Role of Cysteine Sulfenic Acid Formation in Compartmentalization of VEGF Signalin

描述(申请人提供)：活性氧物种(ROS)通过可逆地氧化特定目标蛋白中的活性半胱氨酸(Cys)残基，作为信号分子发挥作用。由于ROS是高度可扩散的，开发新的探针来检测和定量ROS或最初的关键半胱氨酸氧化产物半胱氨酸磺酸(Cys-OH)，具有高空间和时间分辨率，对于理解氧化还原信号的分子机制是必不可少的。血管生成依赖于ROS，对治疗缺血性心脏/肢体疾病非常重要，这些疾病是导致发病率和死亡率的常见原因。因此，增强血管生成反应的有效性可能是治疗缺血性血管疾病的有效方法。血管内皮生长因子诱导VEGFR2的自磷酸化(VEGFR2-Py)，最初部分发生在小窝/脂筏(C/LR)，随后在细胞内进一步激活，导致内皮细胞(EC)增殖。我们证明了NADPH氧化酶来源的ROS是血管内皮生长因子诱导的VEGFR2-Py、EC增殖和出生后血管生成所必需的。此外，我们发现“IQGAP1”是一种新的VEGFR2结合支架蛋白，参与ROS依赖的VEGFR2信号转导，与EC增殖和缺血后新生血管有关。然而，ROS和Cys-OH的形成如何调节血管内皮生长因子信号并促进出生后血管生成仍不清楚。使用新开发的半胱氨酸检测探针的初步研究发现，血管内皮生长因子促进人内皮细胞IQGAP1、VEGFR2和PTP1B的半胱氨酸形成及其二硫键的形成，这一作用可被硫醇抗氧化剂N-乙酰半胱氨酸抑制，并在小鼠后肢缺血模型中得到证实。我们发现PTP1B对血管内皮生长因子诱导的VEGFR2-Py和EC的增殖具有负性调节作用。虽然研究表明PTP1B被过氧化氢氧化失活，但PTP1B和其他参与VEGF氧化还原信号的蛋白质的半胱氨酸氧化的机制和作用在很大程度上还不清楚。我们的初步数据与假设是一致的，即IQGAP1通过半胱氨酸氧化感受到血管内皮生长因子诱导的ROS信号，促进氧化还原敏感的二硫键与VEGFR2和PTP1B形成氧化还原敏感的二硫键，将ROS依赖的VEGFR2信号区隔，从而促进出生后有效的血管生成。目的1在血管内皮生长因子刺激的血管内皮细胞和后肢缺血模型中，检测NADPH氧化酶来源的ROS是否介导IQGAP1、VEGFR2和PTP1B的半胱氨酸氧化，并确定其半胱氨酸氧化位点。目的2确定半胱氨酸氧化的IQGAP1与血管内皮细胞中的VEGFR2或PTP1B形成的二硫键是否对ROS依赖的VEGFR2在C/LR和ER/内体的激活起重要作用，从而诱导EC的增殖。长期目标是了解ROS调节血管生成的分子机制。这一全新理论的证明将表明，将Cys氧化蛋白和/或它们的分子相互作用靶向于特定的信号微区为提高治疗缺血性心血管疾病所需的治疗性血管生成的疗效提供了一种新的治疗策略。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) function as signaling molecules through reversible oxidation of reactive cysteine (Cys) residues in specific target proteins. Because ROS are highly diffusible, developing new probes to detect and quantify ROS or the initial key Cys oxidation product, cysteine sulfenic acid (Cys-OH), with high degree of spatial and temporal resolution, is essential to understand the molecular mechanisms of redox signaling. Angiogenesis is dependent on ROS and important for treating ischemic heart/limb diseases, common causes of morbidity and mortality. Thus, enhancing efficacy of the angiogenesis response is likely to be an effective therapy for ischemic vascular disease. VEGF induces autophosphorylation of VEGFR2 (VEGFR2-pY), which initially occurs in part at caveolae/lipid rafts (C/LR), followed by its further activation at intracellular compartments, leading to endothelial cell (EC) proliferation. We demonstrated that ROS derived from NADPH oxidase are required for VEGF-induced VEGFR2-pY, EC proliferation and postnatal angiogenesis in vivo. Further, we discovered "IQGAP1" as a novel VEGFR2 binding scaffold protein involved in ROS-dependent VEGFR2 signaling linked to EC proliferation as well as post-ischemic neovascularization. However, how ROS and Cys-OH formation regulate VEGF signaling and can promote postnatal angiogenesis remain unknown. Preliminary studies using newly-developed Cys-OH detecting probes found that VEGF increased Cys-OH formation of IQGAP1, VEGFR2, and PTP1B and their disulfide bond formation in human ECs, which was inhibited by a thiol antioxidant, N-acetylcysteine, and confirmed in mice hindlimb ischemia model. We showed that PTP1B negatively regulates VEGF-induced VEGFR2-pY and EC proliferation. Although oxidative inactivation of PTP1B by H2O2 is shown, the mechanism and role of Cys oxidation of PTP1B and other proteins involved in VEGF redox signaling is largely unknown. Our preliminary data are consistent with the hypothesis that IQGAP1 senses VEGF-induced ROS signal via Cys oxidation, which facilitates redox-sensitive disulfide bond formation with VEGFR2 and PTP1B to compartmentalize ROS-dependent VEGFR2 signaling, thereby promoting effective postnatal angiogenesis. Aim 1 will determine whether NADPH oxidase-derived ROS mediate Cys oxidation of IQGAP1, VEGFR2 and PTP1B in VEGF-stimulated ECs and hindlimb ischemia model in vivo, and identify their Cys oxidation sites. Aim 2 will determine whether disulfide bond formation of Cys oxidized IQGAP1 with VEGFR2 or PTP1B in ECs is essential to compartmentalize ROS-dependent VEGFR2 activation at C/LR and ER/endosomes, thereby inducing EC proliferation. The long-term goal is to understand the molecular mechanisms through which ROS regulate angiogenesis. Proof of this entirely novel theory will suggest that targeting Cys oxidized proteins and/or their molecular interaction at specific signaling microdomains offers a new therapeutic strategy for promoting the efficacy of therapeutic angiogenesis needed for treatment of ischemic cardiovascular diseases.