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）通过特定靶蛋白中活性半胱氨酸（Cys）残基的可逆氧化作为信号分子发挥作用。由于活性氧是高度扩散的，开发新的探针来检测和定量活性氧或初始的关键半胱氨酸氧化产物，半胱氨酸次磺酸（Cys-OH），具有高度的空间和时间分辨率，是必不可少的了解氧化还原信号的分子机制。血管生成依赖于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）非常重要。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VEGFR 2（VEGFR 2-pY）的自磷酸化，其最初部分发生在小窝/脂筏（C/LR）处，随后其在细胞内区室处进一步活化，导致内皮细胞（EC）增殖。我们证明了来源于NADPH氧化酶的ROS是VEGF诱导的VEGFR 2-pY、EC增殖和出生后体内血管生成所必需的。此外，我们发现“IQGAP 1”是一种新型的VEGFR 2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VEGFR 2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然未知。使用新开发的Cys-OH检测探针的初步研究发现，VEGF增加了IQGAP 1，VEGFR 2和PTP 1B的Cys-OH形成及其在人EC中的二硫键形成，这被巯基抗氧化剂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PTP 1B负调控VEGF诱导的VEGFR 2-pY和EC增殖。虽然显示了PTP 1B通过H2 O2的氧化失活，但是PTP 1B和其他参与VEGF氧化还原信号传导的蛋白质的Cys氧化的机制和作用在很大程度上是未知的。我们的初步数据与IQGAP 1通过Cys氧化感知VEGF诱导的ROS信号的假设一致，Cys氧化促进与VEGFR 2和PTP 1B形成氧化还原敏感的二硫键，以区分ROS依赖的VEGFR 2信号传导，从而促进有效的出生后血管生成。目的1在VEGF刺激的内皮细胞和后肢缺血模型中，研究NADPH氧化酶衍生的活性氧是否介导IQGAP 1、VEGFR 2和PTP 1B的Cys氧化，并确定其Cys氧化位点。目的2将确定EC中Cys氧化IQGAP 1与VEGFR 2或PTP 1B的二硫键形成是否对C/LR和ER/内体的ROS依赖性VEGFR 2活化区室化至关重要，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，靶向Cys氧化蛋白和/或它们在特定信号微结构域的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。