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

• 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
• 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

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）：由于高血糖引起的血管细胞死亡引起的视网膜毛细血管损失是糖尿病性视网膜病变（DR）的重要特征，这是糖尿病的常见并发症，是成人失明的主要原因。高血糖诱导的氧化应激（OX）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丧失。随着活性氧（ROS）的形成增加，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剂防御机制受损而导致的。该提案的总体目标是研究糖尿病对硫氧还蛋白的内源性抗氧化剂系统的影响，硫氧还蛋白是细胞氧化还原稳态的中心调节剂，并制定策略以防止其在糖尿病视网膜中发生故障。硫氧还蛋白系统（TRX）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用其氧化还原活性中心降低了氧化的分子，并通过硒仿制半半胱氨酸酶TRXR1将其降低到其“活性”形式，该酶将其用作电子供体。 TXNIP与TRX1的结合代表了系统的停止信号，从而作为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，该分子事件取决于蛋白激酶C三角洲（PKC？），ROS灵敏的PKC同工型上调在暴露于高葡萄糖浓度（HG）的视网膜内皮细胞（REC）中。我们还发现，高血糖会抑制TRXR1的活性，而其表达没有变化。已知氧化和硝化后翻译后修饰会影响TRXR1的酶促活性。蛋白质硝化已显示在糖尿病性视网膜中被上调，也可能导致TRXR1的损害。在这项研究建议中，我们旨在通过关注与TXNIP SERP和TRXR1损伤相关的事件来确定参与高血糖引起的TRX变化的分子机制。我们假设高血糖诱导的TRX损伤涉及TXNIP和TRXR1蛋白硝化的PKC？依赖性丝氨酸磷酸化。我们设计了在暴露于Hg的REC（糖尿病的主要靶标）中进行体外进行的实验，并通过使用链霉菌素诱导的糖尿病大鼠（STZ-鼠（STZ-鼠），一种1型糖尿病的模型）进行体内。 AIM1））检验Hg/糖尿病促进TXNIP的PKC依赖性丝氨酸磷酸化（SERP）的假设，这种作用有助于氧化性视网膜内皮细胞损伤。 AIM 2））检验Hg/糖尿病诱导TRXR1蛋白硝化的假设，这种作用有助于氧化性视网膜内皮细胞损伤。 公共卫生相关性：氧化应激（OX）在糖尿病性视网膜病的发病机理中起关键作用，这是全球失明的主要原因。高血糖诱导的硫氧还蛋白内源性抗氧化剂系统（TRX）的功能障碍与糖尿病诱导的OX和视网膜组织损伤有关。该研究提案着重于确定TRX的糖尿病诱导的分子修饰，因为这些促进了高血糖诱导的OX，视网膜组织损伤和糖尿病性视网膜病的发育。