Development and Maintenance of Lens Transparency

描述(申请人提供)：人类Alphabeta晶体蛋白是小分子热休克蛋白SHSP的原型，参与蛋白质聚集和细丝组装疾病，包括白内障、神经退行性变、心肌病和结蛋白相关肌病。α-β晶状体蛋白之间的相互作用是晶状体细胞内正常的微丝组装和晶状体蛋白组织所必需的。在目标1中，亚基组装、细胞骨架蛋白和人αβ晶体蛋白上靶肽的相互作用位点的表征，将使用蛋白质多针阵列来鉴定人αβ晶体蛋白上相互作用区域的肽序列。相互作用结构域之间的亲和力将通过表面等离子共振(SPR)来量化，并使用体外和体内伴侣活性测定来表征功能。这些结果有望为SHSP亚单位组装成功能复合体以及它们与伴侣靶蛋白以及细胞微丝和细胞骨架元件的相互作用提供新的结构基础。在目标2中，对可能影响转基因小鼠晶状体透明度发育和维持的视网膜-晶状体关系的活体评估，将研究晶状体-视网膜关系对晶状体细胞透明度的正常发育至关重要的历史假设。在选定的动物模型中，视网膜电信号(ERG)和数字裂隙灯记录的混浊将根据晶状体发育期间和白内障形成模型中失去透明度的视网膜功能来量化透明度。最后，目标3将研究晶状体细胞骨架为晶状体纤维结构的发育和维持提供支架的假设，利用共聚焦显微镜和电子显微镜(EM)观察在亚硒酸盐大鼠晶状体发育过程中和在丧失晶状体透明度期间分化晶状体纤维的主要结构蛋白的细胞组织。我们将使用电子显微镜和共聚焦免疫细胞化学技术研究透明晶状体纤维分化过程中细胞骨架和晶状体蛋白的模式和分布。

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.