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：（申请摘要）。 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，与增加的敏感性相关 老年人的压力和能力下降。 这些调查人员有 重点是确定氧化应激和氧化应激之间的拟议联系 衰老过程中观察到的钙调节下降。 基于之前的 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 相应的亚砜导致激活能力降低 PM-Ca-ATPase，以及 CaM 在细胞内信号传导中发挥的关键作用，它们 假设与年龄相关的 CaM 功能下降是造成 在衰老细胞中观察到钙稳态丧失。 的积累 氧化修饰的 CaM (CaMox) 在衰老过程中功能失活 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)，能够在体外修复氧化的 CaM 完全恢复CaMox功能，但在老化过程中可能会受到损害。 同样， 蛋白酶体的功能随着年龄的增长而下降，而蛋白酶体的功能通常会下降 选择性降解氧化蛋白质，可能导致积累 无活性的CaMox。 因此，为了确定导致的分子机制 CaM 功能丧失，以及通常促进的识别功能 Cal修复和营业额，他们提出了以下具体目标：（1） 确定 CaM 中的蛋氨酸氧化如何改变靶蛋白激活，(2) 确定 CaMox（氧化）中促进蛋氨酸生成的识别元素 MsrA 修复亚砜，以及 (3) 发现 CaMox 降解机制 通过蛋白酶体。 这些测量将涉及多学科 方法将结合生化功能的测量 基因工程化的 CaM 突变体改变了对氧化的敏感性 使用 FT-IR、flex、 和核磁共振喷雾。 额外的单分子测量将允许 解决单个 CaMox 分子的结构异质性和 CaM 机制的识别、MsrA 的识别以及 蛋白酶体。 了解改变钙的细胞机制 氧化应激条件下的稳态和 CaM 氧化的作用 修饰靶蛋白激活对于开发 缓解与相关细胞功能下降的新疗法 老化。