Telomere structure and function in Arabidopsis

项目摘要： 活性氧（ROS）是氧代谢的天然副产物。环境污染可以 急剧升高ROS，压倒细胞抗氧化剂的防御，并引发对重要细胞的损害。 大分子，尤其是DNA。氧化损伤与许多疾病状态有关，但尽管如此， 然而，在研究中，基本问题仍然是细胞如何避免ROS的有害影响。越来越多的证据 暗示端粒蛋白在染色体末端以外的功能。端粒1（POT 1）的保护 以及端粒酶催化亚单位TERT进出细胞核的运输，并涉及多个方面 对哺乳动物细胞氧化损伤的反应。然而，揭示潜在的非端粒功能 是有问题的，因为它们在端粒的维持和稳定性中起着关键作用。在此更新申请中， 模式真核生物拟南芥被用来研究端粒相关蛋白（TAPs）如何响应 并减轻氧化应激。拟南芥编码两个高度不同的POT 1旁系同源物AtPOT 1a和AtPOT 1b， 其表现出关于典型端粒生物学的功能分离，因此呈现出独特的 有机会阐明POT 1功能的全部补充。该提案建立在额外的初步 表明AtPOT 1b在细胞质中积累，AtPOT 1b的丢失显著升高了ROS， 激活许多细胞防御氧化应激。AtTERT携带线粒体定位信号， 这表明它也可以调节对ROS的反应。这一提议的核心假设是， AtPOT 1a、AtPOT 1b和AtTERT在细胞防御氧化损伤中起重要作用。这 将通过两个具体目标来检验假设。对于目标1，POT 1功能的广度在促进 基因组完整性将通过测试POT 1a和POT 1b如何调节端粒和端粒中的氧化损伤来评估。 非端粒DNA，以及POT 1b如何与POT 1a和TERT合作以避免基因组不稳定。的作用 将在pot 1b突变体中检查生殖祖细胞中的ATR信号传导。最后，进化的起源 将探索POT 1蛋白的应激反应功能。在目标2中，三个互补的策略将探索 TAP在非端粒途径中的分子机制和相互作用伙伴。这些措施包括监测 响应于应激的TAP的亚细胞运输，鉴定蛋白质结合配偶体的定量质谱， 应激诱导的翻译后修饰，和一种新的抑制筛选，以探索遗传途径， 使POT 1b和TERT能够促进植物发育。这些研究将增加对交叉的理解- TAPs与应激反应之间的对话，为探索POT 1的非典型功能开辟了新的视野， 并提供了一个路线图，以探索人类POT 1的罕见剪接变体，不能与端粒， 但与癌症易感性增加有关。

Project Summary: Reactive oxygen species (ROS) are natural byproducts of oxygen metabolism. Environmental assaults can dramatically elevate ROS, overwhelming the defenses of cellular antioxidants, and triggering damage to essential macromolecules particularly DNA. Oxidative damage is linked to numerous disease states, but despite much research, fundamental questions remain on how cells avert the detrimental impacts of ROS. Mounting evidence implicates telomere proteins in functions beyond the chromosome terminus. Both Protection of Telomeres 1 (POT1) and the telomerase catalytic subunit TERT traffic in and out of the nucleus and are implicated in various aspects of the response to oxidative damage in mammalian cells. However, unmasking potential non-telomeric functions is problematic because of their critical roles in telomere maintenance and stability. In this renewal application, the model eukaryote Arabidopsis thaliana is employed to study how telomere-associated proteins (TAPs) respond to and mitigate oxidative stress. Arabidopsis encodes two highly divergent POT1 paralogs, AtPOT1a and AtPOT1b, which exhibit separation-of-function with respect to canonical telomere biology, and hence present a unique opportunity to elucidate the full complement of POT1 functions. The proposal builds on additional preliminary showing that AtPOT1b accumulates in the cytoplasm, and loss of AtPOT1b significantly elevates ROS and activates numerous cellular defenses against oxidative stress. AtTERT carries a mitochondrial localization signal, suggesting that it too may regulate the response to ROS. The central hypothesis of this proposal is that the TAPs, AtPOT1a, AtPOT1b and AtTERT, serve important roles in the cellular defense against oxidative damage. This hypothesis will be examined through two Specific Aims. For Aim 1, the breadth of POT1 functions in promoting genome integrity will be assessed by testing how POT1a and POT1b regulate oxidative damage in telomeric and non-telomeric DNA, and how POT1b cooperates with POT1a and TERT to avert genome destabilization. The role of ATR signaling in reproductive progenitor cells will be examined in pot1b mutants. Finally, the evolutionary origin of stress response functions in POT1 proteins will be explored. In Aim 2, three complementary strategies will probe the molecular mechanism and interaction partners of TAPs in non-telomeric pathways. These include monitoring the subcellular trafficking of TAPs in response to stress, quantitative mass spec to identify protein binding partners and stress-induced post-translational modifications, and a novel suppressor screen to explore the genetic pathway that enables POT1b and TERT to promote plant development. These studies will increase understanding of the cross- talk between TAPs and the stress response, open new horizons for exploring the non-canonical functions of POT1, and provide a roadmap to explore rare splice variants of human POT1 that cannot associate with telomeres, and yet are associated with increased predisposition to cancer.