Structural Basis of Altered Calcium Homeostasis During Aging
Sponsor: National Institute of Aging
Contact: Tom Squier
Model depicting how methionine oxidation modulates calcium-dependent structural coupling between opposing domains of CaM. Fluorescent probe (red oval) bound to helix A (pink cylinder) in the amino-terminal domain undergoes large amplitude rigid body motions in apo-CaM, which are unaffected upon oxidation of all nine methionines. Calcium activation (black circles) stabilizes the interaction between helix A and helix D (grey shaded cylinders), resulting in the formation of a target peptide binding pocket in the amino-terminal domain. It is suggested that calcium binding to high-affinity sites in the carboxyl-terminal domain induce similar stabilizing interactions between helix H (pink cylinder) and helix E (grey shaded cylinders) that induce the formation of a stable interdomain helix connecting the opposing domains of wild-type (unoxidized) CaM. Upon methionine oxidation functionally sensitive methionines (i.e., Met144 and Met145) in helix H destabilize the interhelical interaction between helix H and Helix E, depicted as the appearance of an exposed binding site (light pink circle), resulting in a less stable interdomain interaction through helices D and E connecting the opposing domains of CaM. Enlarged View
Our long-term goal is to identify the molecular mechanisms that result in the age-dependent loss of calcium regulation in neurons, which correlates with an increased sensitivity to stress and age-related declines in cognitive function. We have focused on identification of the proposed linkage between oxidative stress and decreased calcium regulation observed during aging. Based on our previous findings 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, we hypothesize that age-related decreases in CaM function are primarily 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, age-related 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 CaM repair and turnover, we propose the following specific aims: 1) Identify how methionine oxidation in CaM alters target protein activation, 2) Determine recognition elements in CaMox that promote methionine sulfoxide repair by MsrA, and 3) Discover mechanisms of degradation of CaMox by the proteasome.
These measurements will involve a multi-disciplinary 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, CD, fluorescence, and NMR spectroscopy. Additional single-molecule measurements will permit the resolution of structural heterogeneity in individual CaMox molecules and identification of the mechanisms of CaMox 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 normal biological aging.