This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A limited cellular lifespan is hypothesized to play a crucial role in organismal aging. Despite the putative importance of cellular lifespan in aging, we currently have only a rudimentary understanding of the molecular process. Therefore, elucidation of the molecular mechanisms that control cellular lifespan will have far reaching implications in the fields of aging, cancer biology, and tissue regeneration. When grown in culture, normal human cells divide a limited number of times before entering a state of replicative senescence where they remain viable but are unable to divide further. It is postulated that this limited replicative capacity contributes to the phenotypes associated with aging, such as reduced wound healing and a weakened immune system. Our interest in cellular lifespan is focused on the telomere ?a specialized structure composed of DNA and proteins, which is located at the end of linear chromosomes ?because it plays a key role in controlling cellular lifespan. This proposal will take a novel proteomics approach to identify telomeric binding proteins. While the identification of these proteins will be of great importance, delineation of their function in telomere homeostasis will be paramount to furthering our understanding of cellular aging. We will use these newly identified proteins, and proteins already known to bind the telomere, to set up a number of suppressor screens in human cells. Our expertise in vector-based RNAi systems makes these studies feasible. To date we have developed a novel method to purify telomere binding proteins by crosslinking protein complexes in live cells thus facilitating capture of weak and/or transient interactions, followed by affinity purification of telomeric TIN2 protein tagged with HA and Flag and analysis by mass spectrometric analysis if peptides obtained by in solution digestion. We believe that this approach will allow us to identify novel telomere binding proteins that are likely to be missed by more standard methods. Using this method we have successfully identified all six members of the core telomere binding, Shelterin complex as well as several other known telomere binding proteins (these proteins were not identified in our negative control). In addition, we have identified approximately 30 putative telomere binding proteins and intend to determine what role they play in telomere dynamics.
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