MEMBRANE BIOSYNTHESIS IN NORMAL AND DYSTROPHIC RETINAS

正常和营养不良视网膜中的膜生物合成

• 批准号: 2161092
• 负责人: DAVID S PAPERMASTER
• 金额: $ 26.8万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2161092
• 关键词: Anura; Golgi apparatus; apoptosis; crystallins; gene expression; immunocytochemistry; intracellular transport; laboratory mouse; laboratory rabbit; laboratory rat; membrane biogenesis; membrane proteins; membrane structure; molecular chaperones; molecular pathology; phosphatase inhibitor; phosphorylation; retina; retina degeneration; rhodopsin; tissue /cell culture

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.

意义和具体目标：光感受器的组织是 取决于适当的生物合成、分类、沉积和保留 它的蛋白质在其功能域中。 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 常染色体显性遗传性视网膜色素变性，由基因突变引起 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。 据此，我们正在探索 视紫红质从高尔基体分选到外层的过程 部分。 我们还研究了遗传性细胞死亡的机制 视网膜营养不良并发现其发生过程类似于 程序性细胞死亡或凋亡。 方法和结果：青蛙视网膜 将匀浆分级分离以高尔基体后膜 (PGM) 具有新合成的视紫红质的纯度。 分离的 PGM 上的蛋白质 已被表征。 视觉转导的几种蛋白质 包括转导蛋白和磷酸二酯酶亚基的途径已被 确定。 此外，通过测序和免疫化学相结合 方法，αA2-和αB2-晶状体蛋白都被发现 由视网膜合成并与 携带视紫红质的 PGM。 这为一种非凡的、新的 这些蛋白质除了重要的晶状体外功能之外 在镜头中充当伴侣的角色，帮助维持 透明度。 一组小的 GTP 结合 rab 蛋白，结构上 还发现了与ras癌基因相关的包括rab3、rab6、 rab8 和至少两种可 ADP 核糖基化的 ARF 蛋白。 研究计划：这个rab家族还有几个额外的成员 仍有待通过电泳迁移率、pI 和 免疫化学应该很容易进行，因为它们的结构是 高度保守的哺乳动物同源物序列是已知的。 我们现在 建议通过多种方法评估这些 rab 蛋白的功能 主要是分子方法，包括使用反义结构 通过与合成肽竞争来阻断“效应子”结构域 在体外重建系统中。 晶状体蛋白在转运中的作用 光感受器中的视紫红质将采用类似的技术以及 通过评估磷酸化状态改变的影响 磷酸酶抑制剂，因为只有非磷酸化形式是膜 与 PGM 绑定。 遗传性视网膜变性中的细胞死亡将 通过核酸内切酶原位末端标记新技术进行研究 使用 TDT、生物素 d-UTP 和链霉亲和素缀合物进行 DNA 分析。 我们寻求 继续这些基础研究的额外长期支持 光感受器组织的基础和完成的适度支持 细胞凋亡的初步研究作为对细胞凋亡的基本新见解 视网膜变性中细胞死亡的机制。