Detoxification Role of Retinol Dehydrogenases RDH11 and RDH12

视黄醇脱氢酶 RDH11 和 RDH12 的解毒作用

• 批准号: 7530623
• 负责人: Anne Kasus-Jacobi
• 金额: $ 21.98万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7530623
• 关键词: 11 cis Retinal; Age related macular degeneration; Aldehydes; Antioxidants; Apoptosis; Blindness; Cell Fraction; Cell physiology; Clinical; Clinical Trials; Condition; Cultured Cells; Data; Diabetic Retinopathy; Disease; Drug Metabolic Detoxication; Enzymes; Exposure to; Genes; Hydrogen Peroxide; In Vitro; Incubated; Kinetics; Knock-out; Knockout Mice; Lead; Leber' s amaurosis; Length; Light; Lipid Peroxidation; Mass Spectrum Analysis; Measures; Mediator of activation protein; Methods; Modeling; Mus; Mutation; Names; Numbers; Oral; Oxidative Stress; Pathway interactions; Patients; Photoreceptors; Physiological; Polyunsaturated Fatty Acids; Process; Proteins; Public Health; Reaction; Recycling; Retina; Retinal; Retinal Diseases; Retinal Dystrophy; Retinaldehyde; Retinol dehydrogenase; Role; Source; Specificity; Standards of Weights and Measures; System; Testing; Thinking; Vision; Wild Type Mouse; adduct; base; cell injury; early onset; in vivo; innovation; mutant; novel; novel therapeutics; prevent; protective effect; visual cycle

DESCRIPTION (provided by applicant): This proposal focuses on a novel enzymatic pathway that reduces short-chain aldehydes, eliminating toxic byproducts of oxidative stress in photoreceptor cells. Previous studies have shown that photoreceptor retinol dehydrogenases RDH11 and RDH12 are able to reduce short-chain aldehydes and hydroxyaldehydes of various lengths and unsaturation. These molecules are end products of the lipid peroxidation of polyunsaturated fatty acids. 4-hydroxy-trans-2-nonenal (4-HNE) is one of the major aldehydic products and is an inducer and mediator of oxidative stress. The specificity of RDH11 and RDH12 for the short-chain (hydroxy)aldehydes has been largely overlooked until now and recent findings have prompted us to re-evaluate its significance for the function of these enzymes in the retina. First, it was found that 4-HNE-protein adducts accumulate in photoreceptors during oxidative stress. Then, it was found that the Rdh12 knockout mice are more sensitive to light-induced oxidative damage. Finally, we found a protective effect of RDH11 and RDH12 against 4-HNE-induced apoptosis and adduct formation in cell culture. Based on these findings, we hypothesize that RDH11 and RDH12 are effective at reducing 4-HNE and are therefore an important detoxification system in photoreceptor cells. We will use the Rdh11 and Rdh12 knockout mice to investigate the following Specific Aims: Aim 1 is to characterize the catalytic activities of RDH11 and RDH12 towards 4-HNE in vitro and in mouse retina. We will characterize the kinetics of 4- HNE reduction using microsomal fractions of cells transfected with wild-type Rdh11 and Rdh12 or Rdh12 mutants as a source of enzyme and using a highly accurate and sensitive mass spectrometry method to quantify the substrate and product of the reaction. We will also use microsomal fractions prepared from wild type, Rdh11, and Rdh12 knockout retinas to measure the contribution of each enzyme to the reduction of 4-HNE in vivo. Aim 2 is to determine whether RDH11 and RDH12 are protective against the formation of 4-HNE-protein adducts and the apoptosis of photoreceptors ex vivo and in vivo. Rdh12 knockout mice were found to be more sensitive to light-induced apoptosis of photoreceptors than the wild-type mice, but the mechanism that induces higher sensitivity is unknown. To determine the roles of RHD11 and RDH12 in this process, we will induce oxidative stress in whole retinal cultures with H2O2 and 4-HNE as well as in mice with constant bright light exposure. We will then quantify 4-HNE-protein adducts and photoreceptor apoptosis in these retinas from wild-type, Rdh11, and Rdh12 knockout mice. PUBLIC HEALTH RELEVANCE: If we successfully demonstrate that the physiological role of RDH11 and RDH12 is to detoxify 4-HNE in photoreceptor inner segments, this finding will have a significant clinical impact. First, because it will provide a strategy to treat patients with the early onset retinal dystrophy Leber Congenital Amaurosis, caused by mutations of the RDH12 gene. Second, such detoxification pathways will represent a potential target to slow down the progression of a number of other retinopathies involving oxidative stress.

描述（由申请人提供）：该提案重点关注一种新的酶促途径，该途径可减少短链醛，消除感光细胞中氧化应激的有毒副产物。先前的研究表明，光感受器视黄醇脱氢酶 RDH11 和 RDH12 能够还原各种长度和不饱和度的短链醛和羟基醛。这些分子是多不饱和脂肪酸脂质过氧化的最终产物。 4-羟基-反式-2-壬烯醛 (4-HNE) 是主要的醛类产品之一，是氧化应激的诱导剂和调节剂。迄今为止，RDH11 和 RDH12 对短链（羟基）醛的特异性在很大程度上被忽视，最近的发现促使我们重新评估其对视网膜中这些酶的功能的重要性。首先，我们发现4-HNE-蛋白加合物在氧化应激过程中在光感受器中积累。随后发现Rdh12基因敲除小鼠对光诱导的氧化损伤更加敏感。最后，我们发现 RDH11 和 RDH12 对细胞培养物中 4-HNE 诱导的细胞凋亡和加合物形成具有保护作用。基于这些发现，我们假设 RDH11 和 RDH12 可有效减少 4-HNE，因此是感光细胞中重要的解毒系统。我们将使用 Rdh11 和 Rdh12 敲除小鼠来研究以下具体目标： 目标 1 是在体外和小鼠视网膜中表征 RDH11 和 RDH12 对 4-HNE 的催化活性。我们将使用野生型 Rdh11 和 Rdh12 或 Rdh12 突变体转染的细胞微粒体部分作为酶源，并使用高度准确和灵敏的质谱方法来量化反应的底物和产物，来表征 4-HNE 还原的动力学。我们还将使用由野生型、Rdh11 和 Rdh12 敲除视网膜制备的微粒体级分来测量每种酶对体内 4-HNE 还原的贡献。目标 2 是确定 RDH11 和 RDH12 是否对离体和体内 4-HNE 蛋白加合物的形成以及光感受器细胞凋亡具有保护作用。研究发现 Rdh12 敲除小鼠比野生型小鼠对光诱导的光感受器凋亡更敏感，但诱导更高敏感性的机制尚不清楚。为了确定 RHD11 和 RDH12 在此过程中的作用，我们将在使用 H2O2 和 4-HNE 的整个视网膜培养物以及持续亮光照射的小鼠中诱导氧化应激。然后，我们将量化野生型、Rdh11 和 Rdh12 敲除小鼠视网膜中的 4-HNE 蛋白加合物和光感受器细胞凋亡。 公共健康相关性：如果我们成功证明 RDH11 和 RDH12 的生理作用是解毒感光感受器内节中的 4-HNE，这一发现将产生重大的临床影响。首先，因为它将提供一种治疗由 RDH12 基因突变引起的早发性视网膜营养不良（莱伯先天性黑蒙）患者的策略。其次，这种解毒途径将成为减缓许多其他涉及氧化应激的视网膜病变进展的潜在目标。