Development and Maintenance of Lens Transparency

描述（由申请人提供）：人α β晶体蛋白是小热休克蛋白sHSP的原型，sHSP参与蛋白质聚集和细丝组装疾病，包括白内障、神经变性、心肌病和结蛋白相关肌病。α β晶体蛋白之间的相互作用对于透镜细胞中晶体蛋白的正常细丝组装和组织是必需的。在目的1中，表征人α β晶体蛋白上亚基组装、细胞骨架蛋白和靶肽的相互作用位点，将使用蛋白质多针阵列鉴定人α β晶体蛋白上相互作用结构域的肽序列。相互作用结构域之间的亲和力将使用表面等离子体共振（SPR）进行定量，并使用伴侣蛋白活性的体外和体内测定进行功能表征。这些结果有望为sHSP亚基组装成功能复合物及其与伴侣蛋白靶蛋白、细胞丝和细胞骨架元件的相互作用提供新的结构基础信息。在目的2中，将研究可能影响转基因小鼠中透镜透明度的发育和维持的视网膜-透镜关系的体内评价，即透镜-视网膜关系对于透镜细胞透明度的正常发育是重要的历史假设。在选定的动物模型中，视网膜电图（ERG）和数字裂隙灯不透明度记录将在透镜发育期间和白内障形成模型中透明度丧失期间量化透明度与视网膜功能。最后，假设透镜细胞骨架提供了一个支架的发展和维持透明的透镜纤维结构将在目标3中进行研究，观察主要结构蛋白的细胞组织分化透镜纤维在透镜透明度的发展过程中，在亚硒酸钠大鼠和选定的转基因小鼠模型的透明度损失期间，使用共聚焦显微镜和电子显微镜（EM）。透明透镜纤维分化过程中细胞骨架和晶体蛋白的模式和分布将使用电子显微镜和共聚焦免疫细胞化学进行研究。

DESCRIPTION (provided by applicant): Human alphaBeta crystallin is the archetype for small heat shock proteins, sHSP, that are involved in protein aggregation and filament assembly diseases including cataracts, neurodegeneration, cardiomyopathy and desmin related myopathy. Interactions between alphaBeta crystallin are necessary for normal filament assembly and organization of crystallins in lens cells. In aim 1, characterization of the interactive sites for subunit assembly, for cytoskeletal proteins and for target peptides on human alphaBeta crystallin, the peptide sequences of the interactive domains on human alphaBeta crystallin will be identified using a protein multipin arrays. The affinities between the interactive domains will be quantified using surface plasmon resonance (SPR) and characterized functionally using in vitro and in vivo assays for chaperone activity. The results are expected to provide new information on the structural basis for the assembly of sHSP subunits to functional complexes and for their interaction with chaperone target proteins and with cellular filaments and cytoskeletal elements. In aim 2, in vivo evaluation of retina - lens relationships that may influence development and maintenance of lens transparency in transgenic mice, the historical hypothesis that a lens - retina relationship is important for normal development of lens cell transparency will be studied. Electroretinograms (ERG) and digital slit lamp recordings of opacity in selected animal models will quantify transparency with retinal function during the development of the lens and during loss of transparency in models for cataract formation. Lastly, the hypothesis that lens cytoskeleton provides a scaffold for development and maintenance of transparent lens fiber structure will be investigated in aim 3, observe the cellular organization of major structural proteins in differentiating lens fibers during development of lens transparency and during loss of transparency in the selenite rat and in selected transgenic mouse models using confocal microscopy and electron microscopy (EM). The patterns and distribution of the cytoskeleton and crystallins during differentiation of transparent lens fibers will be investigated using electron microscopy and confocal immunocytochemistry.