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: 2161091
• 负责人: DAVID S PAPERMASTER
• 金额: $ 36.6万
• 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/2161091
• 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.

意义和特定目的：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。 分子的最新进展 几种继承的视网膜变性的生物学基础，包括 常染色体显性视网膜炎色素炎是由突变引起的 Rhodopsin和RDS/外围蛋白基因证明了这一点的重要性 维持人正常视力的概念。 因此，我们正在探索 从高尔基体到外部的视紫红质分类的过程 部分。 我们还研究了遗传中细胞死亡的机制 视网膜营养不良，发现它通过类似的过程进行 程序性细胞死亡或凋亡。 方法和结果：青蛙视网膜 匀浆以分离高尔基膜（PGM）的高度分离 具有新合成的视紫红质的纯度。 分离的PGM上的蛋白质 已经表征了。 视觉转导的几种蛋白质 途径包括转霉素和磷酸二酯酶的亚基 确定。 另外，结合了测序和免疫化学 方法是发现αA2-和alphab2-晶状蛋白是 由视网膜合成，并与 含有视紫红质的PGM。 这提供了一个非凡，新的证据 除了重要的 在镜头中扮演伴侣的角色，在那里他们有助于维护 透明度。 一组小的GTP结合Rab蛋白，结构上 还发现了与RAS癌基因有关的，包括Rab3，Rab6， Rab8和至少两个可以ADP核糖基的ARF蛋白。 研究计划：这个Rab家族还有几个成员 仍然可以通过其电泳迁移率，PI和 免疫化学应该很容易进行，因为它们的结构是 哺乳动物同源物的高度保守和序列是已知的。 我们现在 提议通过多种 方法，主要是分子，包括使用反义构建体 并通过与合成prptides的竞争来阻止“效应子”域 在体外重构系统中。 结晶蛋白在运输中的作用 光感受器中的Rhodopsin将采用类似的技术以及 通过评估其磷酸化状态改变的影响 磷酸酶抑制剂，由于仅未磷酸化形式是膜 绑定到PGM。 遗传性视网膜变性中的细胞死亡将是 通过一种裂解核酸内切酶的原位标记的新技术进行了研究 使用TDT，生物素D-UTP和链霉亲和蛋白结合物的DNA。 我们寻找 额外的长期支持，以继续这些基本研究 组织光感受器和适度支持的基础 凋亡的初步研究是对 视网膜变性中细胞死亡的机制。