Carotinoids in Vision

描述（由申请人提供）：视觉发色团，视网膜的产生和循环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RPE65。RPE65基因突变可导致一系列视网膜营养不良，从莱伯氏先天性黑朦（LCA）到常染色体隐性视网膜色素变性（RP）。除了零突变外，RPE65错义突变还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RPE65的功能和这些突变的后果。最近的生物化学证据表明，rpe65相关的昆虫类胡萝卜素加氧酶NinaB催化了类胡萝卜素的裂解和反式到顺式异构化反应。因此，比较RPE65和NinaB的结构构成了一个独特的挑战，这将有助于我们了解RPE65的功能，并有助于描述这种关键酶中氨基酸取代的后果。NinaB强大的酶活性将允许将结构预测转化为这类蛋白质的反式到顺式异构化、氧化裂解、酶/底物和膜相互作用的关键残基的功能测试。在此背景下，我们提出三个具体目标涉及结构，生化和生理学的方法来分析类维甲酸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分辨率下确定原生RPE65的晶体结构是这一努力的关键突破。现在我们打算确定重组NinaB的结构。比较RPE65和NinaB的结构将有助于鉴定与氧化裂解反应、异构酶反应、底物相互作用和膜结合有关的关键残基。在Aim 2中，将阐明RPE65与NinaB膜结合的结构基础。基于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RPE65的结构保守性，对底物结合区域的比较为鉴定全反式到11顺式异构酶反应所需的氨基酸残基提供了机会。除了从根本上有助于理解视觉的化学性质外，这种方法还将有助于描述与眼部维生素A代谢受损相关的这些酶中氨基酸取代的后果。这些知识也可以转化为改进RPE65致残突变患者的治疗方法。

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.