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。我们对RPE特异性机制感兴趣，在调节和功能水平上，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。Rpe 65敲除小鼠的表型是由于基于RPE的维生素A视觉周期代谢的破坏。因此，在Rpe 65基因敲除小鼠中，存在全反式视黄酯的过度积累和11-顺式视黄酸的完全缺乏。因此，RPE 65的功能似乎与视色素发色团再生中的关键酶视黄醇异构酶的功能相关。我们还继续对β-胡萝卜素15，15 '-单加氧酶（BCMO 1）进行研究。BCMO 1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。我们假设BCMO 1和RPE 65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）我们已经确定了RPE 65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）结合），并将其鉴定为长期寻找的异构水解酶。为了澄清RPE 65？我们在293-F细胞中重建了一个强大的最小视觉周期。仅用RPE 65构建体转染的细胞产生11-顺式类维生素A，但需要与LRAT共表达以实现高水平生产。积累是显着的，总计超过2纳摩尔11-顺式视黄醇每文化。转染与RPE 65的残基中的突变同源的那些所需的相互联系的酶活性和铁的协调相关的酶的构建体废除了这种异构化活性。铁螯合也消除异构化活性。参与RPE 65棕榈酰化的突变半胱氨酸通常对异构化活性影响不大。与Leber先天性黑蒙/早发性失明相关的突变导致异构化活性部分至全部丧失，与其临床效应直接相关。B）我们从BCMO 1中推定的金属结合残基的定点诱变获得的经验数据证明了我们假设参与金属配位的组氨酸和酸性残基在酶活性中的关键作用。这些观察结果，沿着那些关于RPE 65的观察结果（见上文），已经被相关细菌酶集胞藻脱胡萝卜素醛加氧酶的预测结构所证实。根据这种结构，我们正在研究BCMO 1和RPE 65如何进化以利用类胡萝卜素加氧酶家族的基本结构来实现其功能。c）我们已经产生了几种不同的携带RPE 65小基因转基因的转基因小鼠品系，这些转基因小鼠在人类致病性RPE 65中掺入突变，并且这些转基因小鼠已经在Rpe 65敲除背景下繁殖，并且正在测试它们的表型以产生非无效的RPE 65相关视网膜营养不良的模型（如敲除小鼠和Briard狗）。我们发现携带小基因的小鼠比Rpe 65敲除小鼠对光更敏感。该项目的进一步工作将使用同源重组产生的敲入小鼠进行。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。我们已经测试了一个这样的假定因子，ZNF-492，KRAB-锌指蛋白，对RPE 65基因转录有中度影响。

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.