CALMODULIN, AGING AND CALCIUM HOMEOSTASIS

正常的衰老与多种细胞的氧化有关， 蛋白质，并已提出，活性氧（ROS） 选择性地修饰一些蛋白质，最终导致蛋白质的损失。 钙稳态 我们认为这些蛋白质中有两种是CaM， Ca-ATPase。 钙调素（CaM）是一种普遍存在于真核生物中的钙调素， 一种结合蛋白，在扩增中起中介作用， 细胞内钙的瞬时增加，并在 许多细胞过程的调节，包括 神经传递、神经元可塑性、肌肉收缩、细胞骨架 组装，以及参与能量和生物合成的许多反应， 细胞的新陈代谢。 质膜（PM）Ca-ATPase是主要的Ca-ATPase 高亲和力、高容量钙转运蛋白， 维持正常（低）的细胞内钙浓度，通过其 激活钙结合的钙调素。 我们的长期目标是确定 氧化损伤和这些关键钙离子之间的机械关系 调节蛋白和功能。 作为第一步，我们建议确定 钙调素和PM-Ca-ATP酶的敏感性， 相关的活性氧，以及结构和功能的后果， 氧化损伤 在CaM的情况下，我们将机械地将 观察到的氧化修饰钙调素的能力，激活一系列的 生理学相关的靶蛋白（例如，质膜Ca-ATPase， 磷酸二酯酶、钙调蛋白依赖性蛋白激酶和一氧化氮 合成酶）。 对氧化损伤的特定部位的分析将 包括使用HPLC和FAB质谱法解析位点 和部分细胞钙调素和PM-Ca-ATP酶， 被氧化了 与氧化损伤相关的结构后果 将主要通过使用时间分辨荧光进行评估 谱 随后的工作将涉及CaM的重建， 将PM-Ca-ATPase引入生理学相关的模型系统， 模拟与氧化损伤相关的代谢条件。 的 第二个主题，也是该项目的最终目标，是应用这些方法 以确定特定的ROS和相关的功能后果， 与年龄相关的（翻译后）修改这些钙 调节蛋白和相关脂质。 的标识 ROS参与CaM和PM-Ca-ATPase的修饰， 最终提出了可能的治疗方法，可以缓解下降， 与衰老相关的细胞功能。

Normal aging is associated with the oxidation of a wide range of cellular proteins, and it has been proposed that reactive oxygen species (ROS) selectively modify some proteins, ultimately resulting in a loss of calcium homeostasis. We propose that two of these proteins are CaM and the Ca-ATPase. Calmodulin (CaM) is a ubiquitous eukaryotic calcium binding protein that serves as an intermediary in the amplification of transient increases in intracellular calcium, and plays a central role in the regulation of numerous cellular processes, including neurotransmission, neuronal plasticity, muscle contraction, cytoskeletal assembly, and a host of reactions involved in the energy and biosynthetic metabolism of the cell. The plasma membrane (PM) Ca-ATPase is the major high affinity, high capacity calcium transport protein that ultimately maintains normal (low) intracellular calcium concentrations through its activation by calcium-bound CaM. Our long-term goal is to identify mechanistic relationships between oxidative damage and these key calcium regulatory proteins and function. As a first step, we propose to identify both the sensitivity of CaM and the PM-Ca-ATPase to physiologically relevant ROS, and the structural and functional consequences relating to oxidative damage. In the case of CaM, we will mechanistically relate the observed oxidative modifications to CaM's ability to activate a range of physiologically relevant target proteins (e.g., plasma membrane Ca-ATPase, phosphodiesterase, calmodulin-dependent protein kinase, and nitric oxide synthase). The analysis of specific sites of oxidative damage will include the use of HPLC and FAB mass spectroscopy to resolve the site(s) and fraction of cellular calmodulin and PM-Ca-ATPase that have been oxidized. The structural consequences associated with oxidative damage will be assessed primarily through the use of time-resolved fluorescence spectroscopy. Subsequent work will involve the reconstitution of CaM and the PM-Ca-ATPase into physiologically relevant model systems aimed at simulating metabolic conditions associated with oxidative damage. The second theme, and ultimate goal of the project, is to apply these methods to identify the specific ROS and the functional consequences associated with the age-related (post-translational) modification of these calcium regulatory proteins and the associated lipids. An identification of the ROS involved in the modification of CaM and the PM-Ca-ATPase will ultimately suggest possible therapies that could alleviate the decline in cellular functions associated with aging.