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，以及导致人类严重失明的突变。Rpe 65基因敲除小鼠的表型是由于基于RPE的维生素A视觉周期的破坏，其作用是通过维生素A的异构化再生视色素发色团。我们已经表明，在Rpe 65基因敲除小鼠有过度积累的全反式视黄酯和完全没有11-顺式-视网膜，这表明与视色素发色团再生的关键酶视黄醇异构水解酶的作用。我们已经确定了RPE 65在11-顺式视黄醇合成中的催化作用，将其鉴定为长期寻找的视觉循环异构水解酶。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。 因为它们具有相同的结构特征，包括催化组合中相同的残基，我们发现BCMO 1是我们研究RPE 65的机制的有用模型。 在过去一年中，我们取得了以下进展： a）虽然一半的人类RPE 65突变是错义突变，并且其中许多突变并不能完全消除异构酶活性（亚型突变），但我们并不清楚为什么残留的（10%）RPE 65活性允许生命早期的可用视力，同时延迟但不能预防最终的视网膜变性。 我们研究了RPE 65中与不太严重的变性相关的错义突变的影响，以了解它们对蛋白质结构和稳定性的影响。 我们已经发现这些突变体中的许多具有低异构酶活性（5-10%），但它们的携带者可以在生命的第三或第四个十年中保持可用的视力。在与德国临床医生的合作中，他们在一名年轻患者中发现了P25 L突变，我们扩大了RPE 65营养不良表型的临床谱。结果发现，这个年轻的病人有接近正常的视力，结合废除杆功能和夜盲与功能性锥视觉。 RPE 65/P25 L的活性测量为野生型活性的8%。视锥功能的相对保存强烈表明，人类视锥细胞可以选择性地隔离足够的11-顺式视网膜，以保持其功能比杆。我们研究这方面的最终目标是确定这种亚纯型RPE 65突变体的活性是否可以通过药理学试剂调节其稳定性来增强。 此外，鉴于RPE 65基因治疗的最新进展，这些研究为基因治疗的适用性提供了合理的评估。 B）为了补充（a）中报道的工作，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。特别是，他们预计将提供洞察视网膜变性的缓慢进展，如在人类RPE 65突变的不太严重的情况下看到的。 他们还将提供动物模型来测试（a）中开发的药理学策略。 已经发现这些构建体中的第一个是种系的，并且已经建立了敲入系。 第二个已经成功地进行了同源重组，正在等待注入胚胎。 第三种靶向载体目前正在排队等待电穿孔进入ES细胞进行同源重组。 c）之前，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（底物结合，电子转移等）仍不清楚。 根据ACO的结构，我们正在研究BCMO 1，BCMO 2和RPE 65是如何进化的，以利用类胡萝卜素加氧酶家族的基本结构来实现它们的功能。 该家族的所有3个哺乳动物成员都在视网膜和/或RPE中表达，但RPE 65对RPE具有特异性。 在过去的一年中，我们进行了实验，以更好地阐明BCMO 1的机制，通过确定在底物结合隧道中的残基是必要的催化和底物结合活性，并探讨的可能性，即碳阳离子中间体形成的裂解反应过程中，看到许多类异戊二烯生物合成酶。 我们取代保守和底物隧道酪氨酸和酸性残基的定点突变。我们表明，虽然某些酪氨酸残基，本身，所需的活动，在其他人的芳香性的保护是足够的。我们的研究结果是一致的形成的底物碳阳离子中间体和阳离子π稳定化，这是由芳香族残基在底物结合隧道。我们已经建立了一个从头算模型的BCMO 1与安装-胡萝卜素，并证明，残基之间的距离，活性和基板的关键是小于5埃，这是一致的机制，涉及阳离子π稳定。 此外，这些见解可能对理解RPE 65的机制有价值。

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 whose role is to regenerate visual pigment chromophore by isomerization of vitamin A. We have shown that in the Rpe65 knockout mouse there is overaccumulation of all-trans-retinyl esters and total absence of 11-cis-retinal, suggesting a role related to that of retinol isomerohydrolase, the crucial enzyme in visual pigment chromophore regeneration. We have established a catalytic role for RPE65 in the synthesis of 11-cis retinol, identifying it as the long-sought visual cycle isomerohydrolase. 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. Because they share structural features, including identical residues in the catalytic assemblage, we have found BCMO1 to be a useful model for our mechanistic studies addressing RPE65. In the past year we have made the following progress: a) While half of human RPE65 mutations are missense mutations, and many of these do not completely abolish isomerase activity (hypomorphic mutations), we do not have a clear understanding of why residual (10%) RPE65 activity allows usable vision early in life, while delaying but not preventing eventual retinal degeneration. We investigated the effect of missense mutations in RPE65 associated with less severe degenerations to understand their effects on protein structure and stability. We have found that many of these mutants have low isomerase activity (5-10%), but their carriers can retain usable vision into 3rd or 4th decade of life. In a collaboration with German clinicians who identified a P25L mutation in a young patient, we have expanded the clinical spectrum of RPE65 dystrophy phenotype. It was found that this young patient has near normal visual acuity, combining abolished rod function and night-blindness with functional cone vision. The activity of RPE65/P25L was measured at 8% of wildtype activity. The relative preservation of cone function strongly suggests that human cones can selectively sequester enough 11-cis retinal to maintain their function better than rods. The ultimate goal of this aspect of our research is to determine if the activity of such hypomorphic RPE65 mutants can be augmented by modulation of their stability by pharmacologic agents. Also, in light of recent advances in RPE65 gene therapy, such studies provide a rational assessment of suitability for gene therapy. b) To complement the work reported in (a), 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. In particular, they are anticipated to provide insight into the slower progression of the retinal degeneration such as seen in less severe cases of human RPE65 mutations. They also will provide animal models to test pharmacologic strategies developed in (a). The first of these constructs has been found to be germline and the knock-in line has been established. The second has successfully undergone homologous recombination, and is awaiting injection into embryos. The targeting vector for the third is currently in queue for electroporation into ES cells for homologous recombination. c) 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) confirmed a catalytic role for iron in this family of proteins, but other crucial aspects of the mechanism (substrate binding, electron transfer, etc.) remained unclear. In light of the ACO structure we are investigating how BCMO1, BCMO2 and RPE65 have evolved to fulfill their functions utilizing the basic structure of the carotenoid oxygenase family. All 3 mammalian members of the family are expressed in retina and/or RPE, but RPE65 is specific to the RPE. In the past year, we conducted experiments to better elucidate the mechanism of BCMO1 by determining which residues in the substrate binding tunnel are necessary for catalytic and substrate binding activity, and to explore the possibility that a carbocation intermediate is formed during the cleavage reaction, as seen for many isoprenoid biosynthesis enzymes. We replaced conserved and substrate tunnel tyrosines and acidic residues by site-directed mutagenesis. We showed that while certain tyrosine residues, per se, are required for activity, conservation of aromaticity at others is sufficient. Our results are consistent with the formation of a substrate carbocation intermediate and cation-pi stabilization of this by the aromatic residues in the substrate binding tunnel. We have built an ab initio model of BCMO1 with the mounted β-carotene and demonstrated that distances between residues crucial for activity and the substrate are less than 5 angstroms, which is consistent with a mechanism involving cation-pi stabilization. In addition, these insights may be valuable towards understanding the mechanism of RPE65.