Carotinoids in Vision

描述(由申请人提供)：产生和再循环视觉生色团视网膜的代谢途径对视力是必不可少的。在脊椎动物中，关键的循环反应，即反式到顺式异构化，依赖于视网膜色素上皮蛋白RPE65。RPE65基因突变导致一系列视网膜营养不良，从Leber先天性黑色素沉着症(LCA)到常染色体隐性遗传性视网膜色素变性(RP)。除了零突变外，RPE65错义突变还可能影响蛋白质的稳定性、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的功能以及这些突变的后果。最近的生化证据表明，与RPE65相关的昆虫类胡萝卜素加氧酶NINaB催化类胡萝卜素裂解和反式-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，这将有助于我们理解RPE65的功能，并有助于描述这一关键酶中氨基酸替换的后果。NINaB强劲的酶活性将使结构预测转化为对这类蛋白质的反式到顺式异构化、氧化切割、酶/底物和膜相互作用的关键残基的功能测试。在此背景下，我们提出了三个具体的目标，涉及结构，生化和生理方法来分析类维A酸类和类胡萝卜素转换酶的结构和功能。在目标1中，我们将比较RPE65和NINaB的分子结构。在2.14E分辨率下确定天然RPE65的晶体结构是这一努力的关键突破。现在，我们建议确定重组NINaB的结构。通过比较RPE65和NINAB的结构，可以确定与氧化切割反应、异构酶反应、底物相互作用和膜结合相关的关键残基。在目标2中，将阐明RPE65和NINaB膜结合的结构基础。基于对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。在目标3中，将完成对异构酶和加氧酶活性的基于结构的剖析。由于类胡萝卜素加氧酶和RPE65的结构保守，底物结合区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。除了从根本上帮助理解视觉的化学成分，这种方法还将有助于描述这些酶中氨基酸替换的后果，这些氨基酸替换与眼睛维生素A代谢受损有关。这些知识也可以转化为对RPE65基因突变患者的改进治疗。

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.