Detoxification Role of Retinol Dehydrogenases RDH11 and RDH12

描述(由申请人提供)：这项提案侧重于一种新的酶途径，它可以减少短链醛，消除光感受器细胞中氧化应激的有毒副产物。以前的研究表明，光感受器视黄醇脱氢酶RDH11和RDH12能够还原不同长度和不饱和的短链醛和羟醛。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)是一种重要的醛类化合物，是氧化应激的诱导物和中介物。到目前为止，RDH11和RDH12对短链(羟基)醛的特异性在很大程度上被忽视了，最近的发现促使我们重新评估它对这些酶在视网膜中功能的意义。首先，研究发现，在氧化应激过程中，4-HNE-蛋白质加合物在光感受器中积累。后来发现，RDH12基因敲除小鼠对光诱导的氧化损伤更敏感。最后，我们发现RDH11和RDH12对4-HNE诱导的细胞凋亡和加合物形成有保护作用。基于这些发现，我们推测RDH11和RDH12在减少4-HNE方面是有效的，因此是光感受器细胞中一个重要的解毒系统。我们将使用RDH11和RDH12基因敲除小鼠来研究以下特定目的：目的1研究RDH11和RDH12在体外和小鼠视网膜中对4-HNE的催化活性。我们将使用野生型RDH11和RDH12或RDH12突变体转染的细胞的微粒体部分作为酶的来源，并使用高精度和灵敏的质谱学方法来定量底物和反应产物，来表征4-HNE还原的动力学。我们还将使用野生型、RDH11和RDH12基因敲除视网膜制备的微粒体组分，来测量每种酶在体内对4-HNE还原的贡献。目的2确定RDH11和RDH12是否对4-HNE蛋白加合物的形成和体内外光感受器的凋亡具有保护作用。RDH12基因敲除小鼠比野生型小鼠对光诱导的光感受器细胞凋亡更敏感，但诱导更高敏感性的机制尚不清楚。为了确定RHD11和RDH12在这一过程中的作用，我们将用过氧化氢和4-HNE诱导整个视网膜培养物以及持续强光暴露的小鼠的氧化应激。然后，我们将量化这些野生型、RDH11和RDH12基因敲除小鼠视网膜中的4-HNE蛋白加合物和光感受器凋亡。 公共卫生相关性：如果我们成功地证明RDH11和RDH12的生理作用是使光感受器内节段的4-HNE解毒，这一发现将对临床产生重大影响。首先，因为它将为治疗由RDH12基因突变引起的早发性视网膜营养不良Leber先天性黑色素沉着症患者提供一种策略。其次，这种解毒途径将是减缓其他一些涉及氧化应激的视网膜疾病进展的潜在靶点。

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.