Molecular Biology Of Outer Retina-specific Proteins

视网膜色素上皮（RPE）在外视网膜的发育和功能中起着关键作用。我们在调控和功能水平上对RPE特异性机制感兴趣，我们一直在研究RPE65的功能和调控，RPE65是一个基因，其表达仅限于RPE，其突变会导致人类严重失明。Rpe65基因敲除小鼠的表型是由于基于rpe的维生素A视觉循环代谢被破坏。因此，在Rpe65基因敲除小鼠中，全反式视黄醇酯过度积累，11-顺式视网膜完全缺失。因此，RPE65的功能似乎是视黄醇异构酶的功能，视黄醇异构酶是视觉色素发色团再生的关键酶。我们还继续进行了β -胡萝卜素15,15'-单加氧酶（BCMO1）的研究。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)建立了RPE65在合成11-顺式视黄醇中的催化作用（与卵磷脂：视黄醇酰基转移酶（LRAT）一起），并确定了它是长期寻找的异构体水解酶，我们正在研究其酶促机制的细节以及与其他视觉循环蛋白的相互作用。我们研究了与Leber先天性黑朦/早发性失明相关的RPE65错义突变以及小鼠RPE65突变的影响，以了解它们对蛋白质结构和稳定性的影响。b)之前，我们证明了BCMO1（和RPE65）中组氨酸和酸性残基在酶活性中的关键作用，我们假设这些残基参与了金属配位。这些观察结果，结合一种相关细菌酶的预测结构，疏螺旋藻加氧酶（ACO）证实了铁在这一蛋白质家族中的催化作用，但其机制的其他关键方面（电子转移等）仍不清楚。鉴于ACO结构，我们正在研究BCMO1和RPE65如何利用类胡萝卜素加氧酶家族的基本结构进化以实现其功能。c)我们正在通过同源重组产生一组含有小鼠Rpe65基因的拟态敲入小鼠。与基因敲除的极端情况相比，预计这些将为rpe65缺陷表型的可变性提供重要的见解。d)正在寻找与RPE65基因启动子中转录元件结合的推定因子的身份。我们已经测试了一个这样的假设因子，ZNF-492，一种蟹黄锌指蛋白，并表明它对RPE65基因转录有中等影响。ZNF492具有531个氨基酸的开放阅读框，n端被截断，缺少常见的krupp -associated box-A (KRAB-A)，而KRAB-B保持完整，并且具有12个C2H2锌指串联排列。在ZNF492中，我们假设KRAB-A的缺失可能会减少或阻止共抑制因子的结合，从而解释Rpe65基因表达的适度上调。

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 of 11-cis-retinal. The function of RPE65 thus appears to be 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) Having established a catalytic role (in conjunction with lecithin:retinol acyltransferase (LRAT)) for RPE65 in the synthesis of 11-cis retinol, and identifying it as the long-sought isomerohydrolase, we are investigating the details of its enzymatic mechanism and interactions with other visual cycle proteins. We investigated the effect of missense mutations in RPE65 associated with Leber congenital amaurosis/early onset blindness as well as mutations in mouse Rpe65 to understand their effects on protein structure and stability. b) Previously, we demonstrated a crucial role in enzymatic activity for histidine and acidic residues in BCMO1 (and RPE65) that we hypothesized to be involved in metal coordination. These observations, in conjunction with the predicted structure of a related bacterial enzyme, Synechocystis apocarotenal oxygenase (ACO) confirms a catalytic role for iron in this family of proteins but other crucial aspects of the mechanism (electron transfer, etc.) remain unknown. In light of the ACO 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 are generating a panel of hypomorphic knock-in mice in the mouse Rpe65 gene by homologous recombination. It is anticipated that these will provide important insight into the variability of RPE65-deficient phenotypes, in comparison with the extreme case of the knockout. d) The identity of putative factors binding to transcription elements in the RPE65 gene promoter is being sought. We have tested one such putative factor, ZNF-492, a KRAB-zinc finger protein and have shown that it has a moderate effect on RPE65 gene transcription. ZNF492 has an open reading frame of 531 amino acids with a truncated N-terminus and lacks the usual Kruppel-associated box-A (KRAB-A) while KRAB-B remains intact, and has 12 C2H2 zinc-fingers in tandem arrangement. In ZNF492, we postulate that absence of KRAB-A might reduce or prevent co-repressor binding to account for the modest up-regulation of Rpe65 gene expression.