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。我们推测β-CM和RPE 65可能具有相似的作用机制。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经通过定点突变的推定金属结合残基β-CM进行了研究。这些数据表明，假设参与金属配位的残基在酶活性中起着至关重要的作用。不同残基的突变导致铁结合的丧失和/或突变蛋白的Km和Vmax的变化。B）RPE 65含有在所有物种中保守的小窝蛋白相互作用结构域，并且具有功能活性。Caveolin-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。c）β-CM启动子的功能分析表明，它受过氧化物酶体增殖物激活受体（PPAR）反应元件（PPRE）调节。将β-CM启动子-报告基因构建体瞬时转染到猴RPE、ARPE 19和Caco-2人肠上皮细胞系的TC 7和PF 11亚克隆中，证明了该基因的最佳激活需要共有PPRE。电泳迁移率变化实验表明，从猴RPE细胞和TC 7和细胞系的核蛋白结合的PPRE元件，而超迁移实验与抗体的PPARs证明了PPARgamma的结合PPRE网站在这个启动子。此外，我们发现，通过共转染与PPARgamma和RXR α和激活与PPARgamma激动剂LY 17883和格列酮，PPRE元件赋予过氧化物酶体增殖反应通过PPARgamma和类维生素A X受体（RXR）α异二聚体。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。已注意到经处理的小鼠和犬的电生理和行为反应对光的敏感性增加。已研究了一些给药犬中葡萄膜炎的意义。

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. We postulate that beta-CM and RPE65 may share a similar mechanism of action. 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 investigated by site-directed mutagenesis of putative metal binding residues in beta-CM. The data show a crucial role in enzymatic activity for residues hypothesized to be involved in metal coordination. Mutation of the different residues results in loss of iron-binding and/or changes in Km and Vmax of mutant proteins. b) RPE65 contains a caveolin-interaction domain that is conserved in all species and is 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. c) Analysis of the function of the beta-CM promoter demonstrates that it is regulated by a peroxisome proliferator activated receptor (PPAR) response element (PPRE). Transient transfection of beta-CM promoter-reporter constructs into monkey RPE, ARPE19 and the TC7 and PF11 subclones of the Caco-2 human enterocytes cell line demonstrated that a consensus PPRE is required for optimal activation of the gene. Electrophoretic mobility shift experiments showed that nuclear proteins from monkey RPE cells and TC7 and cell line bind to the PPRE element, while supershift experiments with antibodies to PPARs demonstrated the binding of PPARgamma to the PPRE site in this promoter. Furthermore, we showed, by co-transfection with PPARgamma and RXRalpha and activation with PPARgamma agonists LY 17883 and Ciglitazone, that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor (RXR) alpha heterodimer. 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. An increased sensitivity of electrophysiological and behavioral responses to light in treated mice and dogs has been noted. The significance of uveitis in some treated dogs has been investigated.