Development and Maintenance of Lens Transparency

描述（由申请人提供）：拟议研究的主要目的是确定应激蛋白，人？B晶体蛋白，一种小的热休克蛋白（sHSP）和分子伴侣。假设1：人体内多个相互作用序列的活性B晶状体蛋白提供保护，防止蛋白质展开、聚集和老化疾病的毒性，包括白内障、神经变性、AMD和可能的神经肌肉疾病。基于单个相互作用结构域的合成肽？B结晶蛋白介导淀粉样蛋白原纤维的形成，并将用于表征作用于？晶状体蛋白。AIM 1将优化人的功能？B结晶蛋白，所有小热休克蛋白（sHSP）的原型。功能分析将用于描述？B结晶蛋白对体外应激反应及蛋白展开和聚集的保护作用。AIM 2将使用裂隙灯成像技术对神经退行性和神经肌肉疾病转基因小鼠模型中的晶状体进行表型分析。假设2：晶状体透明度的丧失是神经退行性和神经肌肉疾病中蛋白质相互作用改变的敏感指标。晶状体非侵入性光学检查的独特可及性使晶状体细胞非常适合研究活体动物中聚集和淀粉样蛋白形成的基本机制。假设3：负责神经退行性疾病晶状体表型的蛋白质也可能负责神经肌肉疾病晶状体表型，AIM 3将在神经退行性疾病和神经肌肉疾病的转基因小鼠模型中确定负责结构表型的关键蛋白质成分。使用激光捕获显微解剖（LCM）获得的样品的质谱分析将与免疫细胞化学、光学和电子显微镜相结合，将蛋白质成分与结构修饰联系起来，解释在神经退行性和神经肌肉疾病小鼠模型中观察到的表型。对公共卫生的意义：蛋白质展开和淀粉样蛋白/聚集体形成是衰老疾病的特征，包括白内障、年龄相关性黄斑变性（AMD）、神经变性、心肌病和肌肉萎缩症。随着人类寿命的延长，老年性疾病的影响也在急剧增加。确定应激蛋白保护活性的基本机制？B晶体蛋白将是生物医学研究的重大进展，并有可能为衰老疾病提供新的治疗靶点。该镜头是创新功能研究的理想选择。B晶体蛋白在体内发挥作用，可能作为神经退行性和神经肌肉疾病的发生和进展的生物标志物具有重要意义。公共卫生相关性：本应用研究应激蛋白与人？B晶体蛋白负责蛋白质展开和聚集的基本保护机制。该方法是在体外表征相互作用域的活性并量化它们的亲和力，同时使用裂隙灯研究它们对体内晶状体透明结构的影响。该结果将推动生物医学研究，以了解sHSP在神经退行性疾病、心血管疾病、肌肉萎缩症、AMD（年龄相关性黄斑营养不良）和白内障等衰老疾病中的内源性功能。该研究具有潜在的转化应用于开发针对主要衰老疾病的新疗法。透镜体被用于这些研究，因为它具有独特的可及性，可以在体内研究衰老过程中的蛋白质展开和聚集性疾病，以及众所周知的？晶状体蛋白。

DESCRIPTION (provided by applicant): The primary objective of the proposed research is to define the functional mechanism for the protective actions of the stress protein, human ?B crystallin, a small heat shock protein (sHSP) and molecular chaperone. HYPOTHESIS 1: The activity of multiple interactive sequences in human??B crystallin provides protection against protein unfolding, aggregation, and toxicity in aging disorders that include cataract, neurodegeneration, AMD, and possibly, neuromuscular disorders. Synthetic peptides based on the individual interactive domains in ?B crystallin mediate the formation of amyloid fibrils and will be used to characterize the collective mechanism(s) of the interactive domains in the action of ?B crystallin. AIM 1 will optimize the function of human ?B crystallin, the archetype of all small heat shock proteins (sHSP). Functional assays will be used to characterize the effects of new mutants of ?B crystallin on the stress response and protection against protein unfolding and aggregation in vitro. AIM 2 will use slit lamp imaging to conduct phenotype analyses of lenses in transgenic mouse models for neurodegenerative and neuromuscular disorders. This aim addresses HYPOTHESIS 2: The loss of lens transparency is a sensitive indicator of altered protein interactions in neurodegenerative and neuromuscular disorders. The unique accessibility of the lens for non-invasive optical examinations makes lens cells excellent for the study of basic mechanisms of aggregation and amyloid formation in living animals. HYPOTHESIS 3: The proteins responsible for the lens phenotype in neurodegeneration may also be responsible for the lens phenotype in neuromuscular disorders, and AIM 3 will identify critical protein constituents responsible for the structural phenotype in transgenic mouse models for neurodegenerative and neuromuscular disorders. Mass spectrometry of samples obtained using laser capture microdissection (LCM) will be combined with immunocytochemistry, and light and electron microscopy to correlate protein constituents with structural modifications accounting for the phenotype observed in lenses of mouse models for neurodegenerative and neuromuscular disorders. SIGNIFICANCE FOR PUBLIC HEALTH: Protein unfolding and amyloid/aggregate formation characterize disorders of aging that include cataract, age related macular degeneration (AMD), neurodegeneration, cardiomyopathy and muscular dystrophy. With the increase in human longevity, the impact of aging diseases is increasing dramatically. Defining the basic mechanism for the protective activity of the stress protein ?B crystallin will be a major advance in biomedical research and has the potential to provide novel therapeutic targets for aging disorders. The lens is ideal for innovative functional studies of ?B crystallin function in vivo and may have significance as a biomarker for the initiation and progression of neurodegenerative and neuromuscular disease. PUBLIC HEALTH RELEVANCE: This application is a study of the interactions of the stress protein human ?B crystallin responsible for the fundamental protective mechanism(s) against protein unfolding and aggregation. The approach is to characterize the activity of the interactive domains and quantify their affinities in vitro while studying their influence on the transparent structure of the lens in vivo using the slit lamp. The results will advance biomedical research in understanding the endogenous function of sHSP in diseases of aging that include neurodegeneration, cardiovascular diseases, muscular dystrophy, AMD (age related macular dystrophy) and cataract. This research has the potential for translational applications to the development of novel therapeutics against major diseases of aging. The lens is used for these studies because of its unique accessibility for the in vivo investigation of protein unfolding and aggregation diseases in aging and the well known protective effects of ?B crystallin.