Regulation of normal and Leber congenital amaurosis-associated RPE65s

正常和 Leber 先天性黑蒙相关 RPE65 的调节

• 批准号: 8307780
• 负责人: Minghao Jin
• 金额: $ 35.5万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8307780
• 关键词: 11 cis Retinal; 11-cis-Retinol; 26S proteasome; Acyl Coenzyme A; Binding; Biochemical; Biochemical Reaction; Blindness; Cattle; Cells; Childhood; Coenzyme A Ligases; Cyclic GMP; Degenerative Disorder; Development; Enzymes; Esters; Exhibits; Expression Library; Gated Ion Channel; Genes; Goals; Human; Hyperactive behavior; In Vitro; Isomerase; Isomerism; Knock-in Mouse; Knock-out; Knowledge; Lead; Leber' s amaurosis; Light; Link; Membrane; Membrane Proteins; Missense Mutation; Molecular; Mus; Mutant Strains Mice; Mutation; Natural regeneration; Opsin; Pathogenicity; Pathway interactions; Patients; Phenotype; Photoreceptors; Phototransduction; Pigments; Proteins; RPE65 protein; Regulation; Research; Research Project Grants; Retinal Degeneration; Retinal Pigments; Retinoids; Rhodopsin; Role; Screening procedure; Series; Signal Transduction; Site; Structure of retinal pigment epithelium; Testing; Therapeutic Intervention; Ubiquitin; Very Long Chain Fatty Acid; Vision; base; cold temperature; early onset; expression cloning; fatty acid-transport protein; in vivo; inhibitor/antagonist; innovation; loss of function; multicatalytic endopeptidase complex; mutant; novel; photoreceptor degeneration; prevent; response; retinol isomerase; retinyl palmitate; visual cycle

DESCRIPTION (provided by applicant): The visual cycle is a series of enzymatic reactions essential for regenerating 11-cis retinal (11cRAL), which functions as a molecular switch for activating opsin in response to light stimulation. 11cRAL keeps opsin inactive. When light hits the visual pigments, its energy converts the pigments' 11cRAL into all-trans isomer, thus activating opsin. The activated opsin then triggers phototransduction, which converts the light-activated biochemical signal to an electrical energy by closing the cGMP-gated ion channels in the photoreceptors. Since the opsin that lost 11cRAL is no longer responsive to light, 11cRAL must be regenerated via the visual cycle to re-form the light-sensitive visual pigments. The key enzymatic reaction in the visual cycle is the isomerization of the all-trans retinoid to an 11-cis isomer. We have identified RPE65, a retinal pigment epithelium (RPE) specific membrane-associated protein, as the retinoid isomerase. Mutations in the human RPE65 gene have been associated with an early-onset retinal degenerative disease known as Leber congenital amaurosis (LCA). Despite the importance of this protein, the molecular mechanisms that regulate the function of RPE65 are largely unknown. The long-term goals of our research are (1) to define the mechanisms that regulate the enzymatic activity of normal and LCA-associated (LCAA) RPE65s and (2) to develop a novel and effective strategy for rescuing LCAA RPE65s. Identification and characterization of proteins that regulates RPE65 function is the key to defining the mechanisms. By screening of the bovine RPE expression library, we have identified three negative regulators of RPE65. The goal of Specific Aim 1 of this proposal is to elucidate the action mechanisms of the negative regulators in vitro and determine their functional role in the visual cycle in vivo. As a step toward defining the inhibitory mechanisms of RPE65 by the regulators, we will first test if the new regulators bind with RPE65. Next we will test if the new regulator can compete with RPE65 for binding to the substrate of RPE65. We will then determine the role of the new regulator in vision by analyzing the visual cycle-related phenotypes of the mutant mice that lack the new regulator. The goal of Specific Aim 2 of this project is to determine whether the new regulators are involved in the pathological mechanism of the LCAA RPE65 mutations and to define the molecular basis for the pathogenicity of LCAA RPE65s with non-active site missense mutations. The proposed research is innovative because it can establish a new research direction in regulation of isomerase activities of normal and LCAA RPE65s and can lead to the development of a novel rescue strategy for delaying or preventing vision loss and photoreceptor degeneration in patients with LCA that is caused by mutations in the RPE65 gene.

描述（由申请人提供）：视觉循环是再生11-顺式视网膜（11 cRAL）所必需的一系列酶促反应，其作为响应光刺激激活视蛋白的分子开关发挥作用。11 cRAL保持视蛋白不活跃。当光照射到视色素时，其能量将色素的11 cRAL转化为全反式异构体，从而激活视蛋白。激活的视蛋白然后触发光转导，其通过关闭光感受器中的cGMP门控离子通道将光激活的生化信号转化为电能。由于失去11 cRAL的视蛋白不再对光有反应，因此11 cRAL必须通过视觉循环再生以重新形成光敏视色素。视觉循环中的关键酶促反应是全反式维甲酸异构化为11-顺式异构体。我们已经确定RPE 65，视网膜色素上皮（RPE）特异性膜相关蛋白，作为类维生素A异构酶。人类RPE 65基因的突变与称为Leber先天性黑蒙（LCA）的早发性视网膜变性疾病有关。尽管这种蛋白质的重要性，但调节RPE 65功能的分子机制在很大程度上是未知的。我们研究的长期目标是：（1）确定调节正常和LCA相关（LCAA）RPE 65酶活性的机制;（2）开发一种新的有效的拯救LCAA RPE 65的策略。识别和表征调节RPE 65功能的蛋白质是定义机制的关键。通过对牛RPE表达文库的筛选，我们鉴定了RPE 65的三个负调控因子。本提案的具体目标1的目标是阐明体外负调节剂的作用机制，并确定其在体内视觉周期中的功能作用。作为通过调节剂定义RPE 65的抑制机制的一步，我们将首先测试新的调节剂是否与RPE 65结合。接下来，我们将测试新的调节剂是否可以与RPE 65竞争结合RPE 65的底物。然后，我们将通过分析缺乏新调节剂的突变小鼠的视觉周期相关表型来确定新调节剂在视觉中的作用。本项目的具体目标2的目标是确定新的调节因子是否参与LCAA RPE 65突变的病理机制，并确定具有非活性位点错义突变的LCAA RPE 65致病性的分子基础。这项研究具有创新性，因为它可以在调节正常和LCAA RPE 65的异构酶活性方面建立一个新的研究方向，并可以导致开发一种新的拯救策略，用于延迟或预防由RPE 65基因突变引起的LCA患者的视力丧失和感光细胞变性。