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。我们对调控和功能层面的rpe特异性机制都很感兴趣。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，其突变会导致人类严重失明。Rpe65基因敲除小鼠的表型似乎是基于rpe的全反式视黄醇酯到11-顺式视网膜代谢的破坏。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。在过去的一年中，我们取得了以下进展：a)我们确定了RPE65基因在RPE中负责组织特异性表达的调控区域，发现八聚体和E-box转录因子在RPE65基因的转录调控中起着关键作用。b)两项合作研究表明，小鼠RPE65表达水平与视网膜光损伤易感性之间存在直接关系。Rpe65基因敲除小鼠或具有活性较低变体的小鼠对光诱导的视网膜损伤具有抵抗力。c)对衰老Rpe65基因敲除小鼠（两岁以下）的分析显示，与野生型相比，与年龄相关的色素脂褐素（与年龄相关的黄斑变性相关的维生素a的不可逆副产物）的积累显著降低。d)我们已经从小鼠身上鉴定出了第一个哺乳动物β -胡萝卜素15,15'-双加氧酶（β - cd），这是一种在发育和代谢过程中至关重要的酶，控制着从植物来源的β -胡萝卜素中重新进入维生素a。该蛋白是RPE65的同源物，是一个新出现的多种酶家族的成员，包括植物9-顺式环氧类胡萝卜素裂解酶和细菌木本二苯乙烯二氧合酶。e)正在用腺相关病毒构建基因载体，并将对基因转移挽救敲除小鼠进行测试。

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. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal appears to underlie the phenotype of the Rpe65 knockout mouse. 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) We have identified the regulatory region of the RPE65 gene that accounts for tissue-specific expression in the RPE and found that octamer and E-box transcription factors play a critical role in the transcriptional regulation of the RPE65 gene. b) Two collaborative efforts showed a direct relationship between the level of RPE65 expression in mice and susceptibility to retinal light damage. Rpe65 knockout mice or those with a less active variant are resistant to light-induced retinal damage. c) Analysis of aging Rpe65 knockout mice (up to two years old) show a dramatically lowered accumulation of the age-related pigment lipofuscin (an irreversible by-product of vitamin A associated with age-related macular degeneration) compared to wildtype. d) We have identified from mouse the first mammalian beta-carotene 15,15'-dioxygenase (beta-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived beta-carotene. This protein is a homologue of RPE65 and a member of a newly emerging diverse family of enzymes that includes the plant 9-cis-epoxycarotenoid cleavage enzyme and bacterial lignostilbene dioxgenases. e) Gene vector constructs are being made in Adeno-associated virus and will be tested for gene transfer rescue of the knockout mouse.