TELOMERE BINDING PROTEINS AND CELLULAR AGING

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 有限的细胞寿命被假设在有机体衰老中起着至关重要的作用。 尽管细胞寿命在衰老中的重要性，我们目前只有一个 对分子过程的初步了解 因此， 控制细胞寿命的分子机制将具有深远的意义 在衰老、癌症生物学和组织再生领域的研究。 中生长时 正常的人类细胞在进入一种状态之前分裂有限的次数， 复制性衰老的过程中，它们保持活力，但无法进一步分裂。 据推测，这种有限的复制能力有助于表型 与衰老有关，如伤口愈合减少和免疫力减弱 系统 我们对细胞寿命的兴趣集中在端粒上  一个专门 由DNA和蛋白质组成的结构，位于线性结构的末端。 染色体  因为它在控制细胞寿命方面起着关键作用。 这 该提案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， 它们在端粒内稳态中的功能将对我们进一步研究 了解细胞衰老。 我们将使用这些新发现的蛋白质， 已知的蛋白质结合端粒，建立了一些抑制基因， 在人体细胞中进行筛选。 我们在基于载体的RNAi系统方面的专业知识使得这些 可行的研究。 到目前为止，我们已经开发了一种新的方法来纯化端粒 通过交联活细胞中的蛋白质复合物结合蛋白质，从而促进捕获 的弱和/或瞬时相互作用，然后亲和纯化端粒 用HA和Flag标记的TIN 2蛋白，并通过质谱分析进行分析，如果 通过溶液中消化获得的肽。 我们认为，这种做法将使 我们发现了新的端粒结合蛋白，这些蛋白可能被更多的人错过。 标准方法。 利用这种方法，我们成功地识别了所有六个 核心端粒结合的成员，Shelterin复合物以及其他几个 已知的端粒结合蛋白（这些蛋白在我们的阴性对照中未被鉴定）。 对照）。 此外，我们已经确定了大约30个假定的端粒结合 蛋白质，并打算确定它们在端粒动力学中发挥什么作用。

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.