PERIPHERIN/RDS IN PHOTORECEPTOR RENEWAL AND DISEASE

描述(申请人描述)：人类RDS基因缺陷导致 近12种临床上截然不同的视网膜疾病--所有这些疾病都会损害 中央和/或周边视力逐渐增强。RDS基因产物， 外周蛋白/rds，是一种完整的膜蛋白，是形成 光感受器外节(OS)；然而，它在 分子水平上，其在视网膜病理生理学中的作用目前尚不清楚 明白了。我们假设外周/rds中的遗传缺陷可能会影响 特定蛋白质结构域优先抑制光感受器OS盘 形态发生、堆积或脱落--从而产生异质性 疾病表型。具体目标1将检验多面体的假设 外周蛋白/rds中的蛋白质结构域对其有不同的贡献 生物合成、亚细胞定位和体外促进能力 膜融合(融合原性)，使用插入突变方法 与无细胞和异源表达系统相结合。这些研究将 确定外周蛋白/rds个别区域是否以及如何发生突变 选择性地影响蛋白质结构的特定方面和/或在体外 功能。特定目标2将检验外周蛋白/RDS的假设 在体内，OS盘的脱落需要融合。一个异源的 表达系统将用于设计一种功能部分丧失的突变体 只在其催化膜融合的能力方面有缺陷。光和 电子显微镜分析将确定是否阻止了圆盘脱落 在小鼠杆状感光器中表达该转基因的“非融合基因” 外围设备/RDS。具体目标3将检验外周蛋白/RDS的假设 在维护外部段磁盘堆栈的完整性方面发挥作用。一个 异源表达系统将被用来设计一种原本野生型 含有高度特异的凝血因子Xa蛋白酶的外周蛋白/rds变异体 裂解位点；这个变异体将以转基因的形式表达以拯救 视网膜变性缓慢(RDS)小鼠(RDS)的光感受器 快速外围设备/RDS)。Xa因子体外蛋白降解与电子结合的研究 将使用显微镜和蛋白质印迹分析来确定 此外设/RDS变体的特定切割会扰乱磁盘堆栈 转基因小鼠光感受器OSS的组织。总而言之，这项研究 该计划旨在建立正常的功能(S) 操作系统更新中的外围设备/RDS，以开始对 各种进行性致盲疾病背后的病因 来自RDS中的缺陷。

DESCRIPTION (Applicant's Description): Defects in the human RDS gene result in nearly a dozen clinically distinct retinal diseases - all of which impair central and/or peripheral vision progressively. The RDS gene product, peripherin/rds, is an integral membrane protein required for the formation of the photoreceptor outer segment (OS); however, neither its function at the molecular level, nor its role in retinal pathophysiology is currently understood. We hypothesize that inherited defects in peripherin/rds can affect particular protein domains to preferentially inhibit photoreceptor OS disk morphogenesis, stacking, or shedding - and thereby generate heterogeneous disease phenotypes. Specific aim 1 will test the hypothesis that polytopic protein domains in peripherin/rds contribute differentially to its biosynthesis, subcellular localization, and ability to promote in vitro membrane fusion (fusogenicity), by using an insertional mutagenesis approach combined with cell-free and heterologous expression systems. These studies will determine if, and how, mutations in individual regions of peripherin/rds selectively affect particular aspects of protein structure and/or in vitro function. Specific aim 2 will test the hypothesis that peripherin/rds fusogenicity is required in vivo for OS disk shedding. A heterologous expression system will be used to design a partial loss-of-function mutant that is defective only in its ability to catalyze membrane fusion. Light and electron microscopic analyses will determine whether disk shedding is inhibited in mouse rod photoreceptors expressing this transgene for "non-fusogenic" peripherin/rds. Specific aim 3 will test the hypothesis that peripherin/rds plays a role in maintaining the integrity of outer segment disk stacks. A heterologous expression system will be used to design an otherwise wild-type peripherin/rds variant that contains a highly-specific Factor Xa protease cleavage site; this variant will be expressed as a transgene to rescue photoreceptors in the retinal degeneration slow (rds) mouse (rds does not express peripherin/rds). In vitro Factor Xa proteolysis combined with electron microscopic and Western blot analyses will be used to determine whether specific cleavage of this peripherin/rds variant disrupts the disk stack organization of transgenic mouse photoreceptor OSs. In sum, this research program is directed towards establishing the normal function(s) of peripherin/rds in OS renewal, to begin a molecular description of the etiologies behind a wide variety of progressive blinding diseases that result from defects in RDS.