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.

描述：（来自应用程序的摘要）。 长期目标是 确定导致年龄依赖性损失的分子机制 关键细胞功能，与对 压力和老年人能力的减弱。 这些调查人员有 专注于识别氧化应激和 衰老期间观察到的钙调节减少。 基于以前的 结果表明，在衰老期间，多种蛋白 钙调节蛋白钙调蛋白（CAM）被氧化为其修饰 相应的亚氧化甲氧化物导致激活能力降低 PM-CA-ATPase以及CAM在细胞内信号传导中扮演的关键作用，它们是 假设与年龄相关的CAM功能降低是造成的 在衰老细胞中观察到钙稳态的损失。 积累 在衰老期间功能性无活性的氧化修饰的CAM（CAMOX）为 与细胞修复功能降低和降解的功能一致 衰老动物中的酶。 因此蛋氨酸的特定活性 亚氧化氧化物还原酶（MSRA），能够在体外修复氧化的凸轮和 在老化期间，完全恢复的Camox功能可能会受到损害。 同样， 年龄关系的蛋白酶体功能下降，通常 有选择地降解氧化蛋白，可能导致 不活跃的卡诺克。 因此，要确定产生的分子机制 在CAM功能的丧失中，以及通常促进的识别功能 CAL修复和营业额，他们提出以下特定目的：（1） 确定CAM中蛋氨酸氧化如何改变靶蛋白的激活，（2） 确定促进蛋氨酸的Camox（氧化）中的识别元素 通过MSRA修复亚氧化甲氧化物，（3）发现了Camox降解机制 由蛋白酶体。 这些测量将涉及多学科 将结合生化测量功能的方法 具有改变氧化敏感性的基因工程凸轮突变体 使用FT-IR，Flex， 和NMR喷雾。 其他单分子测量将允许 分辨单个camox分子中的结构异质性和 识别CAM机制，MSRA的认可和 蛋白酶体。 了解修饰钙的细胞机制 在氧化应激条件下的稳态和CAM氧化的作用 在修改目标蛋白激活中，对发展至关重要 减轻与细胞功能下降的新疗法 老化。