Development and Maintenance of Lens Transparency

镜头透明度的开发和维护

• 批准号: 7915853
• 负责人: JOHN Irwin CLARK
• 金额: $ 19.42万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7915853
• 关键词: Accounting; Address; Affinity; Age; Age related macular degeneration; Aging; Amyloid; Amyloid Proteins; Amyloidosis; Animals; Binding; Biological Assay; Biological Markers; Biomedical Research; Brain; Cardiomyopathies; Cardiovascular Diseases; Cataract; Cells; Cellular Structures; Chimeric Proteins; Classification; Crystalline Lens; Crystallins; Cytoskeletal Proteins; Deposition; Development; Disease; Dissociation; Electron Microscopy; Filament; Heat shock proteins; Human; Image; In Vitro; Individual; Investigation; Lens Fiber; Life; Light; Link; Literature; Longevity; Maintenance; Mass Spectrum Analysis; Measures; Mediating; Microscopic; Modeling; Modification; Molecular Chaperones; Muscular Dystrophies; Nerve Degeneration; Nervous system structure; Neuromuscular Diseases; Neurons; Optics; Peptides; Phenotype; Phylogenetic Analysis; Proteins; Public Health; Research; Sampling; Structural Protein; Structure; Testing; Therapeutic; Tissues; Toxic effect; Transgenic Mice; aging brain; amyloid fibril formation; amyloid formation; base; biological adaptation to stress; immunocytochemistry; in vitro testing; in vivo; innovation; insight; laser capture microdissection; lens; lens transparency; light scattering; mouse model; mutant; new therapeutic target; novel; novel therapeutics; protective effect; protein B; protein aggregate; protein protein interaction; public health relevance; stress protein; synthetic peptide; Accounting; Address; Affinity; Age; Age related macular degeneration; Aging; Amyloid; Amyloid Proteins; Amyloidosis; Animals; Binding; Biological Assay; Biological Markers; Biomedical Research; Brain; Cardiomyopathies; Cardiovascular Diseases; Cataract; Cells; Cellular Structures; Chimeric Proteins; Classification; Crystalline Lens; Crystallins; Cytoskeletal Proteins; Deposition; Development; Disease; Dissociation; Electron Microscopy; Filament; Heat shock proteins; Human; Image; In Vitro; Individual; Investigation; Lens Fiber; Life; Light; Link; Literature; Longevity; Maintenance; Mass Spectrum Analysis; Measures; Mediating; Microscopic; Modeling; Modification; Molecular Chaperones; Muscular Dystrophies; Nerve Degeneration; Nervous system structure; Neuromuscular Diseases; Neurons; Optics; Peptides; Phenotype; Phylogenetic Analysis; Proteins; Public Health; Research; Sampling; Structural Protein; Structure; Testing; Therapeutic; Tissues; Toxic effect; Transgenic Mice; aging brain; amyloid fibril formation; amyloid formation; base; biological adaptation to stress; immunocytochemistry; in vitro testing; in vivo; innovation; insight; laser capture microdissection; lens; lens transparency; light scattering; mouse model; mutant; new therapeutic target; novel; novel therapeutics; protective effect; protein B; protein aggregate; protein protein interaction; public health relevance; stress protein; synthetic peptide

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.

描述（由申请人提供）：拟议研究的主要目的是定义应激蛋白，人类晶体蛋白，小热激蛋白（SHSP）和分子伴侣的保护作用的功能机制。假设1：人是晶体蛋白中多个相互作用序列的活性提供了蛋白质展开，聚集和毒性的保护，包括白内障，神经变性，AMD，可能是神经肌肉疾病。基于晶体蛋白中的单个交互结构域的合成肽介导淀粉样蛋白原纤维的形成，并将用于表征互动结构域的集体机制。 AIM 1将优化人类Crystallin的功能，Crystallin，这是所有小热激蛋白（SHSP）的原型。功能测定将用于表征晶体蛋白的新突变体对胁迫反应的影响，并保护蛋白质在体外展开和聚集。 AIM 2将使用狭缝灯成像进行转基因小鼠模型中的透镜的表型分析，以进行神经退行性和神经肌肉疾病。该目的解决了假设2：透镜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。透镜在非侵入性光学检查中的独特可访问性使晶状体细胞非常适合研究活动物中聚集和淀粉样蛋白形成的基本机制。假设3：神经退行性中负责晶状体表型的蛋白质也可能导致神经肌肉疾病中的晶状体表型，AIM 3将鉴定负责转基因小鼠模型中负责结构表型的关键蛋白质成分，用于神经退行性和神经肌肉肌肉疾病。使用激光捕获显微解剖（LCM）获得的样品的质谱法将与免疫细胞化学和电子显微镜结合使用，以将蛋白质组成与结构修饰相关联，该结构修饰，该表型在用于神经性和神经肌肉肌肉障碍的小鼠模型中观察到的表型。公共卫生的意义：蛋白质展开和淀粉样蛋白/骨料形成是衰老的疾病，包括白内障，与年龄相关的黄斑变性（AMD），神经变性，心肌病和肌肉营养不良。随着人类寿命的增加，衰老疾病的影响正在急剧增加。定义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步，并有可能为衰老疾病提供新的治疗靶标。该镜头是体内晶体蛋白功能的创新功能研究的理想选择，并且可能是神经退行性和神经肌肉疾病的启动和进展的生物标志物的重要性。公共卫生相关性：此应用是对应激蛋白人类的相互作用的研究，晶体蛋白负责抗蛋白质展开和聚集的基本保护机制。方法是表征交互域的活性并在体外量化其亲和力，同时使用缝隙灯研究它们对体内镜头透明结构的影响。结果将推进生物医学研究，以理解SHSP在衰老疾病中的内源性功能，包括神经退行性，心血管疾病，肌肉营养不良，AMD（与年龄相关的黄斑营养不良）和白内障。这项研究具有转化应用于开发针对主要衰老疾病的新型治疗剂的潜力。该镜头被用于这些研究，因为它具有独特的可及性，可用于体内对蛋白质展开和衰老中蛋白质疾病的研究和众所周知的保护作用。