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: 7689187
• 负责人: Anne Kasus-Jacobi
• 金额: $ 18.31万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7689187
• 关键词: 11 cis Retinal; Age related macular degeneration; Aldehydes; Antioxidants; Apoptosis; Blindness; Cell Culture Techniques; Cell Fraction; Cell physiology; Clinical; Clinical Trials; 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; Oral; Oxidative Stress; Pathway interactions; Patients; Photoreceptors; Physiological; Polyunsaturated Fatty Acids; Process; Proteins; Reaction; Recycling; Retina; Retinal; Retinal Diseases; Retinal Dystrophy; Retinaldehyde; Retinol dehydrogenase; Role; Source; Specificity; System; Testing; Vision; Wild Type Mouse; adduct; base; cell injury; early onset; in vivo; innovation; mutant; novel; novel therapeutics; oxidative damage; prevent; protective effect; public health relevance; 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）：该提案侧重于减少短链醛的新型酶途径，消除感光细胞中氧化应激的有毒副产物。先前的研究表明，光感受器视黄醇还原酶RDH 11和RDH 12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终产物。4-羟基-反式-2-壬烯醛（4-HNE）是一种主要的代谢产物，是氧化应激的诱导剂和介导剂。RDH 11和RDH 12对短链（羟基）醛的特异性迄今为止在很大程度上被忽视，最近的研究结果促使我们重新评估其对这些酶在视网膜中的功能的重要性。首先，发现4-HNE-蛋白加合物在氧化应激期间在光感受器中积累。然后，发现Rdh 12基因敲除小鼠对光诱导的氧化损伤更敏感。最后，我们发现了RDH 11和RDH 12对4-HNE诱导的细胞凋亡和加合物形成的保护作用。基于这些发现，我们假设RDH 11和RDH 12在减少4-HNE方面是有效的，因此是感光细胞中重要的解毒系统。我们将使用Rdh 11和Rdh 12基因敲除小鼠研究以下具体目的：目的1是表征Rdh 11和Rdh 12对4-HNE的体外和小鼠视网膜中的催化活性。我们将使用转染野生型Rdh 11和Rdh 12或Rdh 12突变体的细胞微粒体组分作为酶源，并使用高度准确和灵敏的质谱法定量反应的底物和产物，表征4- HNE还原的动力学。我们还将使用从野生型、Rdh 11和Rdh 12敲除视网膜制备的微粒体组分来测量每种酶对体内4-HNE减少的贡献。目的2是确定RDH 11和RDH 12是否对4-HNE-蛋白加合物的形成和离体和体内光感受器的凋亡具有保护作用。Rdh 12基因敲除小鼠被发现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RHD 11和RDH 12在这一过程中的作用，我们将在整个视网膜培养物中用H2 O2和4-HNE以及在小鼠中用恒定的强光照射诱导氧化应激。然后，我们将量化4-HNE-蛋白加合物和感光细胞凋亡，这些视网膜从野生型，Rdh 11，Rdh 12基因敲除小鼠。 公共卫生关系：如果我们成功地证明RDH 11和RDH 12的生理作用是在感光细胞内节中解毒4-HNE，这一发现将具有重大的临床影响。首先，因为它将提供一种治疗由RDH 12基因突变引起的早发性视网膜营养不良Leber先天性黑蒙患者的策略。其次，这种解毒途径将代表一个潜在的目标，以减缓其他视网膜病变的进展，涉及氧化应激。