Characterization of the thioredoxins system in the diabetic retina

糖尿病视网膜硫氧还蛋白系统的表征

基本信息

批准号：
8400827
负责人：
Folami Lamoke Powell
金额：
$ 4.22万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8400827
关键词：
1,2-diacylglycerol Adult Affect Antioxidants Back Binding Biological Biological Availability Blindness Blood Vessels Blood capillaries Cell Death Cell Death Induction Complications of Diabetes Mellitus Data Defense Mechanisms Development Diabetes Mellitus Diabetic Retinopathy Digestion Diglycerides Dropout Endothelial Cells Event Functional disorder Glucose Goals Homeostasis Hyperglycemia Impairment In Vitro Injury Insulin-Dependent Diabetes Mellitus Lead Mentors Modeling Modification Molecular Monitor Nitrates Oxidation-Reduction Oxidative Stress Pathogenesis Peroxonitrite Phosphorylation Physiological Play Post-Translational Protein Processing Prevention Production Protein Binding Protein Isoforms Protein Kinase C Proteins Reactive Oxygen Species Research Proposals Retina Retinal Selenocysteine Sequence Analysis Serine Signal Pathway Signal Transduction Streptozocin Superoxides System Testing Thioredoxin Tissues Trypsin capillary cell injury delta protein design diabetic diabetic patient diabetic rat effective therapy electron donor in vivo mutant nitration overexpression prevent protein kinase C-delta research study response thioredoxin reductase thioredoxin reductase 1 treatment effect

项目摘要

DESCRIPTION (provided by applicant): Retinal capillary loss, due to hyperglycemia-induced vascular cell death, is a prominent feature of diabetic retinopathy (DR), a common complication of diabetes and the leading cause of blindness in adults. Hyperglycemia-induced oxidative stress (OxS) is thought to result in retinal vascular cell death and consequent capillary loss. Along with increased formation of reactive oxygen species (ROS), oxidative stress in the retinal hyperglycemic milieu can result from the impairment of endogenous anti-oxidant defense mechanisms. The overall goal of this proposal is to investigate the effects of diabetes on the activity of the endogenous anti-oxidant system of the thioredoxin, a central regulator of cellular redox homeostasis, and to develop strategies to prevent its malfunction in the diabetic retina. The thioredoxin system (TrxS) consists of thioredoxin (Trx1), Trx reductase (TrxR1), thioredoxin inhibitory protein (TXNIP) and NAD(P)H. Trx1 reduces oxidized molecules with its redox active center and is reduced back to its "active" form by the selenocysteine enzyme TrxR1, which utilizes NAD(P)H as an electron donor. The binding of TXNIP to Trx1 represents a stop signal for the system, thereby serving as a physiologic negative regulator of the system. We have recently found that hyperglycemia promotes TXNIP serine phosphorylation (SerP). Sequence analysis shows that this molecular event is dependent on Protein Kinase C delta (PKC?), a ROS sensitive PKC isoform up-regulated in the diabetic retina and in retinal endothelial cells (RECs) exposed to high glucose concentrations (HG). We have also found that hyperglycemia inhibits the activity of TrxR1 with no changes in its expression. Oxidative and nitrative post-translational modifications are known to affect the enzymatic activity of TrxR1. Protein nitration has been shown to be up-regulated in the diabetic retina and may also contribute to impairment of TrxR1. In this research proposal, we are aiming at determining the molecular mechanisms involved in hyperglycemia-induced changes in the TrxS by focusing on the events associated with TXNIP SerP and TrxR1 impairment. We hypothesized that hyperglycemia-induced impairment of the TrxS involves PKC?-dependent serine phosphorylation of TXNIP and TrxR1 protein nitration. We designed experiments to be conducted in vitro in RECs (a major target of diabetes) exposed to HG, and in vivo by using streptozotocin-induced diabetic rats (STZ-rats, a model of Type 1 diabetes). Aim1)) Test the hypothesis that HG/diabetes promotes PKC-dependent serine phosphorylation (SerP) of TXNIP and this effect contributes to oxidative retinal endothelial cell injury. Aim 2)) Test the hypothesis that HG/diabetes induces TrxR1 protein nitration and this effect contributes to oxidative retinal endothelial cell injury. PUBLIC HEALTH RELEVANCE: Oxidative stress (OxS) plays a key role in the pathogenesis of diabetic retinopathy, a leading cause of blindness worldwide. Hyperglycemia-induced dysfunction of the endogenous antioxidant system of the thioredoxins (TrxS) has been implicated in diabetes-induced OxS and retinal tissue injury. This research proposal focuses on determining diabetes-induced molecular modifications of the TrxS, as these contribute to hyperglycemia-induced OxS, retinal tissue injury and development of diabetic retinopathy.

描述（由申请人提供）：由于高血糖诱导的血管细胞死亡导致的视网膜毛细血管损失是糖尿病视网膜病变（DR）的一个突出特征，糖尿病视网膜病变是糖尿病的常见并发症，也是成人失明的主要原因。高血糖引起的氧化应激（OxS）被认为会导致视网膜血管细胞死亡和随之而来的毛细血管损失。随着活性氧（ROS）形成的增加，视网膜高血糖环境中的氧化应激可能是由于内源性抗氧化防御机制的受损而导致的。该提案的总体目标是研究糖尿病对硫氧还蛋白（细胞氧化还原稳态的中央调节剂）内源性抗氧化系统活性的影响，并制定预防其在糖尿病视网膜中发生故障的策略。硫氧还蛋白系统（TrxS）由硫氧还蛋白（Trx1）、Trx还原酶（TrxR1）、硫氧还蛋白抑制蛋白（TXNIP）和NAD（P）H组成。 Trx1 以其氧化还原活性中心还原氧化分子，并被硒代半胱氨酸酶 TrxR1 还原回其“活性”形式，该酶利用 NAD(P)H 作为电子供体。 TXNIP 与 Trx1 的结合代表系统的停止信号，从而充当系统的生理负调节剂。我们最近发现高血糖促进 TXNIP 丝氨酸磷酸化 (SerP)。序列分析表明，该分子事件依赖于蛋白激酶 C δ (PKC?)，这是一种 ROS 敏感的 PKC 同工型，在糖尿病视网膜和暴露于高葡萄糖浓度 (HG) 的视网膜内皮细胞 (REC) 中上调。我们还发现高血糖会抑制 TrxR1 的活性，但其表达没有变化。已知氧化和硝基翻译后修饰会影响 TrxR1 的酶活性。蛋白质硝化已被证明在糖尿病视网膜中上调，也可能导致 TrxR1 损伤。在本研究计划中，我们的目标是通过关注与 TXNIP SerP 和 TrxR1 损伤相关的事件来确定高血糖引起的 TrxS 变化的分子机制。我们假设高血糖引起的 TrxS 损伤涉及 TXNIP 的 PKC 依赖性丝氨酸磷酸化和 TrxR1 蛋白硝化。我们设计了在暴露于 HG 的 REC（糖尿病的主要目标）中进行体外实验，并使用链脲佐菌素诱导的糖尿病大鼠（STZ 大鼠，1 型糖尿病模型）进行体内实验。目的 1)) 测试 HG/糖尿病促进 TXNIP 的 PKC 依赖性丝氨酸磷酸化 (SerP) 的假设，并且这种效应导致氧化性视网膜内皮细胞损伤。目标 2)) 测试 HG/糖尿病诱导 TrxR1 蛋白硝化的假设，并且这种效应导致氧化性视网膜内皮细胞损伤。公众健康相关性：氧化应激 (OxS) 在糖尿病视网膜病变的发病机制中发挥着关键作用，而糖尿病视网膜病变是全球失明的主要原因。高血糖引起的硫氧还蛋白 (TrxS) 内源性抗氧化系统功能障碍与糖尿病引起的 OxS 和视网膜组织损伤有关。该研究计划的重点是确定糖尿病诱导的 TrxS 分子修饰，因为这些修饰会导致高血糖诱导的 OxS、视网膜组织损伤和糖尿病视网膜病变的发展。