Molecular Biology Of Outer Retina-specific Proteins

视网膜色素上皮（RPE）在外视网膜的发育和功能中起着关键作用。我们对调控和功能层面的rpe特异性机制都很感兴趣。为此，我们一直在研究RPE65的功能和调控，该基因的表达仅限于RPE，其突变会导致人类严重失明。Rpe65基因敲除小鼠表型的基础似乎是Rpe65基因敲除小鼠的全反式视黄醇酯到11-顺式视网膜的基于rpe的维生素A视觉周期代谢的破坏。因此，RPE65的功能似乎与视黄醇异构酶的功能有关，视黄醇异构酶是视觉色素再生的关键酶。我们还继续研究了β -胡萝卜素15,15'-单加氧酶（β - cm）。β - cm与RPE65密切相关，两者都是新出现的类胡萝卜素裂解酶家族的成员。在过去的一年中，我们取得了以下进展：a)通过对β - cm中假定的金属结合残基进行定点诱变，进一步研究了类胡萝卜素裂解酶家族所有成员（包括β - cm和RPE65）中保守残基的作用。数据证明了一个关键的作用，在酶的活性组氨酸和酸性残基假设参与金属配位。电感耦合等离子体原子发射光谱显示，4个保守组氨酸和一个保守谷氨酸残基的突变导致铁结合的丧失。其他一些非绝对保守残基的突变导致酶活性受损，主要是突变酶的Vmax改变。b) RPE65含有两个潜在的洞穴蛋白相互作用结构域，在所有物种中都是保守的，并且具有功能活性。Caveolin-1是一种已知参与膜运输、脂质转运和信号转导的“支架”蛋白。它与RPE65的相互作用可能涉及部分或全部这些角色。我们发现RPE65结合到caveolin-1的支架结构域和c端结构域。这种相互作用，连同先前显示的与磷脂的相互作用，可能允许RPE65与膜锚定的视觉周期组分相关联，并为视觉周期复合物的研究提供了新的焦点。为了研究这种可能性，已经获得了小窝蛋白敲除小鼠，并正在测试其视觉等方面的表型变化。c)携带含有人类致病性RPE65突变的转基因小鼠已被培育到RPE65敲除背景上，并正在测试其表型。已经生成了几个不同的行。通过这种方式，希望能够产生非无效的RPE65相关视网膜营养不良模型（如敲除小鼠和Briard犬）d)正在寻找与RPE65基因启动子中转录元件结合的推定因子的身份。这些因子的表达克隆正在测试它们对RPE65启动子激活的影响。e)继续合作，通过aav介导的基因转移挽救Rpe65基因敲除小鼠表型和Briard犬Rpe65营养不良。

The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. To this end we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE and mutations in which cause severe blindness in humans. Disruption of the RPE-based vitamin A visual cycle metabolism of all-trans-retinyl esters to 11-cis-retinal appears to underlie the phenotype of the Rpe65 knockout mouse. The function of RPE65 thus appears to be associated with that of the retinol isomerase, the crucial enzyme in visual pigment regeneration. We have also continued studies on beta-carotene 15,15'-monooxygenase (beta-CM). Beta-CM is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. In the past year we have made the following progress: a) The role of residues conserved in all members of the carotenoid-cleavage enzyme family (including beta-CM and RPE65) has been further investigated by site-directed mutagenesis of putative metal binding residues in beta-CM. The data demonstrate a crucial role in enzymatic activity for histidine and acidic residues hypothesized to be involved in metal coordination. Mutation of the 4 conserved histidine and a conserved glutamate residue results in loss of iron-binding as shown by inductively coupled plasma atomic emission spectrometry. Mutation of certain other non-absolutely conserved residues results in impaired but active enzyme, chiefly resulting in Vmax changes of mutant enzymes. b) RPE65 contains two potential caveolin-interaction domains that are conserved in all species and are functionally active. Caveolin-1 is a "scaffolding" protein known to be involved in membrane trafficking, lipid transport and in signal transduction. Its interaction with RPE65 may involve some or all of these roles. We have found that RPE65 binds to both the scaffolding domain and the C-terminal domain of caveolin-1. This interaction, along with the previously shown interaction with phospholipids, may allow for the association of RPE65 with membrane anchored visual cycle components and provides a new focus for study of the visual cycle complex. To investigate such possibilities, caveolin knockout mice have been obtained and are being tested for phenotypic changes in vision, etc. c) Transgenic mice bearing transgenes incorporating mutations in RPE65 identified as pathogenic in humans have been bred onto the Rpe65 knockout background and are being tested as to their phenotypes. Several different lines have been generated. In this way it is hoped to generate models for RPE65-related retinal dystrophy that are not null (like the knockout mouse and the Briard dog) d) The identity of putative factors binding to transcription elements in the RPE65 gene promoter is being sought. Expression clones for these factors are being tested for their effect on activation of the RPE65 promoter. e) Collaboration continues on the rescue of the Rpe65 knockout mouse phenotype and the Briard dog RPE65 dystrophy by AAV-mediated gene transfer.