Metalloproteome of the Aging Rat Brain Mitochondria

衰老大鼠脑线粒体的金属蛋白质组

• 批准号: 7229969
• 负责人: Bruce N Ames
• 金额: $ 15.8万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-15 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7229969
• 关键词: Address; Age; Aging; Binding; Binding Proteins; Brain; Cataloging; Catalogs; Condition; Dancing; Data; Databases; Disease; Elderly; Electrophoresis; Elements; Evaluation; Functional disorder; Genetic; Goals; Homeostasis; Internet; Intervention; Ions; Isoelectric Focusing; Knowledge; Lead; Liquid substance; Localized; Maps; Measures; Metabolic; Metabolism; Metalloproteins; Metals; Methodology; Methods; Microscopy; Mitochondria; Mitochondrial Proteins; Neurodegenerative Disorders; Pattern; Personal Satisfaction; Phase; Physiological; Physiology; Protein Binding; Proteins; Proteome; Proteomics; Protons; Publishing; Rattus; Reporting; Roentgen Rays; Scanning; Signal Transduction; Techniques; Technology; Testing; Time; Trace Elements; Work; age effect; age related; base; cofactor; middle age; novel; tool; transmission process

DESCRIPTION (provided by applicant): Mitochondria are very metal rich. Precise levels of metals are required and typically bind to specific cognate proteins for optimal mitochondrial function. However during aging, genetic and epigentic changes occur that lead to decay in mitochondrial function, including altered metal homeostasis. Fluctuations in metal content and metalloprotein levels can cause subcellular and cellular dysfunction, and eventually disease. To clearly understand the relationship between metal homeostasis and mitochondrial function, a complete description and quantitation of all metals (the metallome) and all metalloproteins (the metalloproteome) in the mitochondria must be made available, yet technical obstacles have hindered progress towards this goal. Recently though, novel methods were developed that merge ultrasensitive elemental analysis with high- throughput proteomic tools resulting in the capacity to separate and identify hundreds of proteins and simultaneously reveal bound metals. This technology is optimally suited to quantify metals and identify metal-binding proteins within discrete proteomes, such as the mitochondria. By measuring the shifting patterns in metal content and metalloprotein expression patterns over time, a more comprehensive understanding of the effects of aging on mitochondrial function can be ascertained. In this preliminary study, (1) metal and metalloprotein content in mitochondria from the rat brain cortex will be quantitated, as cortical regions are a known target of aging and neurodegenerative disease. Then, (2) the hypothesis that mitochondrial metal homeostasis is altered during aging will be tested by mapping the major changes in metal content and metalloprotein patterns from young, middle-aged, and old adult rat cortex. Analysis of expression and elemental content will be performed on mitochondrial protein fractions separated by native liquid-phase isoelectric focusing followed by continuous elution electrophoresis. Protein mass and elemental content will be quantitated with scanning transmission ion microscopy and proton induced X-ray emission, respectively, and then combined with protein identification determined from MALDI-TOF and LC-TOF-MS analyses to generate the metallome and metalloproteome for rat cortical mitochondria. These results will be published as an Internet-based searchable database, highlighting age-related changes. Thus, this work has significant potential to integrate numerous aspects of mitochondrial metabolism and physiology and reveal novel opportunities for combating aging-related decline.

描述（由申请人提供）：线粒体非常富含金属。需要精确的金属水平，通常与特定的同源蛋白结合以达到最佳的线粒体功能。然而，在衰老期间，发生遗传和表观易感性变化，导致线粒体功能腐烂，包括金属稳态改变。金属含量和金属蛋白水平的波动会导致细胞和细胞功能障碍，最终会导致疾病。要清楚地了解金属稳态与线粒体功能之间的关系，必须提供对线粒体中所有金属（金属蛋白）和所有金属蛋白（金属蛋白）（金属蛋白）的完整描述和定量，但是必须提供线粒体中的所有金属蛋白（金属蛋白蛋白），但是技术障碍阻碍了该目标的进度。但是，最近开发了新的方法，将超敏感性分析与高吞吐量蛋白质组学工具合并，从而使能力分离和识别数百种蛋白质并同时揭示结合金属。该技术最适合量化金属并鉴定离散蛋白质组（例如线粒体）内的金属结合蛋白。通过测量金属含量和金属蛋白表达模式的转移模式，可以确定对衰老对线粒体功能的影响的更全面的理解。在这项初步研究中，（1）将从大鼠脑皮质中的线粒体中的金属和金属蛋白含量进行定量，因为皮质区域是已知的衰老和神经退行性疾病的靶标。然后，（2）通过映射金属含量和金属蛋白质模式的主要变化，从年轻，中年和古老的成年大鼠皮层绘制金属含量和金属蛋白模式的主要变化来测试，即在衰老过程中改变了线粒体金属稳态的假设。对表达和元素含量的分析将对通过天然液态相聚焦分离的线粒体蛋白级分，然后进行连续洗脱电泳。蛋白质质量和元素含量将分别通过扫描透射离子显微镜和质子诱导的X射线发射来定量，然后与由MALDI-TOF和LC-TOF-MS分析确定的蛋白质鉴定结合，以生成金属蛋白质组和金属蛋白酶用于Rat rat cortical-Ratical Mitochria。这些结果将作为基于Internet的可搜索数据库发布，突出了与年龄有关的变化。因此，这项工作具有整合线粒体代谢和生理学的许多方面的巨大潜力，并揭示了打击与衰老相关的下降的新机会。