Cone opsins in photoreceptor degeneration

描述(申请人提供)：维甲酸异构酶，RPE65，和卵磷脂-视黄醇酰基转移酶(LRAT)在视网膜色素上皮(RPE)中11顺式视网膜循环中起重要作用。任何一种基因的突变都会导致Leber先天性黑色素沉着症(LCA)，这是一种遗传性视网膜退行性疾病，特征是儿童时期严重丧失视力，视锥细胞早期退化，随后是视杆细胞。RPE65-/-和Lrat-/-两个模型概括了主要的病理特征，其中缺少11顺式视网膜。然而，在小鼠模型和人类患者中，导致锥体早期退化的机制还不是很清楚。具体地说，尚不清楚为什么小鼠模型中的腹锥体和中央锥体比背锥体死亡得更快。同样，目前尚不清楚患者早期丧失蓝锥功能的原因。本应用的目的是使用LRAT-/-模型来确定LCA中锥体快速退变的机制。我们的中心假设是视锥视蛋白决定了LCA中视锥感光感受器退行性变的速度。我们根据我们的初步数据提出了这一假设，该假说表明短波长的OPTIN比中/长波长的OPTIN更容易聚集/聚集，从而触发内质网(ER)应激。在目标1中，我们将通过基因缺失S视蛋白或M-视蛋白来分析S和M-视蛋白在大鼠视锥变性中的相对作用。在目标2中，我们将确定生色团剥夺导致S视蛋白聚集而不是M视蛋白聚集的结构基础。我们在短波长视蛋白家族(SW1)中发现了一个富含苯丙氨酸的区域，但在中/长波长视蛋白家族中缺失。由于芳香族残基通过相互作用引导自组装在蛋白质的聚集中发挥重要作用，我们假设S视蛋白中的苯丙氨酸富集区负责其在Lrat-/-锥体中的聚集。我们设计了两个子目标来检验这一假设。分目标2A；通过基因打靶将S视蛋白(70-125)的苯丙氨酸富集区与M-视蛋白(86-141)的同源区互换。子目标2B，通过破坏苯丙氨酸富集区之间的相互作用，抑制S视蛋白与N-(1，4-二氢-1，4-二氧代-2-萘基)-L色氨酸(NQTrp)的聚集。我们期望这些研究能够明确为什么在RPE65/LRAT-LCA中S锥体比M-锥体退化更快的机制。 公共卫生相关性：这些研究将确定为什么在RPE65/LRAT-LCA中S锥体比M-锥体退化更快的机制。所提出的视锥蛋白聚集导致视锥细胞快速变性的机制可用于设计新的治疗LCA的药物。例如，NQTrp有可能通过阻断视锥细胞视蛋白聚集而成为开发新型治疗LCA药物的先导。

DESCRIPTION (provided by applicant): Retinoid isomerase, RPE65, and Lecithin-retinol acyltransferase (LRAT) are important in recycling 11-cis- retinal in retinal pigment epithelium (RPE). Mutations in either gene lead to Leber congenital amaurosis (LCA), an inherited retinal degenerative disease characterized by severe loss of vision in childhood and early degeneration of cones followed by rods. The main pathologic features are recapitulated by two models, Rpe65-/- and Lrat-/- in which 11-cis-retinal is lacking. However, the mechanisms responsible for early cone degeneration in both mouse models and human patients are not well understood. Specifically, it is unclear why ventral and central cones in mouse models die much more rapidly than dorsal cones. Similarly, it is unclear why blue cone function is lost early in patients. The objective of this application is to define the mechanism responsible for the rapid cone degeneration in LCA using the Lrat-/- model. Our central hypothesis is that cone opsin determines the rate of cone photoreceptor degeneration in LCA. We formulated this hypothesis based on our preliminary data showing the short-wavelength (SW) opsins are prone to aggregation/accumulation triggering endoplasmic reticulum (ER) stress than the medium/long-wavelength opsin. In Aim 1, we will dissect the relative contributions of S and M opsin to cone degeneration in Lrat-/- mice by genetically deleting S-opsin or M-opsin. In Aim 2, we will determine the structural basis on why chromophore deprivation causes S-opsin but not M-opsin aggregation. We identified a phenylalanine-rich region in the short-wavelength opsin family (SW1) but absent from the medium/long-wavelength opsin family. Since aromatic residues play a significant role in the aggregation of proteins by directing self-assembly via ¿-¿ interactions, we hypothesize that the phenylalanine-rich region in the S-opsin is responsible for its aggregation in Lrat-/- cones. We designed two sub-aims to test this hypothesis. Sub-aim 2A; swap the phenylalanine-rich region in the S-opsin (70-125) with the homologous region of M-opsin (86-141) by gene targeting. Sub-aim 2B, inhibit S-opsin aggregation with N- (1, 4-dihydro-1, 4-dioxo-2-naphthalenyl)-L-tryptophan (NQTrp) by disrupting the ¿-¿ interactions between the phenylalanine-rich region. We expect these studies will define the mechanism on why S-cones degenerate faster than M-cones in RPE65/LRAT-LCA. PUBLIC HEALTH RELEVANCE: These studies will define the mechanism on why S-cones degenerate faster than M-cones in RPE65/LRAT- LCA. The proposed mechanism that cone opsin aggregation is responsible for rapid cone degeneration can be exploited in designing novel therapeutic agents in the treatment of LCA. For example, NQTrp has the potential to serve as a lead for developing a new class of therapeutic drugs for LCA by blocking cone opsin aggregation.