Probing TRAP150/GSK3-mediated regulation of the aging-related protein PSF

描述（由申请人提供）：衰老是一种复杂的、尚未完全理解的现象，它在许多层面上对人类健康产生重大影响。在分子水平上，衰老过程的主要特征包括基因组稳定性和基因表达的变化，这些变化源于核心核过程（如染色质维护、DNA损伤修复和RNA加工）的效率降低或重编程。最终，这些有害的变化导致与衰老相关的疾病的发展，如癌症、免疫功能障碍和神经变性。为了避免这些有害状态，细胞必须严格调节负责控制关键核操作的多功能核蛋白。尽管它们在促进细胞健康方面很重要，但我们对这些蛋白质如何以信号响应方式调节的理解仍然很大程度上不完整。本提案的目标是通过了解如何调节核因子PSF以控制其功能来帮助纠正这种情况。PSF （SFPQ）是一种含有707个氨基酸的蛋白质，在DNA损伤反应、转录和RNA生物发生途径的几个步骤中发挥作用。Lynch实验室最近的工作已经确定PSF是丝氨酸/苏氨酸激酶GSK3的下游靶点。GSK3对PSF T687的磷酸化促进了PSF与TRAP150的相互作用，这种相互作用消除了PSF结合至少一种已知RNA靶点的能力。然而，目前尚不清楚磷酸化如何调节TRAP150的关联，也不清楚TRAP150如何改变PSF的功能。为了充分理解这种调控机制，我将首先通过使用共免疫沉淀和下拉试验来确定PSF-TRAP150相互作用的分子特征，以定义最小的复杂界面。接下来，我将采用拉下、电泳迁移率转移测定和紫外交联实验来生化分析GSK3磷酸化和TRAP150关联对PSF结合其他生物学上重要的蛋白质伴侣和DNA和RNA靶标的能力的影响。最后，我将使用有限蛋白水解、质谱和氢/氘交换质谱来研究磷酸化、TRAP150关联和核酸结合如何影响PSF构象。总之，这些实验将使我能够开发可测试的调节模型，这将指导我们长期努力研究PSF和其他核因子在DNA双链断裂修复和mRNA选择性剪接等过程中的作用，这些过程对衰老和衰老相关疾病具有影响。

DESCRIPTION (provided by applicant): Aging is a complex, incompletely understood phenomenon that substantially impacts human health on numerous levels. Major hallmarks of the aging process at the molecular level include changes in genomic stability and gene expression that stem from the decreased efficiency or reprogramming of core nuclear processes such as chromatin maintenance, DNA damage repair and RNA processing. Ultimately, these deleterious changes lead to the development of aging-related disorders such as cancer, immune dysfunction and neurodegeneration. To avert these harmful states, cells must tightly regulate the multifunctional nuclear proteins responsible for controlling critical nuclear operations. Despite their importance in promoting cell health, our comprehension of how these proteins are regulated in a signal-responsive manner remains largely incomplete. The goal of this proposal is help remedy this situation by understanding how one such nuclear factor, PSF, is regulated to control its functional capabilities. PSF (SFPQ) is a 707 amino acid protein that plays a role in, among other things, the DNA damage response, transcription, and several steps of the RNA biogenesis pathway. Recent work in the Lynch lab has identified PSF as a downstream target of the serine/threonine kinase GSK3. Phosphorylation of PSF T687 by GSK3 promotes interaction of PSF with TRAP150, and this interaction abrogates PSF's ability to bind at least one known RNA target. However, it is so far unclear how phosphorylation regulates TRAP150 association or how mechanistically TRAP150 alters PSF's function. To fully understand this regulatory regime, I will first determine the molecular features that underpin PSF-TRAP150 interaction by using co-immunoprecipitation and pulldown assays to define the minimal complex interface. I will next employ pulldowns, electrophoretic mobility shift assays and UV crosslinking experiments to biochemically analyze the effects of GSK3 phosphorylation and TRAP150 association on PSF's ability to bind other biologically important protein partners and DNA and RNA targets. Finally, I will use limited proteolysis, mass spectrometry, and hydrogen/deuterium exchange mass spectrometry to investigate how phosphorylation, TRAP150 association, and nucleic acid binding affect PSF conformation. Together, these experiments will allow me to develop testable models of regulation that will guide us in our long term efforts to study the role of PSF and other nuclear factors in processes such DNA double strand break repair and mRNA alternative splicing that have implications for aging and aging-related disorders.