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特异性机制很感兴趣，我们一直在研究RPE65的功能和调控，RPE65是一种基因，其表达仅限于RPE，以及导致人类严重失明的突变，称为Leber先天性黑朦2 （LCA2）。LCA2已成为成功的体细胞RPE65基因治疗试验的目标。RPE65基因缺陷（LCA2）和RPE65基因敲除小鼠（全反式视黄醇酯的过度积累和11-顺式视网膜的完全缺失，导致对光极度不敏感）共同的表型是，基于RPE65的维生素A视觉周期被破坏，导致维生素A异构化以再生视觉色素色团。我们已经确定了RPE65在合成11-顺式视黄醇中的催化作用，并将其确定为长期寻找的视觉循环异构水解酶。我们也一直在研究β -胡萝卜素15,15'-单加氧酶（BCMO1），我们首先根据与RPE65的相似性确定了它。BCMO1与RPE65密切相关，两者都是一个新出现的多样化类胡萝卜素切割酶家族的成员。我们假设BCMO1和RPE65具有相似的潜在作用机制。由于它们具有相同的结构特征，包括催化组合中的相同残基，我们发现BCMO1是我们研究RPE65机制的有用模型。

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, known as Leber Congenital Amaurosis 2 (LCA2). LCA2 has been the target of successful somatic RPE65 gene therapy trials. Disruption of the RPE-based vitamin A visual cycle for isomerization of vitamin A to regenerate visual pigment chromophore is the common phenotype shared by humans with RPE65 gene defects (LCA2) and the Rpe65 knockout mouse (overaccumulation of all-trans-retinyl esters and total absence of 11-cis retinal, resulting in extreme insensitivity to light). 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 been studying beta-carotene 15,15'-monooxygenase (BCMO1), which we first identified based on similarity to RPE65. BCMO1 is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid cleaving enzymes. We postulate that BCMO1 and RPE65 share a similar underlying 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) In the past year we made novel insights into the mechanism of RPE65. The mechanism of retinol isomerization in the vertebrate retina visual cycle has been controversial for 20 years, since its enzymatic nature was established. Does it operate via nucleophilic addition at C11 of the all-trans substrate, or via a carbocation mechanism? A nucleophilic addition mechanism would predict a clean isomerase making only 11-cis isomer, while a carbocation mechanism could be leaky, making not just 11-cis, but other isomers also. Indeed, the Rdh5 knockout accumulates not only 11-cis but aberrant levels of 13-cis retinoids as well. The identity of RPE65 as the isomerase/isomerohydrolase has only been definitively known since 2005. We reasoned that the correct mechanism for retinol isomerization would be the one employed by RPE65, as the isomerase enzyme. We had made the observation that wildtype RPE65 in vitro could produce the 13-cis and 11-cis isomers equally robustly. Also, mutations affecting RPE65 activity coordinately depress 11-cis and 13-cis isomer production but diverge as 11-cis decreases to zero while 13-cis reaches a plateau consistent with thermal isomerization. This suggested that RPE65 could be a leaky isomerase. We asked the question whether this is a legitimate property of RPE65 and how it is related to the structure of RPE65. To determine these points, we modeled the RPE65 substrate cleft on the apocarotenal oxygenase structure template to identify residues that might interact with substrate and/or intermediate and analyzed them with panels of mutations. We identified 4 residues for further study (F103, T147, Y239, and W331) All Y239 mutations abolish activity, suggesting a tyrosine-specific role. Mutation of W331 reduced its activity, in W331Y to 25% of wildtype, in W331F to 8% of wildtype, and to zero with W331L or W331Q, establishing a requirement for aromaticity. This could be consistent with cation-pi carbocation stabilization. Two cleft residues modulate isomerization specificity: T147 is important, as replacement by Ser increases 11-cis relative to 13-cis by 40% compared to wildtype. F103 mutations are opposite in action: F103L dramatically reduces 11-cis synthesis relative to 13-cis synthesis compared to wildtype. T147 and F103 thus may be pivotal in controlling RPE65 specificity and changes to them directly affect product fidelity. Lastly, experiments using labeled retinol showed exchange at 13-cis retinol C15 oxygen, thus confirming enzymatic isomerization for both isomers. Thus, we concluded that RPE65 is not inherently 11-cis specific and can produce both 11-cis and 13-cis isomers, supporting a carbocation (or radical cation) mechanism for isomerization. These results also specify that visual cycle selectivity for 11-cis isomers is not entirely dependent on the isomerase (RPE65) but partly resides downstream, attributable to mass action by CRALBP, RDH5, and high affinity of opsin apoproteins for 11-cis retinal. b) BCMO1, RPE65 and related enzymes share histidine and acidic residues that are involved in iron coordination crucial to activity of these enzymes, but other crucial aspects of the mechanism (substrate binding, intermediate formation, etc.) remain unclear. In the past year, we completed 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. However, a tyrosyl radical mechanism was ruled out as replacement of any of these tyrosines did not completely abolish activity. 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 cleft of BCMO1. 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. c) To complement the work on hypomorphic mutations, such as P25L, in human disease we have been 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, we hope 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. We have established the knock-in line for the first of these constructs, and it is currently undergoing phenotyping. We are currently breeding chimeras for a second construct to see if they are germline. A third has successfully undergone homologous recombination, and is awaiting injection into embryos.