Carotinoids in Vision

类胡萝卜素对视力的影响

基本信息

批准号：
8037864
负责人：
Johannes Friedrich von Lintig
金额：
$ 37.83万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8037864
关键词：
11 cis Retinal Active Sites Address Affect Age related macular degeneration Amino Acid Substitution Amino Acids Animals Binding Biochemical Biological Carotenoids Catalysis Cell Line Characteristics Chemicals Chemistry Cleaved cell Diet Dimerization Dissection Enzymes Event Exposure to Family Family member G-Protein-Coupled Receptors Gene Targeting Genes Genetic Polymorphism Human Image Insecta Integral Membrane Protein Intervention Isomerase Knowledge Lead Leber&apos s amaurosis Light Lipid Bilayers Lipids Mediating Membrane Membrane Proteins Metabolic Pathway Metabolism Missense Mutation Modification Molecular Molecular Structure Mutation Opsin Oxygenases Pathway interactions Patients Pharmacotherapy Photoreceptors Phototransduction Physiological Prevention Process Production Proteins RPE65 protein Reaction Recombinants Recycling Research Resolution Retinal Retinal Diseases Retinal Dystrophy Retinal Pigments Retinitis Pigmentosa Retinoids Role Site-Directed Mutagenesis Stimulus Structure Structure of retinal pigment epithelium Substrate Interaction Testing Translating Variant Vertebrates Vision Visual Vitamin A base chromophore design enzyme substrate gene replacement human disease immunocytochemistry improved insight membrane activity mutant null mutation palmitoylation response retinol isomerase visual cycle

项目摘要

DESCRIPTION (provided by applicant): Metabolic pathways for production and recycling of the visual chromophore, retinal, are essential for vision. In vertebrates, the key recycling reaction, namely trans-to-cis isomerization, depends on the retinal pigmented epithelium protein, RPE65. Mutations in RPE65 lead to a spectrum of retinal dystrophies ranging from Leber congenital amaurosis (LCA) to autosomal recessive retinitis pigmentosa (RP). Aside from null mutations, RPE65 missense mutations may affect protein stability, catalytic activity and membrane association. However, without sufficient structural information it is impossible to truly understand the functioning of RPE65 and the consequences of these mutations. Recent biochemical evidence indicates that the RPE65-related insect carotenoid-oxygenase, NinaB, catalyzes a combined carotenoid cleavage and trans-to-cis isomerization reaction. Thus, comparing the structures of RPE65 and NinaB constitutes a unique challenge that will contribute to our understanding of RPE65 function and help delineate the consequences of amino acid substitutions in this critical enzyme. The robust enzymatic activity of NinaB will allow translating structural predications into functional testing of key residues for trans-to-cis-isomerization, oxidative cleavage, enzyme/substrate and membrane interactions for this class of proteins. In this context we propose three specific aims involving structural, biochemical and physiological approaches to analyze the structure and function of retinoid- and carotenoid-converting enzymes. In Aim 1, we will compare the molecular structures of RPE65 and NinaB. Determining the crystal structure of native RPE65 at 2.14 E resolution was a breakthrough critical to this endeavor. Now we propose to determine the structure of recombinant NinaB. Comparing structures of RPE65 and NinaB will allow identification of critical residues related to the oxidative cleavage reaction, isomerase reaction, substrate interactions and membrane association. In Aim 2, the structural basis for membrane association of RPE65 and NinaB will be elucidated. Based on the structural analysis of RPE65, we will test the roles of palmitoylation, hydrophobic protein/membrane interactions and dimerization. In Aim 3, a structure-based dissection of isomerase and oxygenase activities will be accomplished. Due to structural conservation of carotenoid-oxygenases and RPE65, comparisons of the substrate binding regions provide an opportunity to indentify amino acid residues required for the all-trans to 11-cis isomerase reaction. Aside from contributing fundamentally to understanding the chemistry of vision, this approach will help delineate the consequences of amino acid substitutions in these enzymes that are associated with impaired ocular vitamin A metabolism. Such knowledge also could translate into improved therapies for patients with disabling mutations in RPE65. PUBLIC HEALTH RELEVANCE: Comprehensive knowledge of biological structures and molecular events that comprise their mechanism of action is needed to improve our understanding and treatment of retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). But molecular understanding of key enzymes in the visual cycle is still lacking. The objective of this research is to elucidate the pathway for chromophore production from carotenoids including its trans-to-cis isomerization.

描述（由申请人提供）：视网膜视觉成色团生产和回收的代谢途径对于视觉至关重要。在脊椎动物中，关键的回收反应，即反式 - cis异构化，取决于视网膜色素上皮蛋白RPE65。 RPE65的突变导致视网膜营养不良的范围从Leber先天性症（LCA）到常染色体隐性视网膜炎色素（RP）。除无效突变外，RPE65错义突变可能会影响蛋白质稳定性，催化活性和膜关联。但是，如果没有足够的结构信息，就无法真正理解RPE65的功能和这些突变的后果。最近的生化证据表明，与RPE65相关的昆虫类胡萝卜素氧合酶（Ninab）催化了类胡萝卜素裂解和反式 - cis异构化反应。因此，比较RPE65和NINAB的结构构成了一个独特的挑战，这将有助于我们对RPE65功能的理解，并有助于描述这种关键酶中氨基酸取代的后果。 NINAB的鲁棒酶活性将允许将结构谓词转化为关键残基的功能测试，以进行反式 - Cis-异构化，氧化性裂解，酶/底物和此类蛋白质的膜相互作用。在这种情况下，我们提出了三个涉及结构，生化和生理方法的特定目的，以分析类维生素类和类胡萝卜素转化酶的结构和功能。在AIM 1中，我们将比较RPE65和NINAB的分子结构。在2.14 E分辨率下确定天然RPE65的晶体结构对于这项工作至关重要。现在，我们建议确定重组NINAB的结构。比较RPE65和NINAB的结构将允许鉴定与氧化裂解反应，异构酶反应，底物相互作用和膜关联有关的关键残基。在AIM 2中，将阐明RPE65和NINAB的膜关联的结构基础。根据RPE65的结构分析，我们将测试棕榈酰化，疏水蛋白/膜相互作用和二聚化的作用。在AIM 3中，将完成基于结构的异构酶和氧合酶活性的解剖。由于类胡萝卜素 - 氧化酶和RPE65的结构保守性，底物结合区的比较为缩进全反式氨基酸对11-CIS异构酶反应所需的氨基酸残基提供了机会。除了从根本上为了解视力的化学作用做出贡献外，这种方法还将有助于描述这些酶中与眼部维生素A代谢受损相关的氨基酸取代的后果。这种知识还可以转化为改善RPE65残疾突变患者的疗法。公共卫生相关性：需要对构成其作用机制的生物结构和分子事件的全面知识，以提高我们对视网膜炎色素炎（RP）（RP）和与年龄相关的黄斑变性（AMD）等视网膜疾病的理解和治疗。但是在视觉周期中对关键酶的分子理解仍然缺乏。这项研究的目的是阐明从类胡萝卜素（包括其反式 - cis异构化）产生发色团的途径。