Abstract
AbstractPost‐translational modification (PTM) with ADP‐ribose and poly(ADP‐ribose) using nicotinamide adenine dinucleotide (NAD+) as substrate is involved in the regulation of numerous cellular pathways in eukaryotes, notably the response to DNA damage caused by cellular stress. Nevertheless, due to intrinsic properties of NAD+ e.g., high polarity and associated poor cell passage, these PTMs are difficult to characterize in cells. Here, two new NAD+ derivatives are presented, which carry either a fluorophore or an affinity tag and, in combination with developed methods for mild cell delivery, allow studies in living human cells. We show that this approach allows not only the imaging of ADP‐ribosylation in living cells but also the proteome‐wide analysis of cellular adaptation by protein ADP‐ribosylation as a consequence of environmental changes such as H2O2‐induced oxidative stress or the effect of the approved anti‐cancer drug olaparib. Our results therefore pave the way for further functional and clinical studies of the ADP‐ribosylated proteome in living cells in health and disease.
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