CALMODULIN, AGING AND CALCIUM HOMEOSTASIS

正常衰老与多种细胞的氧化有关。 蛋白质，并提出了活性氧自由基(ROS)。 选择性地修饰一些蛋白质，最终导致 钙稳态。我们推测其中的两个蛋白质是CaM和 Ca-ATPase。钙调素(CaM)是真核生物中普遍存在的钙 结合蛋白在扩增过程中起中介作用的结合蛋白 细胞内钙的短暂增加，并在 对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，以及与能量和生物合成有关的一系列反应 细胞的代谢。质膜(PM)Ca-ATPase是主要的 高亲和力、高容量的钙转运蛋白最终 通过其功能维持正常(低)细胞内钙浓度 钙结合钙调素的激活。我们的长期目标是确定 氧化损伤与这些关键钙的机制关系 调节蛋白和功能。作为第一步，我们建议确定 CaM和PM-Ca-ATPase对生理的敏感性 相关的RO，以及与以下相关的结构和功能后果 氧化损伤。在Cam的情况下，我们将机械地将 观察到CaM的氧化修饰激活了一系列 生理上相关的靶蛋白(例如，质膜Ca-ATPase， 磷酸二酯酶、钙调蛋白依赖的蛋白激酶和一氧化氮 合酶)。对氧化损伤的特定部位的分析将 包括使用高效液相色谱和FAB质谱学来解析位置(S) 以及细胞钙调蛋白和PM-Ca-ATPase的部分 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 光谱学。后续工作将涉及CAM和CAM的重建 将PM-Ca-ATPase引入生理相关模型系统，旨在 模拟与氧化损伤相关的新陈代谢条件。这个 第二个主题，也是项目的最终目标，是应用这些方法 确定特定的ROS和相关的功能后果 与年龄相关的(翻译后)这些钙的修饰 调节蛋白和相关的脂类。一种身份识别 ROS参与CaM和PM-Ca-ATPase的修饰 最终建议可能的治疗方法，以缓解 与衰老有关的细胞功能。

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.