Molecular Biology Of Outer Retina-specific Proteins

外视网膜特异性蛋白质的分子生物学

• 批准号: 8339747
• 负责人: Thomas Redmond
• 金额: $ 183.56万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8339747
• 关键词: 11 cis Retinal; 11-cis-Retinol; Address; Affect; Aftercare; Age related macular degeneration; Alkalinization; All-Trans-Retinol; Animal Model; Apoptosis; Apoptotic; Beta Carotene; Binding; Biochemistry; Blindness; Breeding; Carotenoids; Catalytic Domain; Cations; Cell Culture Techniques; Cells; Chemistry; Cleaved cell; Clinical Trials; Collaborations; Complement; Cysteine; DNA; Data; Defect; Development; Docking; Due Process; Enzymes; Esters; Family; Fluoroacetates; Future; Genes; Genetic; Genetic Engineering; Genetic Transcription; Goals; Homology Modeling; Human; Imidazole; Isomerase; Isomerism; Kinetics; Knock-in Mouse; Knock-out; Knockout Mice; Label; Learning; Leber' s amaurosis; Light; Literature; Macular degeneration; Mass Spectrum Analysis; Membrane; Messenger RNA; Metabolic; Methods; Mitochondria; Mixed Function Oxygenases; Modeling; Molecular Biology; Molecular Genetics; Mus; Mutation; Natural regeneration; Oxides; Oxygenases; Paper; Pathway interactions; Peptides; Phenotype; Phenylalanine; Play; Polyenes; Process; Production; Property; Protein Biochemistry; Proteins; Publishing; RPE65 protein; Regulation; Reporting; Research; Retina; Retinal; Retinal Degeneration; Retinal Pigments; Retinoids; Role; Shapes; Site-Directed Mutagenesis; Spin Trapping; Stargardt' s disease; Structural Models; Structure; Structure of retinal pigment epithelium; Surface; Testing; Therapeutic; Toxic effect; Transgenic Organisms; Vision; Vitamin A; Work; base; beta-Carotene 15,15' -dioxygenase; chromophore; cytotoxicity; gene therapy; homologous recombination; human tissue; in vitro activity; in vivo; inhibitor/antagonist; insight; interest; lipid metabolism; member; mouse model; mutant; operation; palmitoylation; phenyl-N-tert-butylnitrone; prevent; protective effect; research study; structural biology; visual cycle

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 successfully treated by somatic RPE65 gene therapy. 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. 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. Because they share structural features, including identical residues in the catalytic assemblage, BCMO1 is a useful model for our mechanistic studies addressing RPE65. In the past year we have made the following progress: a) We identified inhibitors of RPE65 that could have future therapeutic benefit. We previously showed that RPE65 does not specifically produce 11-cis retinol only but also 13-cis retinol, supporting a carbocation or radical cation mechanism of isomerization. The intrinsic properties of conjugated polyene chains result in facile formation of radical cations in oxidative conditions. We hypothesized that such radical intermediates, if involved in the mechanism of RPE65, could be stabilized by spin traps. We tested a variety of hydrophilic and lipophilic spin traps for their ability to inhibit RPE65 isomerohydrolase activity. We found that the aromatic lipophilic spin traps such as N-tert-butyl-alpha-phenylnitrone (PBN), 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO) and nitrosobenzene (NB) strongly inhibit RPE65 isomerohydrolase activity in vitro, while non-aromatic or hydrophilic spin traps had no inhibitory activity. This supports the hypothesis of a radical intermediate in the enzymatic mechanism of RPE65. We also determined that the mode of inhibition was uncompetitive. As PBN is relatively non-toxic and well-tolerated and has been tested in clinical trials, it may provide a means to reduce RPE65 activity in vivo, without complete inhibition of activity, such as in Stargardt macular dystrophy and age-related macular degeneration where bisretinoid accumulation is a concern. A paper describing these results was published during this reporting period. We are also studying another class of RPE65 inhibitor, unrelated to spin traps. b) We extended our understanding of how the structure of RPE65 directs isomerization. We built a structural model of the substrate-bound form of RPE65 and used site-directed mutagenesis to analyze invariant aromatic residues for isomerase activity. Substrate docking predictions of wildtype and mutant RPE65 were modeled. Homology models reveal a large, hydrophobic cleft lined by aromatic residues. The cleft opens out to the surface, allowing a direct conduit for membrane-bound substrate to access the RPE65 catalytic core. We analyzed several cleft residues, mainly phenylalanines. Previous, unexpected, results from F103 mutants showed that RPE65 is a leaky isomerase that can produce not only 11-cis retinol, but also biologically irrelevant 13-cis retinol. This implicated a delocalized bond-order retinyl intermediate (carbocation or radical cation) in the RPE65 mechanism and has already guided the discovery of specific inhibitorrs of RPE65 (see above). We identified other residues, mutations of which also yielded enzymes preferentially producing 13-cis isomer. Substrate docking models concurred with these experimental results. We conclude that these aromatic residues constrain cleft shape and volume to favor production of 11-cis retinol. A temporal model for progression of isomerization by RPE65 can be built based on these findings. Thus, by strategic placement of aromatics to modulate the geometry and chemistry of the catalytic cleft, RPE65 has evolved from a carotenoid oxygenase to an isomerase role to drive the retinal visual cycle. c) We began a 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 a bioorthogonal method to determine if RPE65 is acylated by metabolic labeling in a physiologically relevant cell culture model. Existence of labeled cysteine(s) will be established by mass spectrometry of RPE65 peptides. We are also beginning experiments to discern how the retinoid visual cycle integrates with overall lipid metabolism in the RPE. d) A project to study the role of A2E in RPE cytotoxicity for Stargardt disease and to study possible therapies was initiated in collaboration with Dr. Brian Brooks and Dr. Nataly Strunnikova (OGVFB). While the toxic effects of A2E accumulation have been shown for high A2E concentrations in a mouse model of recessive Stargardt's macular degeneration, the literature on A2E toxicity to RPE cells at low micromolar concentration varies from DNA protective effects to mitochondrial damage and apoptosis. We believe that this variability may be partially explained by differences in how A2E is synthesized and delivered in these experiments. We found a significant lysosomal pH change after treatment with low micromolar concentrations of A2E fluoroacetate using a pH sensitive lysosensor. However, this treatment with A2E was not apoptotic or proapoptotic. We are investigating further how the use of different counterions affects lysosomal alkalinization and other effects in RPE cells. Targeting this pathway may prevent the deleterious effects of lysosomal alkalinization. Also, we developed a mass spectrometry method to quantitate A2E and applied this to the quantitation of A2E from mouse and human tissues (in collaboration with Crouch/Ablonczy lab at MUSC, Charleston, SC). e) To complement prior work on pathogenic human RPE65 hypomorphic mutations, such as P25L, we have generated, in collaboration with the NEI Genetic Engineering Core, 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 three knock-in lines. To abrogate interference with transcription and/or mRNA processing due to the neo selection cassette, resulting in an effectively null phenotype, we had to breed all three knock-ins with the Zp3-Cre line to remove the neo cassette. The three lines are now being phenotyped. Preliminary data, obtained in collaboration with the NEI Visual Function Core, reveals pronounced changes in the kinetics of regeneration in these knockins, consistent with our predictions.

视网膜色素上皮（RPE）在外视网膜的发展和功能中起关键作用。我们对调节水平和功能水平的RPE特异性机制都感兴趣，并且我们一直在研究RPE65的功能和调控，RPE65的功能和调节，该基因的表达仅限于RPE，以及在人类中引起严重失明的突变，被称为Leber Enceranital Amaurosis 2（LCA2）。 LCA2已通过体细胞RPE65基因疗法成功治疗。基于RPE的维生素的破坏是视觉色素发色子的视觉周期再生是具有RPE65基因缺陷（LCA2）和RPE65敲除小鼠的常见表型（全反性元素 - 元素 - 元素 - 元素 - 元素 - 元素过度，总缺乏11- cis的视网膜，导致了极端的效果）。我们已经在11-CIS视黄醇的合成中确定了RPE65的催化作用，将其确定为长期追求的视觉周期等异羟氢集酶。我们还一直在研究β-胡萝卜素15,15'-单加氧酶（BCMO1），我们首先根据与RPE65的相似性识别出来。 BCMO1与RPE65密切相关，两者都是新出现的类胡萝卜素裂解酶的成员。由于它们具有结构特征，包括催化组合中相同的残基，因此BCMO1是我们针对RPE65的机械研究的有用模型。 在过去的一年中，我们取得了以下进展： a）我们确定了RPE65的抑制剂，这些抑制剂可能会带来未来的治疗益处。我们先前表明，RPE65不专门产生11-CIS视黄醇，而是13-CIS视黄醇，支持异构化的碳定位或自由基阳离子机理。 共轭多烯链的固有特性导致在氧化条件下易于形成自由基阳离子。我们假设这种自由基中间体如果参与RPE65的机制，可以通过自旋陷阱稳定。我们测试了各种亲水性和亲脂性自旋陷阱，以抑制RPE65等杂化酶活性的能力。 We found that the aromatic lipophilic spin traps such as N-tert-butyl-alpha-phenylnitrone (PBN), 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO) and nitrosobenzene (NB) strongly inhibit RPE65 isomerohydrolase activity in vitro, while non-aromatic or hydrophilic spin陷阱没有抑制活性。 这支持了RPE65酶促机理中一个自由基中间体的假设。我们还确定抑制方式是非竞争的。由于PBN相对无毒且耐受性良好，并且在临床试验中已经进行了测试，因此它可以提供一种减少体内RPE65活性的方法，而没有完全抑制活性，例如在Stargardt黄斑营养不良症和年龄相关的黄斑变性中，双氨基素的积累是您的问题。在此报告期间发表了一篇描述这些结果的论文。我们还研究了另一种与自旋陷阱无关的RPE65抑制剂。 b）我们扩展了对RPE65结构如何指导异构化的理解。我们构建了RPE65底物结合形式的结构模型，并使用定位的诱变来分析异构酶活性的不变芳族残基。 对野生型和突变体RPE65的底物对接预测进行了建模。同源性模型显示出大型的疏水性裂缝，衬有芳香族残基。裂口可以打开表面，从而使膜结合的底物的直接导管进入RPE65催化核心。我们分析了几种裂口残基，主要是苯丙氨酸。以前的F103突变体的出乎意料的结果表明，RPE65是一种泄漏的异构酶，不仅可以产生11 cis视黄醇，而且可以生物学上无关的13张视黄醇。这意味着RPE65机制中的一个离域键 - 视网膜基因中间体（碳定位或自由基阳离子），并且已经指导发现RPE65的特定抑制剂（见上文）。我们确定了其他残基，其突变也产生了酶优先产生13-CIS异构体。底物对接模型与这些实验结果一致。我们得出的结论是，这些芳族残基会限制裂缝的形状和体积以有利于生产11-CIS视黄醇。可以根据这些发现构建通过RPE65进行异构化进展的时间模型。 因此，通过策略放置芳香族以调节催化裂解的几何形状和化学，RPE65已从类胡萝卜素氧合酶演变为同构酶的作用，以驱动视网膜视觉循环。 c）我们开始了一项研究，以建立（或以情况为代表）RPE65半胱氨酸的棕榈酰化，这是RPE65生物化学的有争议的方面。不同的组已经使用质谱法确定RPE65是棕榈酰，或者不是。 显然，这些替代方案中只有一个是真实的。我们正在使用生物正交方法来确定RPE65是否通过在生理相关的细胞培养模型中的代谢标记来酰化。 标记的半胱氨酸的存在将通过RPE65肽的质谱法建立。 我们还开始进行实验，以辨别性类维生素类似的视觉周期如何与RPE中的总脂质代谢相结合。 d）与Brian Brooks博士和Nataly Strunnikova（OGVFB）合作启动了一个研究A2E在RPE细胞毒性中的作用并研究可能疗法的项目。 尽管已在隐性星形阳性黄斑变性的小鼠模型中显示了A2E积累的毒性作用，但在低微摩尔浓度下对RPE细胞的毒性文献从DNA保护效应到线粒体损伤和凋亡。我们认为，这种可变性可能是通过在这些实验中合成和交付A2E的差异来部分解释的。我们发现，使用pH敏感的溶酶体传感器，用低微摩尔浓度的A2E氟乙酸盐处理后发现明显的溶酶体pH变化。但是，这种用A2E治疗不是凋亡或促凋亡的。 我们正在进一步研究不同柜台的使用如何影响RPE细胞中的溶酶体碱化和其他影响。针对该途径可能会阻止溶酶体碱化的有害作用。此外，我们开发了一种质谱法来定量A2E，并将其应用于小鼠和人体组织的A2E（与SC Charleston，SC MUSC的Crouch/Ablonczy Lab合作）。 e）为了补充致病性人RPE65型肌电65突变（例如p25L）的先前工作，我们与Nei Genetic Engineering Core合作生成了小鼠RPE65基因中的型型遗传工程核心，通过同源重组。可以预计，与敲除的极端情况相比，这些将为RPE65缺陷表型的变异性提供重要的见解。特别是，我们希望能够深入了解视网膜变性的较慢，例如在不太严重的人类RPE65突变中所见。 他们还将提供动物模型来测试药理策略。我们已经建立了三条敲门线。为了消除因新选择盒引起的转录和/或mRNA加工的干扰，从而有效地表型，我们不得不用ZP3-CRE系列繁殖所有三个敲门件，以去除新盒式盒子。 现在，这三条线正在表型。 与NEI视觉函数核心合作获得的初步数据揭示了这些敲门蛋白的再生动力学的明显变化，这与我们的预测一致。