MECHANISMS OF PHOTORECEPTOR CELL DEGENERATION

在常染色体显性遗传性视网膜色素变性(ADRP)和各种形式的黄斑营养不良(MD)的发病机制中，编码视盘边缘特异性蛋白的基因突变被认为是有关联的。外周蛋白/RDS在盘沿结构的形态发生和维持中起重要作用。这种作用是由与其他蛋白质的相互作用支持的。这项研究的主要目的是确定外周蛋白/rds及其相互作用蛋白的分子作用基础和功能特性，并研究在人类视网膜疾病转基因模型中导致光感受器退化的分子异常。具体目标1、2和3是利用结构、电生理和生化研究来评估表达外周蛋白/rds基因不同突变的转基因视网膜。这些突变包括：(1)导致人类MD的ARG172TP突变，(2)确定分子间二硫键在组装功能性外周蛋白/rds-rom-1复合体中的作用的CYS214Ser突变。我们将分析这些突变对盘膜形态发生的影响，以及外周蛋白/rds和rom-1对复合体形成的调控。我们可以通过比较Arg172Trp和Cys214Ser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。对Cys214和Cys150Ser转基因小鼠的比较将有助于深入了解分子间和分子内二硫键形成在外周蛋白/rds功能中的作用。在特定的im4中，我们将使用酵母双杂交系统来鉴定外周蛋白/rds和rom-1形成多聚体复合体所涉及的相互作用位点。该系统为检测蛋白质之间的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，以寻找参与组装功能外周蛋白/rds-rom-1复合体的其他蛋白质。我们已经确定了所有已知的外周蛋白/RDS与位于大盘内环的ROM-1之间的高度同源性区域。我们假设这些区域调节外周蛋白/rds-rom-1复合体之间的相互作用，并需要以其扁平的形状保持杆状圆盘或圆锥形薄片。这些相互作用可能是通过亚基复合体的直接结合或通过其他蛋白质间接调节的。将进行诱变研究以评估这些相互作用。这些研究将深入了解外周蛋白/rds在正常和疾病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.