Current research on aging has largely focused the molecular mechanisms of age-related diseases, and mitochondrial dysfunction has been associated with several human diseases of aging. However, the cellular mechanisms of mitochondrial dysfunction and how they lead to age-related diseases are not known. Chemical modifications to mitochondrial proteins control several aspects of mitochondrial function, and the long-term goal of this project is to understand how these modifications are regulated by the NAD(+)-dependent sirtuin deacylases and influence the diseases of aging. The objective of this proposal is to define the role of SIRT5 in the molecular mechanisms of aging. The central hypothesis is that SIRT5 regulates mitochondrial function by removing a newly discovered acyl modification from mitochondrial proteins. In the absence of SIRT5, mitochondrial proteins will become hyper-acylated, have reduced mitochondrial function, and exhibit several markers of accelerated aging. The rationale for this hypothesis is based on preliminary data, which suggests an important role for SIRT5 in the cellular mechanisms of aging and disease. To test these hypotheses, three specific aims will be pursued: 1. Determine the changes in protein acylation as a function of age, using a proteomic strategy to quantify hepatic mitochondrial protein acylation from young, middle-aged, and old mice; 2. Determine the effect of this novel acyl modification on protein function by using a combination of cell and murine models, in order to measure the changes in protein activity of specific proteins, as well as the overall function of mitochondria; 3. Determine role of SIRT5 on aging by measuring several physiological parameters of aging using both in vitro cellular assays and in vivo assays in mice, to determine how SIRT5 maintains mitochondrial and cellular homeostasis during aging. This study combines a novel protein modification, a comprehensive experimental design, and an innovative conceptual framework. Furthermore, this study will build a foundation for this early-stage investigator and ensure a successful research program focused on the cellular mechanisms of aging and disease. Importantly, the proposed research is significant because it is expected to advance and expand understanding how mitochondrial dysfunction can lead to age-related diseases. Ultimately such knowledge has the potential to inform the development of new therapies against several diseases of aging.