MECHANISMS OF PHOTORECEPTOR CELL DEGENERATION

编码盘缘特异性蛋白（peripherin/rds）的基因突变与常染色体显性视网膜色素变性（ADRP）和各种形式的黄斑营养不良（MD）的发病机制有关。Peripherin/rds在盘缘结构的形态发生和维持中起重要作用。这种作用是通过与其他蛋白质的相互作用来支持的。本研究的主要目标是确定外周蛋白/rds和这些相互作用蛋白的作用和功能特性的分子基础，并研究导致人类视网膜疾病转基因模型中光感受器变性的分子异常。具体目的1、2和3是使用结构、电生理和生物化学研究来评价在外周蛋白/rds基因中表达不同突变的转基因视网膜。这些突变包括：（1）导致人类MD的ARG 172 TP突变，（2）CYS 214 Ser突变，以确定分子间二硫键在功能性外周蛋白/rds-rom-1复合物组装中的作用。我们将分析这些突变对椎间盘膜形态发生的影响，以及对外周蛋白/rds和rom-1调控复合物形成的影响。我们可以通过比较Arg 172 Trp和Cys 214 Ser突变对视杆细胞和视锥细胞外段结构和功能的影响来评估外周蛋白/rds在视杆细胞和视锥细胞中所起的不同作用。Cys 214和Cys 150 Ser转基因小鼠的比较将提供深入了解外周蛋白/rds功能中分子间和分子内二硫键形成的作用。在具体的im 4，我们将使用酵母双杂交系统，以确定参与形成的多聚体复合物的外周蛋白/rds和rom-1的相互作用的网站。该系统为检测蛋白质-蛋白质相互作用提供了强大的遗传机制。我们还将使用酵母双杂交系统来筛选视网膜cDNA文库中参与功能性外周蛋白/rds-rom-1复合物组装的其他蛋白质。我们已经确定了所有已知的外周蛋白/rds和rom-1之间高度同源的区域，它们位于大的椎间盘内环。我们假设这些区域介导外周蛋白/rds-rom-1复合物之间的相互作用，并需要保持杆盘或锥板在其扁平的形状。这些相互作用可以通过亚基复合物的直接缔合或通过其他蛋白质间接介导。将进行致突变研究以评价这些相互作用。这些研究将提供深入了解外周蛋白/rds在正常和病变视网膜中的功能作用。

Mutations in the gene encoding the disk rim specific protein, peripherin/rds, have been implicated in the pathogenesis of both autosomal dominant retinitis pigmentosa (ADRP) and various forms of macular dystrophy (MD). Peripherin/rds plays an important role in the morphogenesis and maintenance of the disk rim structure. This role is supported by interactions with other proteins. The primary goals of the proposed research are to determine the molecular basis of action and functional properties of peripherin/rds and these interacting proteins, and to investigate the molecular abnormalities that lead to photoreceptor degeneration in transgenic models of human retinal diseases. Specific Aims 1, 2 and 3 are to use the structural, electrophysiological and biochemical studies to evaluate transgenic retinas expressing different mutations in the peripherin/rds gene. These mutations include: (1) the ARG172TP mutation that causes MD in humans, (2) the CYS214Ser mutation to determine the role of intermolecular disulfide bonds in the assembly of functional peripherin/rds-rom-1 complexes. We will analyze the effects of these mutations on morphogenesis of the disc membrane and on regulation of complex formation by peripherin/rds and rom-1. We can evaluate the different roles played by peripherin/rds in rods versus cones by comparing the effects of the Arg172Trp and Cys214Ser mutations on the structure and function of rod and cone outer segments. Comparisons of the Cys214 and Cys150Ser transgenic mice will provide insight into the role of inter- and intramolecular disulfide bond formation in peripherin/rds function. In Specific im 4, we will use a yeast two-hybrid system to identify the sites of interaction involved in formation of multimeric complexes by peripherin/rds and rom-1. This system provides a powerful genetic mechanism for detecting protein-protein interactions. We will also use the yeast two-hybrid system to screen a retinal cDNA library for other proteins that are involved in the assembly of functional peripherin/rds-rom-1 complexes. We have identified regions of high homology between all known peripherin/rds and rom-1 that are located in the large intradiscal loop. We hypothesize that these regions mediate the interactions between peripherin/rds-rom-1 complexes and are required to hold the rod discs or cone lamellae in their flattened shape. These interactions may be mediated by direct association of the subunit complexes or indirectly through other proteins. Mutagenesis studies will be performed to evaluate these interactions. These studies will provide insight into the functional role of peripherin/rds in normal and diseased retinas.