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特有的调节和功能水平的机制感兴趣，我们一直在研究RPE65的功能和调节，RPE65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是由于以RPE为基础的维生素A视觉循环代谢的中断。因此，在RPE65基因敲除的小鼠中，全反式视黄酸酯过度积累，完全缺乏11顺式视网膜。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生色团再生的关键酶。我们还在继续研究β-胡萝卜素15，15‘-单加氧酶(BCMO1)。BCMO1与RPE65密切相关，两者都是新出现的类胡萝卜素裂解酶家族的成员。我们假设BCMO1和RPE65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)我们已经建立了RPE65在11-顺式视黄醇合成中的催化作用(与卵磷脂：视黄醇酰基转移酶(LRAT))，并确定它是人们长期寻找的异构体水解酶。为了阐明RPE65、S在异构化中的作用，我们在293-F细胞中重构了一个健壮的最小视觉周期。只有转RPE65构建体的细胞才能产生11-顺式维甲酸，但需要与LRAT共表达才能高水平生产。积累量显著，每次培养产生2毫摩尔以上的11-顺式视黄醇。用含有RPE65残基突变的构建体转染，该构建体与链接酶活性所需的同源，并且相关酶中的铁配位取消了这种异构化活性。铁的络合作用也消除了异构化活性。RPE65棕榈酰化所涉及的半胱氨酸突变通常对异构化活性影响不大。与Leber先天性黑色素/早发性失明相关的突变可导致部分或全部异构化活性丧失，这与其临床效果直接相关。B)我们对BCMO1中可能的金属结合残基进行定点突变的经验数据表明，我们假设参与金属配位的组氨酸和酸性残基的酶活性起着至关重要的作用。这些观察结果，以及关于RPE65(见上文)的观察结果，已经被一种相关细菌酶--聚球藻无果糖加氧酶的预测结构所证实。根据这种结构，我们正在研究BCMO1和RPE65是如何利用类胡萝卜素加氧酶家族的基本结构进化来实现其功能的。C)我们已经产生了几种不同的转基因小鼠，这些转基因小鼠携带RPE65微小基因转基因，在人类中整合了RPE65致病基因的突变，这些转基因小鼠被培育到RPE65基因敲除背景上，并正在接受表型测试，以产生非零的RPE65相关视网膜营养不良模型(如基因敲除小鼠和Briard狗)。我们发现，携带迷你基因的小鼠比RPE65基因敲除小鼠对光更敏感。该项目的进一步工作将使用通过同源重组产生的敲入小鼠进行。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。我们已经测试了一个这样的假定因子ZNF-492，它是一种KRAB-锌指蛋白，对RPE65基因转录有适度的影响。

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, and 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. The phenotype of the Rpe65 knockout mouse is due to disruption of the RPE-based vitamin A visual cycle, metabolizing. Consequently, in the Rpe65 knockout mouse there is overaccumulation of all-trans-retinyl esters and total absence of11-cis-retinal. The function of RPE65 thus appears to be associated with that of the retinol isomerase, the crucial enzyme in visual pigment chromophore regeneration. We have also continued studies on beta-carotene 15,15'-monooxygenase (BCMO1). BCMO1 is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that BCMO1 and RPE65 share a similar mechanism of action. In the past year we have made the following progress: a) We have established a catalytic role (in conjunction with lecithin:retinol acyltransferase (LRAT)) for RPE65 in the synthesis of 11-cis retinol, and identify it as the long-sought isomerohydrolase. To clarify RPE65?s role in isomerization we reconstituted a robust minimal visual cycle in 293-F cells. Only cells transfected with RPE65 constructs produced 11-cis retinoids but co-expression with LRAT was needed for high level production. Accumulation was significant, amounting to more than 2 nmoles 11-cis retinol per culture. Transfection with constructs harboring mutations in residues of RPE65 homologous to those required for interlinked enzymatic activity and iron coordination in related enzymes abolished this isomerization activity. Iron chelation also abolished isomerization activity. Mutating cysteines implicated in palmitoylation of RPE65 had generally little effect isomerization activity. Mutations associated with Leber congenital amaurosis/early onset blindness caused partial to total loss of isomerization activity in direct relation to their clinical effect. b) Our empirical data from site-directed mutagenesis of putative metal binding residues in BCMO1 demonstrated a crucial role in enzymatic activity for histidine and acidic residues we hypothesized to be involved in metal coordination. These observations, along with those regarding RPE65 (see above), have been corroborated by the predicted structure of a related bacterial enzyme, Synechocystis apocarotenal oxygenase. In light of this structure we are investigating how BCMO1 and RPE65 have evolved to fulfill their functions utilizing the basic structure of the carotenoid oxygenase family. c) We have generated several different lines of transgenic mice bearing RPE65 minigene transgenes that incorporate mutations in RPE65 pathogenic in humans and these have been bred onto the Rpe65 knockout background and are being tested as to their phenotypes to generate models for RPE65-related retinal dystrophy that are not null (like the knockout mouse and the Briard dog). We find that the minigene-carrying mice are slightly more light-sensitive than Rpe65 knockout mice. Further work on this project will be carried out using knock-in mice generated by homologous recombination. 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. We have tested one such putative factor, ZNF-492, a KRAB-zinc finger protein that has a moderate effect on RPE65 gene transcription.