Molecular Biology Of Outer Retina-specific Proteins

视网膜色素上皮(RPE)在视网膜外层的发育和功能中起着关键作用。我们对RPE特有的机制感兴趣，无论是在监管层面还是在功能层面。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和导致人类严重失明的突变。RPE65基因敲除小鼠的表型似乎是破坏基于RPE的维生素A视觉周期代谢，将全反式视黄酸酯代谢为11顺式视黄醇。因此，RPE65的功能似乎与视黄醇异构酶有关，视黄醇异构酶是视觉色素再生的关键酶。我们还在继续研究β-胡萝卜素15，15‘-单加氧酶(β-CM)。β-CM与RPE65密切相关，两者都是一个新出现的多样化的类胡萝卜素裂解酶家族的成员。我们推测，β-CM和RPE65可能具有类似的作用机制。在过去的一年里，我们取得了以下进展：a)通过对β-CM中可能的金属结合残基进行定点突变，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。数据表明，假设与金属配位有关的残基在酶活性中起着至关重要的作用。不同残基的突变会导致铁结合丧失和/或突变蛋白的Km和Vmax发生变化。B)RPE65含有一个小窝蛋白相互作用结构域，该结构域在所有物种中都是保守的，并且具有功能活性。小窝蛋白-1是一种“脚手架”蛋白，参与膜转运、脂质转运和信号转导。它与RPE65的交互可能涉及到这些角色中的一些或全部。我们发现RPE65既能与小窝蛋白-1的支架结构域结合，又能与小窝蛋白-1的C末端结构域结合。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成分相关联，并为视觉周期复合体的研究提供了一个新的焦点。C)对β-CM启动子的功能分析表明，它受一个过氧化物酶体增殖物激活受体(PPAR)反应元件(PPRE)的调控。将β-CM启动子-报告基因瞬时导入猴RPE、ARPE19以及Caco-2人肠上皮细胞系的TC7和PF11亚克隆，结果表明，该基因的最佳激活需要一个一致的PPRE。电泳迁移率改变实验表明，来自猴RPE细胞、TC7和细胞系的核蛋白与PPRE元件结合，而抗PPAR抗体的超平移实验证明PPARGamma与该启动子中的PPRE结合。此外，我们还发现，通过与PPARγ和RXRα共转染，以及用PPARγ激动剂LY 17883和Ciglitazone激活，PPRE元件通过PPARγ和维甲酸X受体(RXR)α异二聚体提供了过氧化体增殖物的反应性。D)正在寻找与RPE65基因启动子中的转录元件结合的假定因子的身份。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)在通过AAV介导的基因转移挽救RPE65基因敲除小鼠表型和Briard犬RPE65营养不良方面的合作仍在继续。在治疗的小鼠和狗中，电生理和行为反应对光的敏感度增加。对一些治疗犬葡萄膜炎的意义进行了研究。

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.