TELOMERE BINDING PROTEINS AND CELLULAR AGING

端粒结合蛋白与细胞衰老

• 批准号: 8168727
• 负责人: Sheila A Stewart
• 金额: $ 0.97万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168727
• 关键词: Affinity Chromatography; Aging; Binding; Binding Proteins; Cancer Biology; Cell Aging; Cells; Chromosomes; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; Digestion; Funding; Grant; Homeostasis; Human; Immune system; Institution; Life; Longevity; Methods; Molecular; Peptides; Phenotype; Play; Process; Proteins; Proteomics; RNA Interference; Research; Research Personnel; Resources; Role; Solutions; Source; Structure; System; TINF2 gene; Telomere-Binding Proteins; Time; United States National Institutes of Health; Wound Healing; base; interest; member; novel; protein complex; protein crosslink; telomere; tissue regeneration; vector

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 假设有限的细胞寿命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推定重要性，但我们目前对分子过程只有基本的理解。 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领域具有很大的影响。 在培养物中生长时，正常的人类细胞在进入复制衰老状态之前会划分有限的次数，但它们保持可行，但无法进一步分裂。 据推测，这种有限的复制能力有助于与衰老相关的表型，例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，该结构由DNA和蛋白质组成，该结构位于线性染色体的末端，因为它在控制细胞寿命方面起着关键作用。 该建议将采用一种新型的蛋白质组学方法来鉴定端粒结合蛋白。 尽管这些蛋白质的鉴定将非常重要，但描述它们在端粒稳态中的功能将至关重要，这对于增进我们对细胞衰老的理解至关重要。 我们将使用这些新鉴定的蛋白质，以及已经已知可以结合端粒的蛋白质来在人类细胞中建立许多抑制筛选。 我们在基于向量的RNAi系统方面的专业知识使这些研究可行。 迄今为止，我们已经开发了一种新的方法来纯化端粒结合蛋白，通过在活细胞中交联蛋白质复合物，从而促进捕获弱和/或瞬态相互作用，然后通过质谱分析在溶液中获得质谱分析，然后通过质谱分析对端粒TIN2蛋白进行亲和力纯化。 我们认为，这种方法将使我们能够鉴定出新型的端粒结合蛋白，这些蛋白可能会被更多的标准方法遗漏。 使用这种方法，我们成功地确定了核心端粒结合，庇护素复合物以及其他几种已知的端粒结合蛋白的所有六个成员（在我们的阴性对照中未鉴定这些蛋白）。 此外，我们已经确定了大约30个推定的端粒结合蛋白，并打算确定它们在端粒动力学中的作用。