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是视觉周期的关键视黄醇异构酶，一个基因的表达仅限于RPE，以及导致人类严重失明的突变，称为Leber先天性黑内障2 （LCA2）。RPE65基因缺陷（LCA2）和RPE65基因敲除小鼠（全反式视黄醇酯过度积累和11-顺式视网膜完全缺失，导致对光极度不敏感）的共同表型是RPE65基因缺陷（LCA2）的人类和RPE65基因敲除小鼠共有的。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。目前的工作重点是建立RPE65催化的分子机制，以及其在视网膜发育和疾病中的调节和活性。我们也在研究视觉周期的类双维甲酸副产物（如A2E）对RPE溶酶体代谢的影响。

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, the key retinol isomerase enzyme of the visual cycle, 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). Disruption of the RPE-based vitamin A visual cycle blocking regeneration of 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. Current work is focused on establishing the molecular mechanism of RPE65 catalysis, as well as its regulation and activity in the context of retinal development and in disease. We are also studying the effects of bisretinoid byproducts of the visual cycle (e.g., A2E) on RPE lysosomal metabolism. In the past year we have made the following progress: a) We have published several papers over the past few reporting periods focused on determining how RPE65 catalyzes all-trans to cis isomerization of retinol. We have presented experiments that support a retinyl cation-mediated mechanism causing general polyene bond delocalization rather than a nucleophilic substitution mechanism targeting only the C11-C12 double bond. Our analysis specifically favors a radical cation intermediate rather than a carbocation intermediate as the former alternative allows for the early loss of bond order crucial for docking of cis retinyl esters. We are currently addressing another aspect of the complex mechanism of RPE65, that of the O-alkyl bond cleavage that results in leaving of the fatty acid moiety. It is our hypothesis that this is the acquired primary enzymatic activity of RPE65, with cis isomerization being a secondary but crucial function. This O-alkyl bond cleavage is simplistically, but erroneously, characterized as a hydrolase reaction. We are now focusing on a) defining the mechanism of the O-alkyl cleavage and b) determining the first fate of the palmitate product. Methods have been established in this reporting period to test possible mechanisms for cleavage and disposition of the palmitate product. Experiments are currently underway to answer these questions. b) We continued a project to study to establish (or disprove, as the case may be) palmitoylation of RPE65 cysteine(s), a controversial aspect of RPE65 biochemistry. Different groups have used mass spectrometry to definitively establish that RPE65 is palmitoylated, or that it is not. Clearly, only one of these alternatives is true. We are using bioorthogonal methods to determine if RPE65 is acylated by metabolic labeling in vitro, as well as in physiologically relevant cell culture models. Existence of labeled cysteine(s) will be established by labeling of protein and secondarily by mass spectrometry of RPE65 peptides. Two separate approaches will be used to validate the presence or absence of a palmitoyl group. c) We completed a project to establish the origin of the vertebrate visual cycle. This question has been somewhat controversial and inadequately addressed. There has been speculation whether more primitive chordates, such as tunicates and cephalochordates, anticipated this feature. We concluded that the crucial transition from the typical carotenoid double bond cleavage functionality (BCMO) to the isomerase functionality (RPE65), coupled with the origin of LRAT, occurred subsequent to divergence of the more primitive chordates (tunicates, etc.) in the last common ancestor of the jawless and jawed vertebrates. A manuscript describing these data was published this reporting period in PLoS One. Further work in this area addresses other enzymatic functions to provide insight into how a retinoid isomerase evolved from the carotenoid double bond cleavage functionality. d) We previously generated a panel of hypomorphic knock-in mice in the mouse Rpe65 gene by homologous recombination. We continued phenotypic analysis of these mice in this reporting period. The P25L knockin mouse models the mild phenotype of a homozygous P25L LCA2 patient with well-preserved cone vision. Cone development and maintenance is highly dependent on an adequate supply of 11-cis retinal (RAL) and suffers, more so than rods, when this is absent such as in RPE65 null mutations. Milder human RPE65 missense mutations have better preserved cone function. Also, preserving cone function is a key concern in managing RPE65 retinal dystrophy, and an important objective of RPE65 gene therapy. Existing mouse models of Rpe65 retinal dystrophy (including 2 null and 1 knockin), exhibit early (null) to midstage (R91W) cone loss. We wished to establish a knock-in mouse to model milder RPE65 mutations and to determine the lower limit of 11-cis RAL for long-term preservation of cone structure and function. The P25L line had RPE65 mRNA levels identical to wildtype (WT) but RPE65 protein levels were significantly lower in P25L mice compared to WT. The retinas of P25L homozygotes were normal at 2 months and 8 months compared to WT. We found that under typical mouse husbandry and light conditions, P25L electrophysiological rod and cone function were close to WT. Importantly, there was no evidence of cone opsin mislocalization in P25L retina at 7 months suggestive of extended cone viability, unlike in the Rpe65 KO where this occurs by 1 month. However, compared to WT, P25L mice were protected against severe light damage, suggesting that visual cycle chromophore turnover was negatively affected by the P25L mutation, despite its close to normal physiology under a lower light level regime. e) We continued a study on homeostatic responses of RPE to lipofuscin accumulation. Daily phagocytosis of outer segments (OS) leads to the accumulation of storage bodies in the RPE containing autofluorescent lipofuscin, which consists of lipids and the bisretinoids, such as A2E and its oxidation products, that are byproducts of the visual cycle and that are implicated in the pathogenesis of several retinal degenerative diseases. However, A2E accumulates in RPE during normal aging. Therefore, we developed a cell model to determine the homeostatic mechanisms of RPE cells in response to A2E accumulation. To distinguish between pathologic and normal response of RPE to A2E accumulation we treated ARPE-19 cells with low micromolar amounts of A2E over several weeks. We compared lysosomal function, lysosomal pH, degree of phagocytosis and melanization of treated to untreated differentiated ARPE-19 cells in response to a challenge of purified rod OS. We found that differentiated post-confluent ARPE-19 cells uptake, accumulate and partially degrade A2E under dim light conditions. The A2E uptake in lysosomes leads to an increase in lysosomal pH. Upon challenge with ROS, A2E-treated ARPE-19 cells showed an increase in melanin pigment. In addition, the activities of the lysosomal enzymes cathepsin D and lysosomal acid phosphatase were impaired in A2E treated cells. We found that these cells responded to A2E treatment by producing a melanized lysosome fraction and therefore do not become impaired in OS phagocytosis. Thus, while A2E treatment leads to lysosomal alkalinization of ARPE-19 cells, as has been previously reported, a potential homeostatic mechanism may protect them from death.