Aging is a complicated and multifactorial phenomenon. Model systems involving the induction of replicative senescence in cultured cells have been indispensable in elucidating some of the mechanisms underlying this complex process. An understanding of how and why cellular senescence occurs is thus critical to the field of aging research. Despite the fact that there is significant correlative evidence suggesting a connection between poly(ADP-ribose) and mammalian longevity, no studies have been done to explore a possible role for PARP-1 - the enzyme responsible for synthesis of 90% of cellular poly(ADP-ribose) - in senescence. Furthermore, most methods currently being used for analysis of protein poly(ADP-ribosylation are incapable of making convincing distinctions between covalent modification and non-covalent association, and are thus fraught with imprecision. We propose to address these weaknesses first by developing novel mass spectrometric methods for the unambiguous analysis of in vitro poly(ADP-ribosylation. We will then apply and further refine these techniques for in vivo use by characterizing the poly(ADP-ribosylation) response to oxidative damage in a model system. Finally, we will test the hypothesis that PARP-1 is an integral player in mechanisms of cellular senescence by using a combination of mass spectrometry and conventional methods to analyze PARP-1 post-translational modifications, protein levels, and enzymatic activity in aging human fibroblasts grown in culture. These studies will for the first time establish a specific and direct connection between PARP-1 and human aging. Normal human cells growing in culture can divide only a set number of times before they become incapable of further proliferation. This phenomenon, known as replicative senescence, is a widely-used model in aging studies. The proposed project will analyze the role of the nuclear enzyme PARP-1 in mechanisms of cellular senescence, in order to better understand how and why humans age. ? ? ?
