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.

在大多数真核生物中，聚（ADP-ribos）ylation（Parylation）是翻译后的主要修饰和信号传导事件。诸如DNA修复和转录之类的基本过程由这种瞬时聚合物协调，并且与蛋白质结合。 ADP-核糖基转移酶（ARTS）将复杂的ADP-核糖链从NAD+构建到各种受体蛋白上。修饰的复杂性和缺乏合适的工具使得挑战性的分子研究。在我们成功的早期工作的基础上，该协作项目的主要目的是为DNA光损伤诱导的蛋白质parylation Dynamics的定量研究建立手段，并在具有高临时和空间分辨率的活细胞中为蛋白质parylation Dynamics建立。我们的方法基于新型合成NAD+基于NAD+的工具和高级光学显微镜技术的开发。基于初步结果，马克思组将开发出通过细胞膜传递改良的NAD+ - 动物的手段，而不会干扰细胞活力。设想了两种修饰的NAD-Analogues，可以通过处理Desthiobiotin修饰的NAD+Analogues进行修饰（通过荧光染料修饰）和蛋白质的亲和力富集。 Zumbusch组将进一步开发光学实验，以优化NIR微辐照，从而可以控制特定类型的DNA损伤的诱导。为此，我们将建立能够独立控制用于微辐射的NIR激光的平均功率的方法。同时，将研究用于应用合成NAD+的工具和PAR形成的光学显微镜的实验方案，并研究对细胞健康的影响最小。这些将通过在DNA造影损伤之后对选定蛋白的荷脂化动力学的活细胞显微镜进行评估，并为将来的应用提供道路。