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；以前被描述为双加氧酶)。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)用野生型全反式视黄醇和RPE65基因敲除小鼠进行的急性放射性标记联合研究表明，在48小时的测试期内，两者的肝脏和血清摄取大致相似。然而，在视网膜中，这种组织以视黄醛的形式摄取标记在野生型中很快，而在RPE65基因敲除中根本看不到。在RPE中，视黄酸酯的积累在野生型中是暂时的，但在基因敲除中是在48小时内逐渐积累的。分析类维A酸类物质流入RPE的数据，得出结论：在RPE65基因敲除的RPE中没有发生野生型的强健外流。这些数据表明，11-顺式维甲酸刺激全反式视黄醇在RPE的基底膜外流。野生型视网膜中的数据进一步证实了后进/先出？全反式视黄醇加工为11-顺式视黄醇的假说，其中视黄醇在活跃的视觉周期通量中不会穿过存储的视黄酯池。B)通过对β-CM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。C)免疫荧光显微镜和RT-PCR检测发现，β-CM在多种动物(小鼠、牛、猴和人)的视网膜和RPE中低水平和多种多样地表达，而在小鼠的RPE中不表达。不同RPE细胞系的RT-PCR结果显示，在1个猴RPE细胞系中有表达，而在3个人RPE细胞系中未见表达。我们的结论是，它在这些组织中的功能不太可能是视网膜或RPE特有的功能。D)我们分析了小鼠β-CM基因的基因组结构，发现它有11个外显子。我们已经克隆了5？将该基因的侧翼区插入到荧光素酶报告质粒中，并产生了一系列可能的启动子的嵌套缺失。将其瞬时导入猴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共同的PPAR反应元件(PPRE)。电泳迁移率改变实验表明，猴RPE细胞、TC7和细胞系的核蛋白与PPRE元件结合。PPAR特异性抗体的超移位实验证明了PPARGamma与该启动子中的PPRE位点结合。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。E)视网膜特异性抗原(如Arrestin、IRBP和视紫红质)诱导的实验性自身免疫性葡萄膜视网膜炎(EAU)是人类眼部炎症性疾病(如葡萄膜炎)的模型。我们继续与李在RPE65抗原诱导的EAU方面进行合作。这种疾病在后段更活跃，对前段的影响不像arrestin诱导的Eau那样严重，而且与后者不同的是，没有松果体炎。疾病从免疫动物到幼稚动物的过继转移表明，该疾病与其他形式的EAU一样，是由细胞介导的。F)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。尽管功能有所改善，但形态挽救一直是有限的。

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; formerly described as a dioxygenase). 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) A collaborative study by acute radiolabeling with all-trans-retinol of wildtype and Rpe65 knockout mice showed that liver and serum uptake was broadly similar in both, over the tested period of 48 hours. In the retina, however, uptake of label by this tissue as retinaldehyde was rapid in wildtype and not seen at all in the Rpe65 knockout. In the RPE, accumulation as retinyl ester was transient in the wildtype, but progressive over 48 hours in the knockout. Analysis of the retinoid influx data into the RPE, gave rise to the conclusion that a robust efflux that occurs in the wildtype does not occur in the Rpe65 knockout RPE. These data suggest that 11-cis retinoids stimulate the efflux of all-trans retinol at the basolateral membrane of the RPE. The data in wildtype retina further corroborate the ?last in/first out? hypothesis of all-trans retinol processing to 11-cis retinol, where retinoid in the active visual cycle flux does not traverse the stored pool of retinyl ester. b) 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 beta-CM. The data show a crucial role in enzymatic activity for residues hypothesized to be involved in metal coordination. c) Beta-CM expression was found by immunofluorescence microscopy and RT-PCR to be low and variable in retina and RPE of a variety of species (mouse, bovine, monkey and human) in the mouse inner retina and not in the mouse RPE. RT-PCR of various RPE cell lines revealed expression in a monkey RPE cell line but not in 3 human RPE cell lines. We conclude that its function in these tissues is not likely to be a retina- or RPE-specific one. d) We have analyzed the genomic structure of the mouse beta-CM gene and found it to have 11 exons. We have cloned the 5? flanking region of the gene into a luciferase reporter plasmid and generated a series of nested deletions of the putative promoter. Transient transfection of these into monkey RPE, ARPE19 and the TC7 and PF11 subclones of the Caco-2 human enterocytes cell line demonstrated that a consensus peroxisome proliferator activated receptor (PPAR) response element (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. Supershift experiments with antibodies specific 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. e) Experimental autoimmune uveoretinitis (EAU) induced by retina-specific antigens (e.g., arrestin, IRBP and rhodopsin) is a model for human ocular inflammatory diseases such as uveitis. We have continued our collaboration with LI on EAU induced with RPE65 antigen. The disease is more active in the posterior segment and does not affect the anterior segment to the same extent as arrestin-induced EAU, and, unlike the latter, there is no pinealitis. Adoptive transfer of the disease from immunized to naive animals indicates that the disease is cell-mediated, like other forms of EAU. f) 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. In spite of functional improvements, morphological rescue has been limited.