PERIPHERIN/RDS IN PHOTORECEPTOR RENEWAL AND DISEASE

描述（申请人描述）：人RDS基因缺陷导致 近十二种临床上不同的视网膜疾病-所有这些疾病都损害了 中央和/或周边视觉逐渐增强。RDS基因产物， 外周蛋白/rds是一种完整的膜蛋白， 光感受器外节（OS）;然而，无论是其功能在 分子水平，也不是它的作用，在视网膜病理生理学是目前 明白我们假设外周蛋白/RDS的遗传缺陷可以影响 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 疾病表型具体目标1将检验多胞体 外周蛋白/RDS中的蛋白质结构域对其 生物合成、亚细胞定位和体外促进能力 膜融合（融合原性），通过使用插入诱变方法 与无细胞和异源表达系统组合。这些研究将 确定外周蛋白/RDS的单个区域是否以及如何突变 选择性地影响蛋白质结构的特定方面和/或在体外 功能具体目标2将检验外周蛋白/RDS 融合性是体内OS盘脱落所必需的。异源 表达系统将用于设计部分功能丧失突变体， 只在催化膜融合的能力上有缺陷。光和 电子显微镜分析将确定盘脱落是否被抑制 在表达这种“非融合”转基因的小鼠视杆细胞中， 外周蛋白/rds.具体目标3将检验外周蛋白/RDS 在保持外部段磁盘堆栈的完整性方面起作用。一 异源表达系统将用于设计另外的野生型 含有高度特异性Xa因子蛋白酶的外周蛋白/rds变体 切割位点;该变体将表达为转基因以拯救 视网膜变性缓慢（RDS）小鼠（RDS不 express peripherin/RDS）。体外Xa因子蛋白水解联合电子 将使用显微镜和Western印迹分析来确定是否 这种外周蛋白/RDS变异体的特异性切割破坏了磁盘堆叠 转基因小鼠光感受器OS的组织。总之，这项研究 程序是针对建立正常的功能（S） 外周蛋白/rds在OS更新，开始的分子描述 导致各种进行性致盲疾病的病因 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.