Mechanistic analysis of ATR signaling

ATR信号传导机制分析

基本信息

批准号：
10004100
负责人：
MATTHEW MICHAEL
金额：
$ 33万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-08 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10004100
关键词：
ATR gene Address Binding Biochemical Biochemistry Bioinformatics Biological Assay Books Cancer Biology Cell Cycle Cell Cycle Progression Cells Clinical Trials Closure by clamp Complex Crowding Cultured Cells DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair DNA biosynthesis Data Development Event Feedback Genotoxic Stress Goals Human In Vitro Learning Lesion Link Maps Modeling Molecular Conformation Normal Cell Nuclear Proteins Pathologic Pathway interactions Patients Pharmaceutical Preparations Phosphorylation Phosphotransferases Preclinical Testing Protein Kinase Proteins Reporting Research Signal Transduction Site Slide Stress System TOPBP1 Gene TREX1 gene Testing Thinking Time Travel Work Xenopus anti-cancer therapeutic base cancer cell cell transformation design egg exhaustion experimental study in vivo inhibitor/antagonist physical property preclinical study prevent programs protein complex protein protein interaction recruit replication stress response scaffold tumorigenesis

项目摘要

Project Summary/Abstract The ATR protein kinase sits atop a complex signaling cascade that is activated by DNA replication and hyper- activated by replication stress or DNA double-strand breaks. Activation of ATR by genotoxic stress is essential for the ability of cells to survive stress and represents a barrier to transformation of normal cells to a pathological condition that promotes tumorigenesis. The downstream effectors and consequences of ATR signaling are now being understood, however early events in signaling, such as the initial activation of ATR kinase at sites of gentoxic stress, are still poorly understood. This proposal focuses on the biochemical mechanism for ATR activation at sites of DNA damage. This proposal features the TOPBP1 protein, which physically interacts with ATR at sites of DNA damage and is required for ATR activation during the replication stress and DNA damage responses. TOPBP1 is a BRCT repeat containing protein that likely acts as a scaffold to link checkpoint proteins together into active signaling centers. In this proposal we combine biochemical studies using purified factors, functional studies in Xenopus egg extracts, and in vivo studies in cultured cells to mount an in-depth exploration of how TOPBP1 activates ATR at sites of DNA damage. Recent studies on the ETAA1 protein have shown that TOPBP1 is not alone in its ability to activate ATR, and our preliminary studies suggest that a third and possibly more ATR activators are present in human cells. In this proposal we will also study these new activators, with the long-term goal of building a comprehensive, systems-level view of how ATR signaling is initiated. The work is divided into three Aims. In Aim 1, we examine how active ATR signaling centers form at sites of damage. The recruitment mechanism is complex, and likely involves multiple protein-protein interactions between TOPBP1 and its binding partners. Recent work from our group has shown that soluble TOPBP1 is held in an auto-inhibitory conformation and thus an additional goal of Aim 1 is to determine how this auto- inhibition is resolved at sites of damage. In Aim 2 we examine how ATR may use a negative feedback loop to regulate assembly of additional signaling centers at sites of damage. We will also explore the possibility that an ATR signaling center is mobile on DNA. In the final Aim we will define a rule-book for how different ATR activators are utilized, how their functions are related (or not), and how their activities are integrated during an ATR response.

项目摘要/摘要 ATR蛋白激酶位于一个复杂的信号级联上，该级联由DNA复制和高度激活通过复制应力或DNA双链断裂而激活。通过遗传毒性应激激活ATR是必不可少的为了使细胞能够生存应力并表示正常细胞转化为A的障碍促进肿瘤发生的病理状况。 ATR的下游效应子和后果现在已经了解了信号传导，但是信号传导中的早期事件，例如ATR的初始激活在绅士胁迫部位的激酶仍然对此知之甚少。该提议着重于生化 DNA损伤部位ATR激活的机制。该建议的特征是TopBP1蛋白，该蛋白在DNA损伤部位与ATR进行物理相互作用，并且在复制过程中激活ATR需要压力和DNA损伤反应。 TOPBP1是含有蛋白质的BRCT重复，可能充当脚手架将检查点蛋白链接到活动信号中心。在此提案中，我们结合了生化使用纯化因子，爪蟾卵提取物中的功能研究以及培养细胞中的体内研究要深入探索TopBP1如何在DNA损伤部位激活ATR。最近关于 etaa1蛋白表明，TopBP1并不孤单地激活ATR，我们的初步研究表明人类细胞中存在三分之一甚至更多的ATR激活剂。在这个建议中，我们也将研究这些新的激活剂，其长期目标是建立全面的系统级别的观点启动ATR信号。工作分为三个目标。在AIM 1中，我们检查了如何在损害。募集机制很复杂，可能涉及多种蛋白质 - 蛋白质相互作用在TopBP1及其具有约束力的合作伙伴之间。我们小组的最新工作表明可溶性TOPBP1已举行在自动抑制构象中，因此目标1的另一个目标是确定这种自动在损害部位解决抑制作用。在AIM 2中，我们检查了ATR如何使用负面反馈循环调节损坏部位的其他信号转导中心的组装。我们还将探讨 ATR信号中心是在DNA上移动的。在最终目标中，我们将定义一本规则手册，以了解ATR的不同使用激活剂，其功能如何相关（或不相关），以及如何整合其活动。 ATR响应。