MEMBRANE BIOSYNTHESIS IN NORMAL AND DYSTROPHIC RETINAS

意义和具体目标：光感受器的组织是 依赖于适当的生物合成，分选，沉积和保留 其蛋白质的功能域。 分子生物学的最新进展 几种遗传性视网膜变性的生物学基础，包括 常染色体显性视网膜色素变性引起的突变， 视紫红质和RDS/外周蛋白基因证明了这一点的重要性。 维持人类正常视力的概念。因此，我们正在探索 视紫红质从高尔基体向外部的分选过程 片段 我们还研究了遗传性肿瘤细胞死亡的机制， 视网膜营养不良，并发现它通过一个类似于 程序性细胞死亡或凋亡。 方法和结果：蛙视网膜 将匀浆分级以分离高浓度的高尔基体后膜（PGM）。 新合成的视紫红质。 分离的PGM上的蛋白 已经被定性。 几种视觉信号转导蛋白 包括转导素亚基和磷酸二酯酶的途径已经被 鉴定 此外，通过测序和免疫化学的结合， 方法，已经发现α A2-和α B2-晶体蛋白都是 由视网膜合成，并特别与 带有视紫红质的PGM。 这为一个非同寻常的、新的 这些蛋白质的晶状体外功能除了它们重要的 作为透镜中的伴侣，它们帮助维持 透明度 一组小GTP结合rab蛋白，结构上 也发现了与ras癌基因相关的包括Rab 3，Rab 6， rab8和至少两种可被ADP核糖基化的ARF蛋白。 研究计划：这个rab家族还有几个成员 仍需通过其电泳迁移率、pI和 由于它们的结构 哺乳动物同源物的高度保守和序列是已知的。 我们现在 建议通过多种方法评估这些rab蛋白的功能， 方法，主要是分子，包括使用反义构建体 以及通过与合成肽竞争来阻断"效应"域 在体外重组系统中。 晶体蛋白在药物转运中的作用 光感受器中的视紫红质将采用类似的技术， 评估其磷酸化状态改变的影响， 磷酸酶抑制剂，因为只有未磷酸化的形式是膜 与PGM绑定。 遗传性视网膜变性中的细胞死亡 用酶切后原位末端标记的新技术研究了 使用TDT、生物素d-UTP和链霉亲和素缀合物的DNA。 我们寻求 提供额外的长期支持，以继续进行这些基础研究， 光感受器组织的基础和适度的支持，以完成 细胞凋亡的初步研究作为一个基本的新的见解， 视网膜变性中细胞死亡的机制。

Significance and Specific Aims: The organization of the photoreceptor is dependent on the proper biosynthesis, sorting, deposition and retention of its proteins in their functional domains. Recent advances in the molecular biological basis of several inherited retinal degenerations, including autosomal dominant retinitis pigmentosa arising from mutations of the rhodopsin and rds/peripherin genes demonstrate the importance of this concept in maintaining normal vision in man. Accordingly, we are exploring the process of sorting of rhodopsin from the Golgi apparatus to the outer segment. We have also studied the mechanism of cell death in inherited retinal dystrophies and discovered that it proceeds by a process resembling programmed cell death or apoptosis. Methods and Results: Frog retinal homogenates are fractionated to isolate post-Golgi membranes (PGM) in high purity that bear newly synthesized rhodopsin. The proteins on isolated PGM have been characterized. Several proteins of the visual transduction pathway including subunits of transducin and phosphodiesterase have been identified. In addition, by a combination of sequencing and immunochemical approaches, both alphaA2- and alphaB2-crystallins have been found to be synthesized by the retina and to be specifically associated with the rhodopsin-bearing PGM. This provides evidence for an extraordinary, new extralenticular function for these proteins in addition to their important role as chaperones in the lens where they assist in maintaining transparency. A set of small GTP binding rab proteins, structurally related to the ras oncogene have also been discovered including rab3, rab6, rab8 and at least two ARF proteins that can be ADP ribosylated. Research Plan: There are several additional members of this rab family still to be identified by their electrophoretic mobility, pI, and immunochemistry which should proceed readily since their structures are highly conserved and sequences of mammalian homologues are known. We now propose to evaluate the function of these rab proteins by a variety of approaches, primarily molecular, including the use of antisense constructs and blocking of "effector" domains by competition with synthetic prptides in reconstituted systems in vitro. The role of crystallins in transport of rhodopsin in photoreceptors will employ similar techniques as well as evaluating the impact of alteration of their phosphorylation state by phosphatase inhibitors, since only the unphosphorylated forms are membrane bound to PGM. Cell death in inherited retinal degenerations will be studied by a new technique of in situ end labeling of endonuclease cleaved DNA using TDT, biotinyl d-UTP and streptavidin conjugates. We seek additional long-term support to continue these studies of the fundamental basis of organization of photoreceptors and modest support to complete the initial studies of apoptosis as a fundamental new insight into the mechanism of cell death in retinal degenerations.