Role of Cysteine Sulfenic Acid Formation in Compartmentalization of VEGF Signalin

描述（由申请人提供）：活性氧（ROS）作为信号分子通过在特定靶蛋白中的活性半胱氨酸（Cys）残基的可逆氧化起作用。由于ROS具有高度的可扩散性，开发新的探针来检测和量化ROS或初始关键的Cys氧化产物半胱氨酸磺酸（Cys- oh），具有高度的空间和时间分辨率，对于了解氧化还原信号的分子机制至关重要。血管生成依赖于活性氧，对治疗缺血性心脏/肢体疾病、发病率和死亡率的常见原因很重要。因此，增强血管生成反应的疗效可能是缺血性血管疾病的有效治疗方法。VEGF诱导VEGFR2的自磷酸化（VEGFR2- py），最初部分发生在小泡/脂筏（C/LR），随后在细胞内室进一步激活，导致内皮细胞（EC）增殖。我们证明了来自NADPH氧化酶的ROS是vegf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，参与与EC增殖和缺血后新生血管相关的ros依赖性VEGFR2信号传导。然而，ROS和Cys-OH的形成如何调节VEGF信号并促进出生后血管生成尚不清楚。利用新开发的Cys-OH检测探针进行的初步研究发现，VEGF增加了人内皮细胞中IQGAP1、VEGFR2和PTP1B的Cys-OH形成及其二硫键的形成，这一作用被巯基抗氧化剂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负调控vegf诱导的VEGFR2-pY和EC增殖。虽然H2O2可以使PTP1B氧化失活，但Cys氧化PTP1B和其他参与VEGF氧化还原信号的蛋白的机制和作用在很大程度上是未知的。我们的初步数据与假设一致，即IQGAP1通过Cys氧化感知vegf诱导的ROS信号，促进与VEGFR2和PTP1B形成氧化还原敏感的二硫键，从而区分开ROS依赖性的VEGFR2信号，从而促进有效的出生后血管生成。目的1将确定在体内vegf刺激的ECs和后肢缺血模型中，NADPH氧化酶衍生的ROS是否介导IQGAP1、VEGFR2和PTP1B的Cys氧化，并确定其Cys氧化位点。Aim 2将确定Cys氧化的IQGAP1与VEGFR2或PTP1B在EC中形成二硫键是否对区隔ros依赖性的C/LR和ER/内体的VEGFR2激活至关重要，从而诱导EC增殖。长期目标是了解ROS调节血管生成的分子机制。这一全新理论的证据将表明，在特定信号微域靶向Cys氧化蛋白和/或其分子相互作用，为促进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.