TELOMERE BINDING PROTEINS AND CELLULAR AGING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 有限的细胞寿命被认为在生物体衰老中起着至关重要的作用。尽管假设细胞寿命在衰老过程中很重要，但我们目前对分子过程只有一个初步的了解。因此，阐明控制细胞寿命的分子机制将在衰老、癌症生物学和组织再生等领域具有深远的意义。当在培养中生长时，正常的人类细胞在进入复制衰老状态之前会分裂有限的次数，在这种状态下它们仍然活着，但无法进一步分裂。据推测，这种有限的复制能力导致了与衰老相关的表型，如伤口愈合减少和免疫系统减弱。我们对细胞寿命的兴趣集中在端粒上，端粒是一种由DNA和蛋白质组成的特殊结构，位于线性染色体的末端，因为它在控制细胞寿命方面发挥着关键作用。这项建议将采用一种新的蛋白质组学方法来鉴定端粒结合蛋白。虽然这些蛋白质的鉴定将是非常重要的，但描绘它们在端粒稳态中的功能将是进一步了解细胞衰老的关键。我们将使用这些新发现的蛋白质，以及已知的结合端粒的蛋白质，在人类细胞中建立一些抑制屏幕。我们在基于载体的RNAi系统方面的专业知识使这些研究成为可能。到目前为止，我们已经发展了一种新的方法来纯化端粒结合蛋白，方法是在活细胞中通过交联蛋白复合体来捕获弱的和/或瞬时的相互作用，然后亲和纯化标记有HA和FLAG的端粒TIN2蛋白，并通过溶液中消化获得的多肽进行质谱分析。我们相信，这种方法将使我们能够识别新的端粒结合蛋白，这些蛋白可能会被更标准的方法遗漏。使用这种方法，我们已经成功地鉴定了核心端粒结合蛋白复合体的全部六个成员以及其他几个已知的端粒结合蛋白(这些蛋白在我们的阴性对照中没有被鉴定)。此外，我们已经确定了大约30个可能的端粒结合蛋白，并打算确定它们在端粒动力学中所起的作用。

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. 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.