New Approaches for Probing PARylation in living Cells
Final Report Abstract
Poly(ADP-ribos)ylation (PARylation) is a major posttranslational modification and signalling event in most eukaryotes. Fundamental processes like DNA repair and transcription are coordinated by this transient polymer and its 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. The major aim of this collaborative project was to establish means for quantitative studies of DNA photodamage induced protein PARylation dynamics in live cells with high temporal and spatial resolution. The approach was based on the development of novel synthetic NAD+-based tools and advanced optical microscopy techniques. The Marx group has successfully developed means to deliver modified NAD+-analogues through the cell membrane that do not interfere with the cell viability. By employment of these probes, first insights into the PARylome in living cells were obtained. The Zumbusch group first investigated the influence of PAR length and branching on its interaction with various proteins. Subsequently, an optical experiment was established that allows to follow the interaction of proteins or of proteins with other biomolecules in living cells. This is based on the spatially resolved measurement of the decrease of fluorescence lifetime of a labelled protein that transfers its excitation energy to a second labelled molecule that is in close proximity (FLIM-FRET microscopy). This experiment was first employed to image the glycosylation status of proteins in living cells. Then a pulsed laser source suited for inducing DNA photodamage in a highly controlled manned was integrated into the FLIM setup. Using this experiment and the modified NAD+ molecules from the Marx group, it was finally possible to study the PARylation kinetics of proteins involved in DNA damage repair in live cells.
Publications
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Profiling of the ADP‐Ribosylome in Living Cells. Angewandte Chemie International Edition, 61(18).
Lehner, Maike; Rieth, Sonja; Höllmüller, Eva; Spliesgar, Daniel; Mertes, Bastian; Stengel, Florian & Marx, Andreas
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Influence of chain length and branching on poly(ADP-ribose)–protein interactions. Nucleic Acids Research, 51(2), 536-552.
Löffler, Tobias; Krüger, Annika; Zirak, Peyman; Winterhalder, Martin J.; Müller, Anna-Lena; Fischbach, Arthur; Mangerich, Aswin & Zumbusch, Andreas
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A desthiobiotin labelled NAD+ analogue to uncover Poly(ADP‐ribose) polymerase 1 protein targets. ChemBioChem, 25(5).
Rieth, Sonja; Spliesgar, Daniel; Orth, Jan; Lehner, Maike; Kasprzyk, Renata; Stengel, Florian & Marx, Andreas
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Next‐Generation Metabolic Glycosylation Reporters Enable Detection of Protein O−GlcNAcylation in Living Cells without S‐Glyco Modification. Angewandte Chemie, 136(20).
Kufleitner, Markus; Haiber, Lisa Maria; Li, Shuang; Surendran, Harsha; Mayer, Thomas U.; Zumbusch, Andreas & Wittmann, Valentin
