Molecular Biology Of Outer Retina-specific Proteins

视网膜色素上皮（retinal pigment epithelium，RPE）在外层视网膜的发育和功能中起着关键作用。我们感兴趣的RPE的具体机制，在监管和功能水平。为此，我们一直在研究RPE 65的功能和调节，RPE 65是一种表达仅限于RPE的基因，其突变导致人类严重失明。基于RPE的维生素A视觉周期代谢的全反式视黄酯的11-顺式-视网膜的破坏似乎是Rpe 65敲除小鼠表型的基础。因此，RPE 65的功能似乎与视色素再生中的关键酶视黄醇异构酶的功能相关。我们还继续研究β-胡萝卜素15，15 '-单加氧酶（β-CM）。β-CM与RPE 65密切相关，两者都是新出现的多样化类胡萝卜素裂解酶家族的成员。在过去的一年里，我们取得了以下进展：a）在所有成员的类胡萝卜素裂解酶家族（包括β-CM和RPE 65）保守的残基的作用已被进一步研究的定点诱变推定的金属结合残基在β-CM。这些数据表明，假设参与金属配位的组氨酸和酸性残基在酶活性中起着至关重要的作用。突变的4个保守的组氨酸和保守的谷氨酸残基的铁结合的损失，如电感耦合等离子体原子发射光谱法所示。某些其他非绝对保守残基的突变导致受损但活性的酶，主要导致突变酶的Vmax变化。B）RPE 65含有两个潜在的小窝蛋白相互作用结构域，其在所有物种中是保守的并且是功能活性的。小窝蛋白-1是一种“支架”蛋白，已知参与膜运输、脂质转运和信号转导。它与RPE 65的相互作用可能涉及这些作用中的一些或全部。我们已经发现RPE 65结合到caveolin-1的支架结构域和C-末端结构域。这种相互作用，沿着先前显示的与磷脂的相互作用，可能允许RPE 65与膜锚定的视觉周期组分的关联，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，并且正在测试其视觉等的表型变化。c）携带转基因的转基因小鼠，所述转基因并入了在人类中被鉴定为致病的RPE 65中的突变，已经将其繁殖到Rpe 65敲除背景上，并且正在测试其表型。已经生成了几条不同的线。以这种方式，希望产生非无效的RPE 65相关视网膜营养不良的模型（如敲除小鼠和Briard狗）d）正在寻找与RPE 65基因启动子中的转录元件结合的推定因子的身份。正在测试这些因子的表达克隆对RPE 65启动子激活的影响。e）继续合作通过AAV介导的基因转移拯救Rpe 65敲除小鼠表型和Briard狗Rpe 65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.