Protein Disulfide Isomerase as Novel Redox Sensor in VEGF Signaling

项目总结 由NADPH氧化酶(NOX)产生的过氧化氢等活性氧物种(ROS)起信号分子的作用 促进血管内皮生长因子诱导的内皮细胞血管生成和缺血后新生血管的形成。根本问题 仍然是“如何有效地传递可扩散的过氧化氢信号以促进治疗性血管生成。” ROS的信号转导功能是通过氧化活性半胱氨酸残基生成“半胱氨酸-磺酸”。 它参与二硫键的形成和氧化还原信号的传递。蛋白质二硫键异构酶(PDI) 根据氧化还原环境的不同，起到了氧化酶、还原酶和异构酶的作用。“PDIA1”是一种主要的PDI亚型 氧化还原活性区域含有四个活性半胱氨酸残基。鉴于PDI的氧化还原特性，PDI可用作 氧化还原传感器在ROS依赖的血管内皮生长因子信号通路中促进治疗性血管生成和维持内皮功能 新陈代谢状态。初步数据发现，PDIA1+/-小鼠或糖尿病小鼠PDIA1表达减少 显示受损的修复性血管生成，表明PDIA1在体内的意义。在原代内皮细胞中，血管内皮生长因子 刺激增加了各种蛋白质的Cys-OH形成，而PDIA1 siRNA显著减少了这种形成。 利用二维凝胶实验和寻找PDIA1结合伙伴的实验发现，PDIA1的功能是 ROS依赖的血管内皮生长因子信号通路中的氧化还原传感器促进关键调节因子AMPK的半胱氨酸氧化/激活 通过二硫键的形成，影响细胞代谢和血管生成。此外，在静止的基底内皮细胞中，PDIA1 基因敲除意外诱导线粒体断裂和内皮细胞衰老而未诱导内质网应激 通过增加关键的裂变型GTP酶Drp1的半胱氨酸氧化。因此，我们假设PDIA1充当密钥 DRp1的氧化还原适配器/还原酶在静止内皮细胞中维持线粒体动力学以及氧化还原 半胱氨酸氧化传感器转导血管内皮生长因子诱导的过氧化氢信号促进氧化激活 通过二硫键形成AMPK，从而促进血管内皮细胞代谢和血管生成 ECS。这是缺血性血管疾病中充分新生血管所必需的。目标1将决定 PDIA1感知血管内皮生长因子诱导的H_2O_2信号促进EC代谢的分子机制 通过氧化激活AMPK的血管生成，而AMPK在糖尿病血管内皮细胞中受损。AIM2将检查是否 PDIA1通过与Drp1结合来维持线粒体的动力学，使其处于静止的还原/非活动状态 ECS，从而防止糖尿病患者线粒体断裂和ECS功能障碍。目标3将决定 内皮细胞PDIA1在糖尿病受损的ROS依赖的修复性新生血管中的作用。 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； 线粒体动力学成像；EC特异性PDIA1/-或糖尿病小鼠；EC靶向半胱氨酸的基因转移 PDIA1、AMPK和Drp1氧化缺陷突变体。我们的建议将为赛斯提供新的见解 还原/氧化蛋白质和半胱氨酸氧化介导的分子相互作用潜在的治疗作用 缺血性心血管代谢性疾病的治疗目标。

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.