New Approaches for Probing PARylation in living Cells

探测活细胞中 PARylation 的新方法

• 批准号: 430576836
• 负责人: Professor Dr. Andreas Marx
• 金额: --
• 依托单位: Arbeitsgruppe Physikalische Chemie (AG Zumbusch)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/430576836?language=en
• 关键词: New; Approaches; Probing; PARylation; living

Poly(ADP-ribos)ylation (PARylation) is a major post-translational modification and signalling event in most eukaryotes. Fundamental processes like DNA repair and transcription are coordinated by this transient polymer and it’s binding to proteins. ADP-ribosyltransferases (ARTs) build complex ADP-ribose chains from NAD+ onto various acceptor proteins. The complexity of the modification and the absence of suitable tools render molecular studies of PARylation challenging. Building on our successful earlier work that already lead to several joint publications, the major aim of this collaborative project is to establish means for quantitative studies of DNA photodamage induced protein PARylation dynamics in live cells with high temporal and spatial resolution. Our approach is based on the development of novel synthetic NAD+-based tools and advanced optical microscopy techniques. Based on the preliminary results, the Marx group will develop means to deliver modified NAD+-analogues through the cell membrane that do not interfere with the cell viability. Two kinds of modified NAD-analogues are envisioned that will allow imaging (through modification with a fluorescent dye) and affinity enrichment of protein that are modified through processing of desthiobiotin-modified NAD+-analogues. The Zumbusch group will further develop optical experiments to optimize NIR microirradiation such that controlled induction of specific types of DNA damages becomes possible. To this end, we will establish means to independently control pulse energy and average power of the NIR laser used for microirradiation. At the same time, experimental protocols for the application of synthetic NAD+-based tools and optical microscopy of PAR formation with minimized effects on cell health will be investigated. These will be evaluated by live cell microscopy of PARylation dynamics of selected proteins after DNA photo-damage and path the way for future applications.

聚 (ADP-核糖) 化 (PARylation) 是大多数真核生物中的主要翻译后修饰和信号传导事件，由这种瞬时聚合物协调，并与蛋白质结合。从 NAD+ 到各种受体蛋白的复杂 ADP-核糖链修饰的复杂性和缺乏合适的工具使得 PARylation 的分子研究具有挑战性。我们早期的成功工作已经导致了几篇联合出版物，这个合作项目的主要目标是建立以高时间和空间分辨率定量研究活细胞中 DNA 光损伤诱导的蛋白质 PARylation 动力学的方法。基于新型合成 NAD+ 的工具和先进的光学显微镜技术，Marx 小组将开发通过细胞膜传递修饰的 NAD+ 类似物的方法，这些类似物不会干扰细胞活力。类似物预计将允许对通过脱硫生物素修饰的 NAD+-类似物进行处理而修饰的蛋白质进行成像（通过荧光染料修饰）和亲和富集。Zumbusch 小组将进一步开发光学实验来优化 NIR 微辐射，从而控制特异性的诱导。为此，我们将建立独立控制用于微照射的近红外激光的脉冲能量和平均功率的方法，同时，建立用于合成的实验方案。将研究基于 NAD+ 的 PAR 形成工具和光学显微镜，对细胞健康的影响最小化。这些将通过活细胞显微镜对 DNA 光损伤后选定蛋白质的 PARylation 动力学进行评估，并为未来的应用铺平道路。