STRUCTURAL BASIS--ALTERED CALCIUM HOMEOSTASIS OF AGING

结构基础——改变衰老的钙稳态

• 批准号: 6509739
• 负责人: THOMAS Comey SQUIER
• 金额: $ 50.79万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6509739
• 关键词: aging; calmodulin; enzyme activity; fluorescence; genetic manipulation; homeostasis; infrared spectrometry; interferometry; laboratory rat; methionine; mutant; nuclear magnetic resonance spectroscopy; oxidation; oxidative stress; oxidoreductase; posttranslational modifications; proteasome; protein degradation; protein purification; protein structure function; site directed mutagenesis; sulfoxide

DESCRIPTION: (Abstract from the Application). The long term goal is to identify the molecular mechanisms that result in the age-dependent loss of critical cellular functions, which correlate with an increased sensitivity to stress and diminished capabilities of the elderly. These investigators have focused on identification of the proposed linkage between oxidative stress and decreased calcium regulation observed during aging. Based on previous findings which demonstrate that during aging, multiple methionines in the calcium regulatory protein calmodulin (CaM) are oxidatively modified to their corresponding sulfoxides resulting in a reduced ability to activate the PM-Ca-ATPase, and the key role that CaM plays in intracellular signaling, they hypothesize that age-related decreases in CaM function are responsible for the loss of calcium homeostasis observed in senescent cells. The accumulation of oxidatively modified CaM (CaMox) that is functionally inactive during aging is consistent with a decreased function of cellular repair and degradative enzymes in senescent animals. Thus the specific activity of methionine sulfoxide reductase (MsrA), which is able to repair oxidized CaM in vitro and fully restore CaMox function, may be compromised during aging. Likewise, the age relationship decreases in the function of the proteasome, which normally selectively degrades oxidized proteins, may result in the accumulation of inactive CaMox. Therefore, to identify the molecular mechanisms that result in the loss of CaM function, and recognition features that normally promote Cal repair and turnover, they propose the following specific aims: (1) Identify how methionine oxidation in CaM alters target protein activation, (2) Determine recognition elements in CaMox (oxidized) that promote methionine sulfoxide repair by MsrA, and (3) Discover mechanisms of degradation of CaMox by the proteasome. These measurements will involve a multidisciplinary approach that will combine biochemical measurements of the function of genetically engineered CaM mutants with altered sensitivities to oxidative stress and spectroscopic measurements of CaMox structure using FT-IR, flex, and NMR spray. Additional single-molecule measurements will permit the resolution of structural heterogeneity in individual CaMox molecules and identification of the mechanisms of CaM, recognition by MsrA and the proteasome. An understanding of the cellular mechanisms that modify calcium homeostasis under conditions of oxidative stress and the role of CaM oxidation in modifying target protein activation will be important to the development of new therapies to alleviate the decline in cellular functions associated with aging.

说明：（申请书摘要）。 长期目标是 确定导致年龄依赖性丧失的分子机制， 关键的细胞功能，这与增加的敏感性， 老年人的压力和能力下降。 这些调查人员 重点是确定氧化应激与 在衰老过程中观察到钙调节减少。 基于先前 研究结果表明，在衰老过程中， 钙调节蛋白钙调素（CaM）被氧化修饰成它们的 相应的亚砜，导致降低的活化细胞的能力。 PM-Ca-ATP酶，以及CaM在细胞内信号传导中的关键作用， 假设年龄相关的钙调素功能下降是导致 在衰老细胞中观察到钙稳态的丧失。 的积累 氧化修饰的CaM（CaMox）在老化过程中功能失活， 与细胞修复和降解功能下降一致 衰老动物体内的酶 因此蛋氨酸的比活性 亚砜还原酶（MsrA），其能够在体外修复氧化的CaM， 完全恢复CaMox功能，可能会在老化过程中受损。 同样， 蛋白酶体的功能随着年龄的增长而下降， 选择性降解氧化蛋白质，可能导致 不活跃的CaMox。 因此，为了确定导致 CaM功能和通常促进的识别功能的丧失 Cal修复和周转，他们提出以下具体目标：（1） 确定钙调素中的蛋氨酸氧化如何改变靶蛋白的活化，（2） 确定促进甲硫氨酸的CaMox（氧化）中的识别元素 MsrA对亚砜的修复作用;（3）探索CaMox的降解机制 蛋白酶体的作用。 这些测量将涉及多学科 这种方法将联合收割机的功能的生物化学测量 对氧化敏感性改变的基因工程CaM突变体 使用FT-IR、Flex 和核磁共振喷雾 额外的单分子测量将允许 单个CaMox分子中结构异质性的分辨率， 识别CaM的机制，识别MsrA和 蛋白酶体 对改变钙的细胞机制的理解 氧化应激条件下的稳态和CaM氧化的作用 在修饰靶蛋白激活将是重要的发展， 新的治疗方法，以减轻细胞功能的下降， 衰老