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. While there is much correlative evidence to suggest a connection between poly(ADP-ribose) (PAR) 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 PAR — in mechanisms of senescence. Furthermore, many techniques currently used for analysis of protein poly(ADP-ribosyl)ation are fraught with imprecision. We therefore sought to address these issues both by developing methods for the unambiguous analysis of poly(ADP-ribosyl)ation by mass spectrometry, and by exploring the role of PARP-1 in nicotinamide-mediated cellular lifespan extension.
Due to the challenges introduced by PAR's biochemical characteristics, successful mass spectrometric analysis of poly(ADP-ribosylation) will require the use of techniques to reduce the mass, charge, and heterogeneity of the polymer, as well as methods to enrich for poly(ADP- ribosyl)ated protein. To this end, we evaluated the effectiveness of several approaches, including ammonium sulfate fractionation, boronate affinity chromatography, snake venom phosphodiesterase digestion, manipulation of PARP-1 reaction conditions, and immobilized metal affinity chromatography (IMAC) for the preparation of poly(ADP-ribosyl)ated protein samples prior to MS analysis using both MALDI-TOF and Q-TRAP LC-MS. Based on this work, we developed a three-tiered scheme that may provide the first ever identification of poly(ADP- ribosyl)ated peptides from full-length wild-type PARP-1 by mass spectrometry.
Past work in our laboratory has demonstrated that nicotinamide (NAM), a component of vitamin B3, significantly extends the replicative lifespan of human fibroblasts. In order to help elucidate the role of PARP-1 in cellular senescence, we then analyzed the poly(ADP-ribosyl)ation response of aging cells undergoing NAM-mediated lifespan extension. While NAM is a known PARP-1 inhibitor, we found that oxidative stress-induced poly(ADP- ribosyl)ation is increased, not decreased, in NAM-treated cells. We propose that supplemented NAM is taken up by the NAD salvage pathway, ultimately leading to increased cellular NAD and extending replicative lifespan by both preventing PARP-mediated NAD depletion and upregulating SIRT1. We further propose that the demonstrated protective effects of NAM treatment in a number of disease models are due not to PARP-1 inhibition as is commonly assumed, but to upregulation of NAD salvage. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/39180 |
| Date | 02 September 2010 |
| Creators | Edmonds, Yvette M. |
| Contributors | Biochemistry, Helm, Richard F., Kennelly, Peter J., Sible, Jill C., Finkielstein, Carla V. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | Edmonds_YvetteM_D_2010.pdf |
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