Redox Regulation of p21ras in Angiogenesis

描述（由申请人提供）：本实验室的研究已经阐明了信号氧化还原调节在血管细胞功能中的作用。在其他氧化还原敏感蛋白中，我们发现活性氮物种（RNS），包括7NO和低浓度过氧亚硝酸盐（OONO-），对p21ras半胱氨酸-118 （C118）的s -谷胱甘肽化会急剧增加其GTPase活性以及MAP-和pi3激酶信号传导。在组织修复过程中，血管生成所需的内皮细胞（EC）的迁移也由7NO介导，但氧化还原调节的7NO蛋白靶点在介导血管生成中起重要作用尚不清楚。我们的初步研究表明，野生型（WT） p21ras的表达，而不是C118丝氨酸（C118S）突变体诱导EC迁移。此外，glutaredoxin-1 （GRX1）的过表达抑制了VEGF、7NO和WT p21ras刺激的EC迁移，这表明p21ras半胱氨酸-118硫醇的s -谷胱甘肽化是必不可少的。因此，拟议的研究将研究p21ras的氧化还原调控，该调控支持7NO和HMG CoA还原酶抑制剂（他汀类药物）刺激的正常EC迁移。我们的初步研究表明，他汀类药物通过干扰c端半胱氨酸的法尼化，促进p21ras向线粒体的迁移，从而增加线粒体分裂、ROS/RNS的产生，以及p21ras介导的、氧化还原依赖性的血管生成信号。我们还将研究EC暴露于与炎症和代谢性血管疾病相关的氧化剂和反应性脂质是否会破坏正常的p21ras活性和/或亚细胞定位。培养EC的这些研究结果将与我们在喂食高脂高糖（HFHS）饮食的肥胖小鼠的研究相关，我们发现血管生成受到损害。我们的初步研究还表明，GRX1转基因小鼠缺血后肢血管生成减少，提示代谢性疾病中GRX1表达改变可能抑制p21ras和血管生成。通过了解血管生成通常由p21ras和GRX1氧化还原调节的分子机制，我们期望能够确定为什么血管生成在代谢性疾病中受损。

DESCRIPTION (provided by applicant): Studies in this laboratory have elucidated the role of redox regulation of signaling in vascular cell function. Amongst other redox-sensitive proteins, we found that S-glutathiolation of p21ras cysteine-118 (C118) by reactive nitrogen species (RNS), including 7NO and low concentrations of peroxynitrite (OONO-), acutely increases its GTPase activity and MAP- and PI3-kinase signaling. Migration of endothelial cells (EC) which is required for angiogenesis during tissue repair is also mediated by 7NO, but the redox-regulated protein targets of 7NO that are important for mediating angiogenesis are unknown. Our preliminary studies show that expression of wild type (WT) p21ras, but not a C118 serine (C118S) mutant induces EC migration. Furthermore, overexpression of glutaredoxin-1 (GRX1), inhibits EC migration stimulated by VEGF, 7NO, and WT p21ras, suggesting that S-glutathiolation of the p21ras cysteine-118 thiol is essential. The proposed studies will therefore investigate the redox regulation of p21ras that supports normal EC migration which is stimulated by 7NO and HMG CoA reductase inhibitors (statins). Our preliminary studies suggest that statins, by interfering with farnesylation of the C-terminal cysteine which normally keeps p21ras tethered to the plasma membrane, facilitate relocation of p21ras to mitochondria, thereby increasing mitochondrial fission, ROS/RNS production, and p21ras-mediated, redox-dependent angiogenic signaling. We will also investigate whether exposure of EC to oxidants and reactive lipids associated with inflammation and metabolic vascular disease disrupts normal p21ras activity and/or subcellular localization. Results of these studies in EC in culture will be relevant for our studies in obese mice fed a high fat, high sucrose (HFHS) diet in which we find angiogenesis to be impaired. Our preliminary studies also indicate that angiogenesis is diminished in the ischemic hindlimb of GRX1 transgenic mice, suggesting the hypothesis that altered GRX1 expression in metabolic disease may inhibit p21ras and angiogenesis. By understanding the molecular mechanisms by which angiogenesis is normally redox-regulated by p21ras and GRX1, we anticipate being able to determine why angiogenesis is impaired in metabolic disease.