Protein Disulfide Isomerase as Novel Redox Sensor in VEGF Signaling

项目摘要 活性氧物质（ROS），如来源于NADPH氧化酶（NOX）的H2 O2，充当信号分子 以促进VEGF诱导的EC中的血管生成和缺血后的新血管形成。根本问题 仍然是“如何扩散H2 O2信号可以有效地传输，以促进治疗性血管生成。” ROS的信号传导功能是通过反应性Cys残基的氧化以产生“半胱氨酸次磺酸（Cys-100）”。 OH）”，其参与二硫键形成和氧化还原信号传导。蛋白质二硫键异构酶（PDI） 依赖于氧化还原环境，起氧化酶、还原酶和异构酶的作用。“PDIA 1”是主要的PDI同种型， 在氧化还原活性结构域中具有四个反应性Cys残基。考虑到PDI的氧化还原性质，PDI可以用作 ROS依赖性VEGF信号传导中的氧化还原传感器，以增强治疗性血管生成并维持内皮细胞 代谢状态初步数据发现PDIA 1 +/-小鼠或PDIA 1表达降低的糖尿病小鼠 显示受损的修复性血管生成，表明PDIA 1的体内意义。在原发性EC中，VEGF 刺激增加了各种蛋白质的Cys-OH形成，PDIA 1 siRNA显著降低了Cys-OH形成。 使用2D凝胶分析和寻找PDIA 1的结合配偶体的实验发现PDIA 1的功能是 ROS依赖性VEGF信号传导中的氧化还原传感器，以促进Cys氧化/激活AMPK，一种关键调节剂 通过二硫键的形成来控制细胞代谢和血管生成。此外，在静止的基底EC中，PDIA 1 敲除意外地诱导线粒体断裂和EC衰老，而不诱导ER应激 通过增加Cys对Drp 1的氧化，Drp 1是一种关键的裂变GT3。因此，我们假设PDIA 1作为关键的功能， Drp 1的氧化还原适配器/还原酶，以维持静止EC中的线粒体动力学以及氧化还原 当它被Cys氧化以阻断VEGF诱导的H2 O2信号促进氧化活化时， AMPK通过二硫键形成，从而增强内皮代谢和血管生成， EC。这是缺血性血管疾病中完全新生血管形成所必需的。目标1将决定 PDIA 1感知VEGF诱导的H2 O2信号促进EC代谢的分子机制， 通过AMPK的氧化活化，血管生成，AMPK在糖尿病EC中受损。AIM 2将检查是否 PDIA 1通过与Drp 1结合来维持线粒体动力学，以使其在静止时处于减少/失活状态 EC，从而防止糖尿病中的线粒体断裂和EC功能障碍。目标3将决定 内皮PDIA 1在糖尿病受损ROS依赖性修复性新血管形成中的体内作用 我们将使用生物素标记的Cys-OH捕获探针、基于BiFC的分子蛋白质相互作用成像技术; 线粒体动力学成像; EC特异性PDIA 1-/-或糖尿病小鼠;和EC靶向Cys的基因转移 PDIA 1、AMPK和Drp 1的氧化缺陷型突变体。我们的建议将提供新的见解半胱氨酸 作为潜在治疗剂的还原/氧化蛋白质和Cys氧化介导的分子相互作用 用于治疗缺血性心血管代谢疾病的靶点。

PROJECT SUMMARY Reactive oxygen species (ROS), such as H2O2 derived from NADPH oxidase (NOX) act as signaling molecules to promote VEGF-induced angiogenesis in ECs and post-ischemic neovascularization. Fundamental question remains “how diffusible H2O2 signal can be efficiently transmitted to promote therapeutic angiogenesis.” Signaling function of ROS is through oxidation of reactive Cys residues to generate “Cysteine sulfenic acid (Cys- OH)” which is involved in disulfide bond formation and redox signaling. Protein Disulfide Isomerase (PDI) functions as oxidase, reductase and isomerase depending on redox environment. “PDIA1” is a major PDI isoform with four reactive Cys residues in redox active domains. Given redox properties of PDI, PDI may function as redox sensor in ROS-dependent VEGF signaling to enhance therapeutic angiogenesis and maintain endothelial metabolic states. Preliminary Data found that PDIA1+/- mice or diabetes mice with reduced PDIA1 expression show impaired reparative angiogenesis, indicating in vivo significance of PDIA1. In primary ECs, VEGF stimulation increases Cys-OH formation of various proteins, which was markedly decreased by PDIA1 siRNA. Experiments using 2D gel assay and searching for binding partner of PDIA1 discovered that PDIA1 functions as a redox sensor in ROS-dependent VEGF signaling to promote Cys oxidation/activation of AMPK, a key regulator of cell metabolism and angiogenesis, via disulfide bond formation. Moreover, in quiescent basal ECs, PDIA1 knockdown unexpectedly induced mitochondrial fragmentation and EC senescence without inducing ER stress via increasing Cys oxidation of Drp1, a key fission GTPase. We thus hypothesize that PDIA1 functions as key redox adaptor/reductase for Drp1 to maintain mitochondrial dynamics in quiescent ECs as well as redox sensor when it is Cys oxidized to transduce VEGF-induced H2O2 signal to promote oxidative activation of AMPK via disulfide bond formation, thereby enhancing endothelial metabolism and angiogenesis in ECs. This is required for full neovascularization in ischemic vascular disease. Aim 1 will determine the molecular mechanisms by which PDIA1 senses VEGF-induced H2O2 signal to promote EC metabolism and angiogenesis via oxidative activation of AMPK, which is impaired in diabetic ECs. Aim2 will examine whether PDIA1 maintains mitochondrial dynamics via binding to Drp1 to keep it in reduced/inactive state in quiescent ECs, thereby preventing mitochondrial fragmentation and ECs dysfunction in diabetes. Aim 3 will determine the in vivo role of endothelial PDIA1 in ROS-dependent reparative neovascularization, which is impaired in diabetes. We will use biotin-labelled Cys-OH trapping probe; BiFC-based molecular protein interaction imaging; mitochondrial dynamics imaging; EC-specific PDIA1-/- or diabetic mice; and gene transfer of EC-targeted Cys oxidation defective mutants of PDIA1, AMPK and Drp1. Our proposal will provide novel insights into Cys reduced/oxidized proteins and Cys oxidation-mediated molecular interaction as potential therapeutic targets for treatment of ischemic cardiovascular metabolic diseases.