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失。因此，RPE 65的功能似乎是视色素发色团再生中的关键酶视黄醇异构酶的功能。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）已经确定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，我们正在研究其酶促机制和与其他视觉周期蛋白的相互作用的细节。我们研究了与Leber先天性黑蒙/早发性失明相关的RPE 65中的错义突变以及小鼠Rpe 65中的突变的影响，以了解它们对蛋白质结构和稳定性的影响。B）以前，我们证明了BCMO 1（和RPE 65）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金属配位。这些观察结果，结合相关细菌酶的预测结构，集胞藻脱胡萝卜素加氧酶（ACO）证实了铁在这个蛋白质家族中的催化作用，但该机制的其他关键方面（电子转移等）。仍然未知。根据ACO的结构，我们正在研究BCMO 1和RPE 65是如何利用类胡萝卜素加氧酶家族的基本结构来实现其功能的。c）我们通过同源重组在小鼠Rpe 65基因中产生一组亚型敲入小鼠。预计这些将提供重要的洞察RPE 65缺陷表型的变异性，与极端情况下的敲除。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。我们已经测试了这样一个假定的因子，ZNF-492，KRAB-锌指蛋白，并已表明，它对RPE 65基因转录有中度影响。ZNF 492具有531个氨基酸的开放阅读框架，N-末端截短，缺少通常的Kruppel相关盒A（KRAB-A），而KRAB-B保持完整，并且具有串联排列的12个C2 H2锌指。在ZNF 492中，我们假设KRAB-A的缺失可能减少或阻止辅阻遏物结合，以解释Rpe 65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.