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.
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