This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our long-term goal is to identify the molecular mechanisms that result in age-dependent declines in cell function and increased sensitivity to cellular stress and chronic inflammatory responses. We hypothesize that the site-specific oxidation and nitration of critical protein sensors of oxidative stress, such as calmodulin (CaM), modulate cellular responses to chronic inflammation common to aging tissues. The focus of this proposal is to understand a) the normal cellular mechanisms that maintain cellular homeostasis through the efficient repair and degradation of oxidized proteins, and b) how observed age-dependent oxidative modifications to CaM affect cell function. Our focus is to determine the role of CaM and its oxidation on macrophage function, because age-dependent declines in macrophage function contribute to diminished immune responses and the accumulation of nonfunctional cells whose clearance is necessary for healthy aging. Further, we have recently identified a previously unrecognized critical role for CaM in mediating macrophage activation and bacterial killing that involves the coordinate regulation of the oxidative burst through iNOS activation and autocrine signaling involving TNF?-dependent pathways. We expect that age-dependent decreases in CaM abundance and associated age-dependent increases in the oxidized CaM fraction contribute to observed decreases in macrophage function. We suggest that age-dependent increases in the abundances of the circulating cytokines TNF? and IFN- ? associated with macrophage priming induce a chronic oxidative stress that contributes to age-dependent declines in macrophage function through CaM oxidation. We have designed two specific aims:
In Aim 1, levels of chronic inflammation and abundances of repair proteins will be modulated in RAW macrophages. 1: Determine how inflammatory cytokines affect the kinetics of CaM oxidation, repair, and degradation in relationship to cell function. 2: Understand how site-specific oxidation differentially regulates CaM-dependent enzymes.
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