Temporal resolution of phosphorylation-mediated signalling events in DNA Damage Repair

DNA 损伤修复中磷酸化介导的信号事件的时间解析

• 批准号: RGPIN-2020-06612
• 负责人: TrinkleMulcahy, Laura
• 金额: $ 2.62万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741335
• 关键词: Temporal; resolution; phosphorylation; mediated; signalling

The integrity of the genome is constantly threatened by endogenous and exogenous insults that lead to generation of DNA damage. This damage triggers a DNA damage response (DDR) in which activation of a complex signaling network coordinates downstream events that can include cell cycle arrest, DNA repair, senescence or apoptosis. Integrity of this system is critical, as compromised DDR networks found in many cancer cell types allow them to either bypass this damage sensing or evade induction of apoptosis. The DDR pathway comprises sensor molecules that detect the damage, signal transducers that amplify the signal and effector molecules that are recruited/activated to mediate downstream events. Distinct networks are activated depending on the type of damage and cell cycle stage at which it occurs. Reversible phosphorylation has long been known to be a key regulatory event in DDR, but although much is known about the principle Ser/Thr kinases that mediate signalling events, surprisingly little is known about the equally essential roles of their primary counterparts, which include the Ser/Thr phosphatases PP1 and PP2A. Analysis of PP1- and PP2A-mediated dephosphorylation events is complicated by their unique regulatory mechanism, whereby a common catalytic subunit is dynamically distributed between a shared pool of regulatory subunits to form holoenzyme complexes with specific localizations and substrate specificities. We have developed strategies that allow us to monitor the dynamic subcellular distribution of catalytic subunits between holoenzyme complexes and alter the level or activity of individual complexes with high specificity. Our combination of fluorescence imaging with quantitative interactome mapping has armed us with a comprehensive spatial map of nuclear PP1 distribution and identified several complexes directed toward DDR substrates. Standard workflows include complementary quantitative affinity purification/mass spectrometry (AP/MS) and proximity labeling approaches to map spatiotemporal interactomes with high resolution, CRISPR/Cas9-based mutagenesis and quantitative phosphoproteomic mapping to identify the specific residues targeted in substrates. With a long-term goal of building a more complete picture of the signalling dynamics of this complex pathway, the following three short-term objectives will allow us to functionally dissect the contribution of PP1 and PP2A phosphatase complexes to DDR in unprecedented detail: 1. Map changes in the steady-state distribution of PP1 and PP2A catalytic subunits between nuclear holoenzyme complexes in response to distinct type of DDR. 2. Assemble a network map of the spatiotemporal interactomes of DDR-targeted PP1 and PP2A holoenzyme complexes at early, mid and late time points following damage. 3. Assess the effects of disruption of specific complexes on damage detection, pathway choice, repair timing and DDR-associated dephosphorylation events

基因组的完整性不断受到导致DNA损伤产生的内源性和外源性侮辱的威胁。这种损伤触发了DNA损伤响应（DDR），其中复杂信号网络的激活会坐在下游事件，其中可能包括细胞周期停滞，DNA修复，衰老或凋亡。该系统的完整性至关重要，因为在许多癌细胞类型中发现的DDR网络受损，它们可以绕过这种损害感应或逃避凋亡的诱导。 DDR途径包括检测损伤的传感器分子，信号传感器，这些传感器会放大被募集/激活以介导下游事件的信号和效应分子。取决于发生的损伤和细胞周期阶段的类型，将不同的网络激活。长期以来，可逆的磷酸化是DDR中的关键调节事件，但是尽管对介导信号事件的原理Ser/Thr激酶知之甚少，但令人惊讶的是，对其主要对应物的同样重要的作用，包括Ser/Thr Phosparases PP1和PP2A的同样重要的作用知之甚少。 PP1和PP2A介导的去磷酸化事件的分析使其独特的调节机制变得复杂，因此，共同的催化亚基在共享的调节亚基之间动态分布，以形成具有特定局部局部和底物特异性的全酶复合物。我们制定了策略，使我们能够监测全酶复合物之间催化亚基的动态亚细胞分布，并改变具有高特异性的单个复合物的水平或活性。我们将荧光成像与定量相互作用映射的组合结合了我们的核PP1分布的全面空间图，并确定了针对DDR底物的几种复合物。标准工作流程包括互补的定量亲和力纯化/质谱（AP/MS）和映射具有高分辨率，基于CRISPR/CAS9的诱变和定量磷酸蛋白质组映射的时空相互作用的接近性标记方法，以识别源自源带中靶向的特定残基。 With a long-term goal of building a more complete picture of the signalling dynamics of this complex pathway, the following three short-term objectives will allow us to functionally dissect the contribution of PP1 and PP2A phosphatase complexes to DDR in unprecedented detail: 1. Map changes in the steady-state distribution of PP1 and PP2A catalytic subunits between nuclear holoenzyme complexes in response to distinct type of DDR. 2。在损坏后早期，中期和后期时间点组装了靶向DDR靶向PP1和PP2A Holoenzyme复合物的时空相互作用组的网络图。 3。评估特定复合物破坏对损伤检测，途径选择，修复时间和与DDR相关的去磷酸化事件的影响