New Approaches for Probing PARylation in living Cells

多聚(ADP-Ribos)修饰是大多数真核生物中一种重要的翻译后修饰和信号转导事件。DNA修复和转录等基本过程由这种瞬时聚合物协调，并与蛋白质结合。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。修饰的复杂性和合适的工具的缺乏使得对偶联反应的分子研究具有挑战性。在我们早期成功工作的基础上，已经联合发表了几篇论文，该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PAR化动力学的方法。我们的方法是基于新型合成NAD+工具和先进的光学显微镜技术的发展。在初步结果的基础上，马克斯团队将开发出通过细胞膜输送不干扰细胞存活的改良NAD+类似物的方法。设想了两种修饰的NAD-类似物，它们将允许对通过加工去硫代生物素修饰的NAD+-类似物进行修饰的蛋白质进行成像(通过荧光染料修饰)和亲和力浓缩。Zumbusch研究小组将进一步开发光学实验，以优化近红外微照射，使受控地诱导特定类型的DNA损伤成为可能。为此，我们将建立独立控制用于微照射的近红外激光器的脉冲能量和平均功率的手段。同时，还将研究以合成NAD+为基础的工具的应用和对细胞健康影响最小的PAR形成的光学显微镜的应用的实验方案。这些将通过活细胞显微镜对选定的蛋白质在DNA光损伤后的PAR化动力学进行评估，并为未来的应用开辟道路。

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.