PRO-APOPTOTIC GAPDH NUCLEAR ACCUMULATION AND RETINAL CELL DEATH

促凋亡 GAPDH 核积聚和视网膜细胞死亡

• 批准号: 7320954
• 负责人: E Chepchumba K Yego
• 金额: $ 2.84万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7320954
• 关键词: Apoptotic; Biological Assay; Blindness; Caspase-1; Cell Death; Cell Nucleus; Cells; Co-Immunoprecipitations; Complex; Condition; Development; Diabetes Mellitus; Diabetic Retinopathy; Disease; Drug usage; Enzymes; Event; Glucose; Glyceraldehyde-3-Phosphate Dehydrogenases; Homologous Gene; Hyperglycemia; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Inflammatory; Interleukin-1; Interleukin-1 Receptors; Knock-out; Knockout Mice; Laboratories; Ligase; Lipopolysaccharides; Mediating; Modification; Muller' s cell; Mus; Neurodegenerative Disorders; Neuroglia; Neurons; Nuclear; Nuclear Localization Signal; Pathway interactions; Post-Translational Protein Processing; Process; Production; Proteins; Proteomics; Retina; Retinal; Role; Signal Transduction; Testing; Western Blotting; autocrine; cytokine; deprenyl; diabetic; in vivo; macrophage; prevent; receptor; retinal apoptosis; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Diabetic retinopathy is a leading cause of blindness world wide. Currently there are no therapies for the disease. Therefore, understanding mechanisms underlying the development of diabetic retinopathy will help to facilitate the development of effective therapies. Retinal cell death which occurs through an unknown high glucose-induced mechanism contributes to the disease. Nuclear accumulation of the glycolytic enzyme, glyceraldehyde-3- phosphate dehydrogenase (GAPDH), a cytosolic enzyme is an early pro-apoptotic event. Previously, our laboratory has shown that GAPDH nuclear accumulation occurs in retinal Muller (glial) cells under hyperglycemic conditions in vitro and in vivo. Since GAPDH lacks a nuclear localization signal (NLS), the exact mechanism for GAPDH nuclear accumulation is indefinite. However, recent studies have proposed a role for the NLS containing E3 ubiquitin ligase seven in absentia homolog-1 (siah-1) during lipopolysaccharide (LPS)-induced pro-apoptotic GAPDH nuclear accumulation in neurons and macrophages. These studies have suggested that IPS induced post-translational modifications of GAPDH, such as S-nitrosylation, thereby promoting GAPDH/siah-1 interaction and possible complex formation. NLS containing siah-1 then translocates in a complex with GAPDH to the nucleus. This notion is appealing since LPS is a major inducer of pro- inflammatory cytokine production, such as that of interleukin-1 p (IL-1p), and our studies have shown that IL- 1p signaling mediates high glucose-induced GAPDH nuclear accumulation in vitro. It is an intriguing idea that high glucose leads to IL-1 p mediated complex formation of GAPDH and siah-1 and translocation of this complex to the nucleus. Therefore, this project will assess (1) GAPDH/siah-1 complex formation under high glucose conditions in retinal Muller cells possibly mediated by post-translational modifications of GAPDH, and (2) whether IL-1 p mediates high glucose-induced GAPDH/siah-1 complex formation in vitro and in vivo. For theses studies cytosolic and nuclear siah-1 will be evaluated using Western blot and immunofluorescence analysis following high glucose and cytokine treatment. We will also utilize co- immunoprecipitation assays to identify GAPDH/siah-1 complex formation and proteomics analysis to test for possible post-translational modification(s) of GAPDH, which might facilitate complex formation with siah-1. We will also use diabetic caspase-1 and IL-1 receptor knock-out mice to assess the contribution of IL-1-3 to high glucose-induced GAPDH nuclear accumulation in retinal Muller cells in vivo.

描述(申请人提供)：糖尿病视网膜病变是世界范围内导致失明的主要原因。目前还没有治疗这种疾病的方法。因此，了解糖尿病视网膜病变的发病机制将有助于开发有效的治疗方法。视网膜细胞死亡是通过一种未知的高糖诱导机制导致的疾病。糖酵解酶-3-磷酸甘油醛脱氢酶(GAPDH)是一种胞质酶，它的核积聚是一种早期的促凋亡事件。此前，我们的实验室已经证明，在体外和体内的高血糖条件下，视网膜Muller(神经胶质)细胞中存在GAPDH核积聚。由于GAPDH缺乏核定位信号(NLS)，因此GAPDH核积累的确切机制尚不清楚。然而，最近的研究表明，含有E3泛素连接酶7的NLS在内毒素(LPS)诱导的神经元和巨噬细胞中促凋亡的GAPDH核聚集过程中可能起到了缺失同源1(siah-1)的作用。这些研究表明，IPS诱导了谷氨酸脱氢酶的翻译后修饰，如S-亚硝化，从而促进了谷氨酸脱氢酶/siah-1的相互作用和可能的复合体的形成。然后，含有siah-1的NLS在与GAPDH的复合体中移位到细胞核。这一观点很有吸引力，因为内毒素是促炎细胞因子产生的主要诱导者，如白细胞介素1β(IL-1p)，我们的研究表明，IL-1p信号通路在体外介导了高糖诱导的GAPDH核积聚。一个有趣的观点是，高糖导致IL-1β介导的GAPDH和SIAH-1复合体的形成并将该复合体移位到细胞核。因此，本项目将评估(1)高糖条件下视网膜Muller细胞GAPDH/siah-1复合体的形成可能是由GAPDH翻译后修饰介导的，以及(2)IL-1β是否在体外和体内介导高糖诱导的GAPDH/siah-1复合体的形成。对于这些研究，在高糖和细胞因子治疗后，将使用Western印迹和免疫荧光分析来评估胞浆和核SIAH-1。我们还将利用免疫共沉淀实验鉴定GAPDH/SIAH-1复合体的形成，并进行蛋白质组学分析以测试GAPDH可能的翻译后修饰(S)，这可能有助于与SIAH-1形成复合体。我们还将使用糖尿病caspase-1和IL-1受体基因敲除小鼠来评估IL-1-3在体内高糖诱导的视网膜Muller细胞GAPDH核积聚中的作用。