Role of Cysteine Sulfenic Acid Formation in Compartmentalization of VEGF Signalin

半胱氨酸磺酸形成在 VEGF 信号蛋白区室化中的作用

• 批准号: 8620710
• 负责人: Masuko Ushio-Fukai
• 金额: $ 19.54万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8620710
• 关键词: Acetylcysteine; Acute; Address; Affect; Agonist; Antioxidants; Arts; Binding; Biological; Biosensor; Biotin; Blood Vessels; Cardiovascular Diseases; Caveolae; Cell Proliferation; Cells; Cysteine; Data; Disease; Endosomes; Endothelial Cells; Enzymes; Future; G-Protein-Coupled Receptors; Gene Transfer; Genes; Goals; Grant; Growth; Growth Factor; Hindlimb; Human; Hydrogen Peroxide; Image; In Vitro; Ischemia; Laboratories; Limb structure; Link; Mediating; Membrane Microdomains; Modeling; Molecular; Molecular Target; Morbidity - disease rate; Mus; Myocardial Ischemia; NADP; NADPH Oxidase; Oxidation-Reduction; PTPN1 gene; Play; Process; Production; Proteins; Proteomics; Reactive Oxygen Species; Reagent; Reporting; Resolution; Rhodamine; Role; Scaffolding Protein; Signal Transduction; Signaling Molecule; Site; Stimulus; Sulfhydryl Compounds; Testing; Time; Tissues; Vascular Diseases; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; angiogenesis; cell growth; cysteinesulfenic acid; cytokine; dimedone; disulfide bond; effective therapy; in vivo; in vivo Model; innovation; mortality; mutant; neovascularization; new therapeutic target; novel; novel therapeutics; oxidation; postnatal; public health relevance; response; small hairpin RNA; theories; therapeutic angiogenesis; treatment strategy

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.

描述（由申请人提供）：活性氧（ROS）通过特定靶蛋白中活性半胱氨酸（Cys）残基的可逆氧化而充当信号分子。由于 ROS 具有高度扩散性，因此开发具有高度空间和时间分辨率的新探针来检测和量化 ROS 或初始关键 Cys 氧化产物半胱氨酸亚磺酸 (Cys-OH)，对于了解氧化还原信号传导的分子机制至关重要。血管生成依赖于活性氧，对于治疗缺血性心脏/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功效可能是缺血性血管疾病的有效治疗方法。 VEGF 诱导 VEGFR2 (VEGFR2-pY) 的自身磷酸化，最初部分发生在小凹/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，源自 NADPH 氧化酶的 ROS 是 VEGF 诱导的 VEGFR2-pY、EC 增殖和体内出生后血管生成所必需的。此外，我们发现“IQGAP1”作为一种新型 VEGFR2 结合支架蛋白，参与与 EC 增殖以及缺血后新血管形成相关的 ROS 依赖性 VEGFR2 信号传导。然而，ROS 和 Cys-OH 的形成如何调节 VEGF 信号传导并促进出生后血管生成仍不清楚。使用新开发的Cys-OH检测探针的初步研究发现，VEGF增加了人类EC中IQGAP1、VEGFR2和PTP1B的Cys-OH形成及其二硫键的形成，这种作用被硫醇抗氧化剂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 负向调节 VEGF 诱导的 VEGFR2-pY 和 EC 增殖。尽管已显示 H2O2 可导致 PTP1B 氧化失活，但 PTP1B 和其他参与 VEGF 氧化还原信号传导的蛋白质的 Cys 氧化机制和作用在很大程度上尚不清楚。我们的初步数据与以下假设一致：IQGAP1 通过 Cys 氧化感知 VEGF 诱导的 ROS 信号，从而促进与 VEGFR2 和 PTP1B 形成氧化还原敏感的二硫键，以分隔 ROS 依赖性 VEGFR2 信号传导，从而促进有效的产后血管生成。目标 1 将确定 NADPH 氧化酶衍生的 ROS 是否介导 VEGF 刺激的 EC 和体内后肢缺血模型中 IQGAP1、VEGFR2 和 PTP1B 的 Cys 氧化，并确定它们的 Cys 氧化位点。目标 2 将确定 EC 中 Cys 氧化的 IQGAP1 与 VEGFR2 或 PTP1B 形成二硫键是否对于在 C/LR 和 ER/核内体上区分 ROS 依赖性 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表明，针对特定信号微域的Cys氧化蛋白和/或其分子相互作用提供了一种新的治疗策略，用于促进治疗缺血性心血管疾病所需的治疗性血管生成的功效。