MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

视网膜色素上皮(RPE)在视网膜外层的发育和功能中起着关键作用。我们对RPE特有的机制感兴趣，无论是在监管层面还是在功能层面。为此，我们一直在研究RPE65的功能和调控，该基因的表达仅限于RPE和导致人类严重失明的突变。为了更好地了解RPE65的功能及其在RPE/光感受器相互作用中的作用，我们制作了一只RPE65基因敲除小鼠。以RPE为基础的全反式视黄酸酯向11顺式视黄醇代谢的中断似乎是其表型的基础。因此，RPE65的功能似乎与视黄醇异构酶密切相关，视黄醇异构酶是视觉色素再生的关键酶。在过去的一年里，我们取得了以下进展：a)对异构化步骤下游的视觉周期蛋白的分析表明，在RPE65基因敲除中，所有这些蛋白都以正常或接近正常的数量存在。对基因敲除小鼠和野生型小鼠RPE中视黄酸酯的气相色谱/质谱分析表明，两者之间没有化学差异。这意味着在RPE65基因敲除的小鼠中，缺乏11-顺式视网膜并不是由于缺乏异构化产物的后续加工，而是由于缺乏异构化产物，或者由于存在不适当的视黄酸酯(异构酶底物)。B)正在制造腺病毒和慢病毒载体，并进行基因转移拯救基因敲除小鼠的测试。C)对RPE65启动子的进一步分析旨在确定与推测的控制区结合的因素。我们已经制作了更多的转基因动物，以研究这些区域以外可能存在的积极和消极因素。D)在提纯RPE65蛋白方面的进展为生化和结构分析(例如，质谱学和原子吸收光谱学)以及产生针对天然蛋白的抗体提供了材料。视网膜色素上皮；类维甲酸；转基因/基因敲除小鼠；基因调控；视网膜营养不良；蛋白质化学

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. To this end 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. To better understand the function of RPE65 and its role in RPE/photoreceptor interactions we have made an Rpe65 knockout mouse. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal appears to underlie its phenotype. The function of RPE65 thus appears to be closely related to that of the retinol isomerase, the crucial enzyme in visual pigment regeneration. In the past year we have made the following progress: a) Analysis of visual cycle proteins downstream of the isomerization step shows that all are present in normal or close to normal amounts in the Rpe65 knockout. Gas chromatography/mass-spectroscopy of the retinyl esters in the RPE of the knockout and wildtype mice reveals that there is no chemical difference between the two. This implies that in the Rpe65 knockout mouse, lack of 11-cis-retinal is not due to the absence of subsequent processing of the isomerization product but rather to lack of the isomerization product, or due to the presence of inappropriate retinyl esters (isomerase substrate). b) Adenoviral and lentiviral constructs are being made and tested for gene transfer rescue of the knockout mouse. c) Further analysis of the RPE65 promoter was targeted at identifying factors binding to the putative control region. We have made further transgenic animals to investigate possible positive and negative elements outside these regions. d) Progress in purification of the RPE65 protein has provided material for biochemical and structural analyses (e.g., mass- spectroscopy and atomic absorption spectroscopy) and for generation of an antibody to the native protein. - Retinal pigment epithelium; retinoids; Transgenic/knockout mice; gene regulation; retinal dystrophies; protein chemistry