Carotinoids in Vision

描述（由申请人提供）：视觉发色团（视网膜）的产生和回收的代谢途径对视觉至关重要。在脊椎动物中，关键的再循环反应，即反式到顺式异构化，取决于视网膜色素上皮蛋白RPE 65。RPE 65中的突变导致一系列视网膜营养不良，范围从Leber先天性黑蒙（LCA）到常染色体隐性视网膜色素变性（RP）。除了无效突变之外，RPE 65错义突变可能影响蛋白质稳定性、催化活性和膜结合。然而，如果没有足够的结构信息，就不可能真正了解RPE 65的功能和这些突变的后果。最近的生物化学证据表明，RPE 65相关的昆虫类胡萝卜素加氧酶，NinaB，催化组合类胡萝卜素裂解和反式到顺式异构化反应。因此，比较RPE 65和NinaB的结构构成了一个独特的挑战，这将有助于我们理解RPE 65的功能，并有助于描述这种关键酶中氨基酸取代的后果。NinaB的强大酶活性将允许将结构预测转化为这类蛋白质的反式至顺式异构化、氧化裂解、酶/底物和膜相互作用的关键残基的功能测试。在这种情况下，我们提出了三个具体的目标，涉及结构，生化和生理的方法来分析类维生素A和类胡萝卜素转化酶的结构和功能。在目标1中，我们将比较RPE 65和NinaB的分子结构。在2.14 E分辨率下确定天然RPE 65的晶体结构是这一奋进的关键突破。现在我们建议确定重组NinaB的结构。比较RPE 65和NinaB的结构将允许鉴定与氧化裂解反应、异构酶反应、底物相互作用和膜缔合相关的关键残基。在目的2中，将阐明RPE 65和NinaB的膜缔合的结构基础。基于RPE 65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。在目标3中，将完成异构酶和加氧酶活性的基于结构的解剖。由于类胡萝卜素加氧酶和RPE 65的结构保守性，底物结合区的比较提供了一个机会，以确定所需的氨基酸残基的全反式到11-顺式异构酶反应。除了从根本上有助于理解视觉的化学，这种方法将有助于描述这些酶中氨基酸取代的后果，这些酶与眼部维生素A代谢受损有关。这些知识也可以转化为对RPE 65突变患者的改进疗法。 公共卫生相关性：需要全面了解生物结构和分子事件，包括其作用机制，以提高我们的理解和治疗视网膜疾病，如视网膜色素变性（RP）和年龄相关性黄斑变性（AMD）。但是对于视觉循环中关键酶的分子理解仍然缺乏。本研究的目的是阐明类胡萝卜素产生生色团的途径，包括其反式到顺式异构化。

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.