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。我们感兴趣的RPE的具体机制，在监管和功能水平。为此，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，其突变导致人类严重失明。为了更好地理解RPE 65的功能及其在RPE/光感受器相互作用中的作用，我们制作了Rpe 65敲除小鼠。全反式-视黄酯至11-顺式-视黄酸的基于RPE的代谢的破坏似乎是其表型的基础。因此，RPE 65的功能似乎与视色素再生中的关键酶视黄醇异构酶密切相关。在过去的一年里，我们取得了以下进展：a）异构化步骤下游的视觉循环蛋白的分析表明，在Rpe 65敲除中，所有蛋白都以正常或接近正常的量存在。基因敲除小鼠和野生型小鼠RPE中的视黄基酯的气相色谱/质谱分析表明，两者之间没有化学差异。这意味着在Rpe 65敲除小鼠中，缺乏11-顺式-视黄醛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酯（异构酶底物）。B）制备腺病毒和慢病毒构建体并测试其基因转移拯救敲除小鼠。c）RPE 65启动子的进一步分析旨在鉴定与推定的控制区结合的因子。我们已经制作了进一步的转基因动物，以研究这些区域以外可能的阳性和阴性元件。d）RPE 65蛋白纯化的进展为生物化学和结构分析提供了材料（例如，质谱法和原子吸收光谱法）和用于产生天然蛋白质的抗体。- 视网膜色素上皮;类维生素A;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