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.
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