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;以前称为双加氧酶）。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）通过对野生型和Rpe 65基因敲除小鼠进行全反式视黄醇急性放射性标记的合作研究表明，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，该组织对标记的吸收在野生型中是快速的，而在Rpe 65敲除中根本看不到。在RPE中，视黄基酯的积累在野生型中是短暂的，但在敲除中在48小时内进行。类维生素A流入RPE的数据分析得出结论，即在野生型中发生的强流出在Rpe 65敲除RPE中不发生。这些数据表明，11-顺式维甲酸刺激流出的全反式视黄醇在基底外侧膜的RPE。野生型视网膜的数据进一步证实了？后进先出？全反式视黄醇加工成11-顺式视黄醇的假说，其中在主动视觉循环通量中的类维生素A不穿过视黄酯的储存池。B）通过β-CM的定点诱变研究了类胡萝卜素裂解酶家族（包括β-CM和RPE 65）所有成员中保守残基的作用。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。c）通过免疫荧光显微术和RT-PCR发现，在多种物种（小鼠、牛、猴和人）的视网膜和RPE中，在小鼠内层视网膜中而不是在小鼠RPE中，β-CM表达低且可变。各种RPE细胞系的RT-PCR显示在猴RPE细胞系中表达，但在3种人RPE细胞系中不表达。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特异性的。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。我们克隆了5号？将该基因的侧翼区插入荧光素酶报告质粒中，并产生一系列推定启动子的嵌套缺失。将这些瞬时转染入猴RPE、ARPE 19和Caco-2人肠上皮细胞系的TC 7和PF 11亚克隆中，证明了共有过氧化物酶体增殖物激活受体（PPAR）反应元件（PPRE）是基因最佳激活所需的。电泳迁移率变化实验表明，猴RPE细胞和TC 7细胞系的核蛋白与PPRE元件结合。用特异于PPARs的抗体进行的超移位实验证明了PPARgamma与该启动子中的PPRE位点的结合。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。e）由视网膜特异性抗原（例如，抑制蛋白、IRBP和视紫红质）是人眼部炎性疾病如葡萄膜炎的模型。我们继续与LI合作研究RPE 65抗原诱导的EAU。该疾病在后段更活跃，并且不像arrestin诱导的EAU那样影响前段，并且与后者不同，没有松果体。疾病从免疫动物向未免疫动物的连续转移表明该疾病是细胞介导的，与其他形式的EAU一样。f）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。尽管功能上有所改善，但形态学上的挽救仍然有限。

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.