GDP 转化为游离的 463.3 Da 核苷酸硝化产物。质谱和紫外/可见光谱分析表明，该硝化产物是 5-胍基-4-硝基咪唑二磷酸 (NIm-DP)，是 5-硝基-GDP 的降解产物。这些结果表明，NO/O-2 通过将 Ras 结合的 GDP 转化为不稳定的 5-硝基-GDP 来介导 Ras 鸟嘌呤核苷酸交换 (GNE)。 5-硝基-GDP 可以通过 GDP 鸟嘌呤碱基与二氧化氮 ((NO2)-N-) 发生自由基反应来生产。我们还提供证据表明 Ras Phe(28) 侧链在 NO/O-2 诱导的 Ras 5-硝基-GDP 产物的形成中起着关键作用。我们之前提出了NO/O-2介导的Ras GNE机制，其中NO与O-2反应形成(NO2)-N-.，生成Ras Cys(118)硫基自由基(Ras-S-118)中间体。在本研究中，我们为 NO/O-2 介导的 Ras GNE 的自由基机制提供了证据。根据该机理，NO与O-2反应生成(NO2)-N-..(NO2)-N-。然后与 Ras 反应生成 Ras-S-118.，Ras-S-118. 从 Ras 结合的鸟嘌呤核苷酸碱基上夺取一个电子，通过 Phe(28) 侧链生成鸟嘌呤核苷酸二磷酸阳离子自由基 (G(.+)-DP)。 G(.+)-DP随后转化为中性自由基，并可以与另一个(NO2)-N-反应。生产5-硝基-GDP。这种基于自由基的反应过程破坏了 Ras 和鸟嘌呤碱基之间的关键结合相互作用，导致 Ras 释放 GDP 并将其转化为游离的 5-硝基-GDP。这种机制可能是其他包含 NKCD 基序的 Ras 超家族 GTP 酶所共有的，因为 NO/O-2 也促进氧化还原活性 Rap1A 和 Rab3A GTP 酶上的 GNE。 (C) 2005 Elsevier Ltd. 保留所有权利。
Ras proteins cycle between GDP-bound and GTP-bound states to modulate a diverse array of cellular growth processes. In this study, we have elucidated a mechanism by which nitric oxide, in the presence of oxygen (NO/O-2), regulates Ras activity. We show that treatment of Ras with NO/O-2 causes conversion of Ras-bound GDP into a free 463.3 Da nucleotide-nitration product. Mass and UV/visible spectroscopic analyses suggest that this nitration product is 5-guanidino-4-nitroimidazole diphosphate (NIm-DP), a degradation product of 5-nitro-GDP. These results indicate that NO/O-2 mediates Ras guanine nucleotide exchange (GNE) by conversion of Ras-bound GDP into an unstable 5-nitro-GDP. 5-Nitro-GDP can be produced by radical-based reaction of the GDP guanine base with nitrogen dioxide ((NO2)-N-.). We also provide evidence that the Ras Phe(28) side-chain plays a key role in the formation of a NO/O-2-induced Ras 5-nitro-GDP product. We previously proposed a mechanism of NO/O-2-mediated Ras GNE, in which (NO2)-N-., formed by the reaction of NO with O-2, generates a Ras Cys(118) thiyl radical (Ras-S-118) intermediate. In the present study, we provide evidence for a radical-based mechanism of NO/O-2-mediated Ras GNE. According to this mechanism, reaction of NO with O-2 produces (NO2)-N-.. (NO2)-N-. then reacts with Ras to produce Ras-S-118., which withdraws an electron from the Ras-bound guanine nucleotide base to produce a guanine nucleotide diphosphate cation radical (G(.+)-DP) via the Phe(28) side-chain. G(.+)-DP is subsequently converted to a neutral radical, and can react with another (NO2)-N-. to produce 5-nitro-GDP. This radical-based reaction process disrupts key binding interactions between Ras and the guanine base, resulting in release of GDP from Ras and its conversion to free 5-nitro-GDP. This mechanism is likely to be common to other NKCD motif-containing Ras superfamily GTPases, as NO/O-2 also facilitates GNE on the redox-active Rap1A and Rab3A GTPases. (C) 2005 Elsevier Ltd. All rights reserved.