Unravelling the poly(ADP-ribose) code: How does the structural diversity of poly(ADP-ribose) affect cellular functions during genotoxic stress response

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PARP1 catalyzes the posttranslational protein modification with poly(ADP-ribose) (PAR) of variable chain length and branching frequency. Poly(ADP-ribosyl)ation (PARylation) is involved in diverse cellular functions, such as DNA repair, transcription, and regulation of cell death, and contributes to various pathophysiological conditions (Rank et al. Nucleic Acids Res. 2016). Importantly, PARP inhibitors are being employed and further developed in clinical cancer therapy.The overall aim of this proposal is to unravel the biochemical and cellular functions in the structural heterogeneity of PARP1-mediated PARylation and mono-ADP-ribosylation. This includes the objectives:I. What is the role of protein-conjugated mono-ADP-ribose that arises as an intermediate during PAR metabolism?II. What is the function of the heterogeneity in PAR chain length (short vs. long PAR chains)?III. What is the function of the heterogeneity in PAR branching ratios?Within this, the following research questions should be addressed:- Do PAR molecules of different quality exhibit different stabilities with regards to their formation and degradation kinetics?- Do specific readers for the structural diverse PAR molecules exist, i.e., does a PAR code exist?- Does PAR diversity lead to differential regulation of protein complexes and enzymatic functions?- How does the structural diversity of PAR translate into specific cellular functions?Experimentally, these questions will be addressed by using PARP1 mutants with modified enzymatic activities, which result in mono-ADP-ribosylation activity (objective i), and PARP1 mutants generating PAR of altered chain lengths (objective ii) or branching ratios (objective iii). These mutants and the PAR produced by them will be analyzed biochemically using recombinant proteins and in cellular systems using PARP1-reconstituted HAP1 cells, either transiently transfected or CRISPR/Cas-engineered. HAP1 cells are a human, haploid cancer cell line ideally suited for this kind of purpose.The analyses will be performed in a systematic and comparative manner concerning several biochemical and cell biological end-points to decipher the different consequences of altered PARP1 activities with a focus on genotoxic stress response. This includes (i) the detailed analysis of the cellular PARylation and NAD+ metabolism, (ii) identification of PAR-structure-specific interactomes by novel affinity mass spectrometry approaches, (iii) spatio-temporal distribution and trafficking of select PARylation target proteins by advanced bioimaging techniques, and (vi) cellular consequences upon induction of genotoxic stress.In conclusion, using this approach it is expected (i) to obtain a deeper insight into the cellular biochemistry of PARP1, (ii) to improve our understanding on its role in cellular (patho-)physiology, and (iii) in the long run, to help tailor more specific and improved approaches for pharmacological PARP inhibition in cancer therapy.