STRUCTURAL BASIS--ALTERED CALCIUM HOMEOSTASIS OF AGING

结构基础——改变衰老的钙稳态

• 批准号: 6093302
• 负责人: THOMAS Comey SQUIER
• 金额: $ 34.15万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 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/6093302
• 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.

描述：(摘自申请)。长期目标是 确定导致年龄相关性丢失的分子机制 关键的细胞功能，这与增加对 老年人的压力和能力减弱。这些调查人员已经 重点是确定氧化应激和 在衰老过程中观察到的钙调节减少。基于以前的 研究结果表明，在衰老过程中，体内的多种蛋氨酸 钙调节蛋白钙调蛋白(CaM)被氧化修饰为其 相应的亚硫醚导致激活的能力降低 PM-Ca-ATPase，以及CaM在细胞内信号转导中发挥的关键作用，他们 假设与年龄相关的凸轮功能下降是导致 在衰老细胞中观察到钙稳态的丧失。积攒的 氧化修饰的CaM(Camox)在老化过程中功能不活跃 与细胞修复和降解性功能下降一致 衰老动物体内的酶。因此，蛋氨酸的比活性 亚砜还原酶(MSRA)在体外对氧化钙调素具有修复作用 完全恢复Camox功能，可能会在老化过程中受损。同样， 年龄关系蛋白酶体的功能下降，正常情况下 选择性地降解氧化蛋白质，可能导致堆积 不活动的卡莫克斯。因此，要确定导致这种现象的分子机制 在失去凸轮功能的情况下，正常促进识别功能 Cal维修和周转，他们提出了以下具体目标：(1) 确定CaM中蛋氨酸氧化如何改变靶蛋白激活，(2) 测定Camox(氧化)中促进蛋氨酸的识别元件 利用MSRA对亚砜进行修复，以及(3)发现Camox的降解机理 通过蛋白酶体。这些测量将涉及多学科 将结合生物化学测量的功能的方法 对氧化敏感性发生改变的转基因CaM突变体 用FT-IR、FLEX、X射线衍射仪测量了CaMOX结构的应力和光谱 和核磁共振喷雾。额外的单分子测量将允许 单个Camox分子的结构异质性的解析 CaM机制的识别、MSRA的识别和 蛋白酶体。对修饰钙的细胞机制的理解 氧化应激条件下的动态平衡及CaM氧化的作用 在修饰靶蛋白的过程中，蛋白的激活对肿瘤的发展具有重要意义 缓解与以下疾病相关的细胞功能下降的新疗法 衰老。