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: 8445715
• 负责人: Masuko Ushio-Fukai
• 金额: $ 23.93万
• 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 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445715
• 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）通过在特定靶蛋白中反应性半胱氨酸（CYS）残基的可逆氧化而起到信号分子的作用。由于ROS是高度扩散的，因此开发新的探针来检测和量化ROS或具有高度空间和时间分辨率的初始关键CYS氧化产物，半胱氨酸硫酸（Cys-OH）对于了解Redox信号的分子机制至关重要。血管生成取决于ROS，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疗法。 VEGF诱导VEGFR2（VEGFR2-PY）的自磷酸化，最初部分发生在小窝/脂质筏（C/LR），然后在细胞内室内进一步激活，从而导致内皮细胞（EC）增殖。我们证明了源自NADPH氧化酶的ROS是VEGF诱导的VEGFR2-PY，EC增殖和体内产后血管生成所必需的。此外，我们发现了“ IQGAP1”是一种新型的VEGFR2结合支架蛋白，涉及与ROS依赖性VEGFR2信号传导，与EC增殖以及缺血后的新血管形成有关。但是，ROS和CYS-OH的形成如何调节VEGF信号传导并促进产后血管生成仍然未知。使用新开发的Cys-OH检测探针进行初步研究发现，VEGF增加了IQGAP1，VEGFR2和PTP1B的Cys-OH形成，及其在人ECS中的二硫键形成，这受到硫醇抗氧化剂，n-乙酰基甲基甲基甲基酸酯的抑制作用，并确认了MICESCHEMBIMBIMB inschembimb。我们表明，PTP1B负调节VEGF诱导的VEGFR2-PY和EC增殖。尽管显示了H2O2对PTP1B的氧化失活，但PTP1B的CYS氧化和参与VEGF氧化还原信号的其他蛋白质的机理和作用在很大程度上未知。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，这促进了用VEGFR2和PTP1B和PTP1B形成氧化还原敏感的二硫键形成，从而使ROS依赖性VEGFR2信号构成有效的后，从而促进了有效的后肾上腺远产后。 AIM 1将确定在VEGF刺激的ECS中，NADPH氧化酶衍生的ROS是否介导了IQGAP1，VEGFR2和PTP1B的CYS氧化，并在体内介导Hindimb缺血模型，并识别其CYS氧化位点。 AIM 2将确定在ECS中用VEGFR2或PTP1B氧化的Cys的二硫键形成对于在C/LR和ER/内体时将ROS依赖性VEGFR2激活划分至关重要。长期目标是了解ROS调节血管生成的分子机制。这种完全新颖的理论的证明将表明，针对Cys氧化蛋白质和/或在特定信号微区下的分子相互作用为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提供了一种新的治疗策略。