22 September 2021

Post-translational modifications of proteins are indispensable drivers and regulators of essentially all cellular processes. They do so by affecting protein properties such as folding, trafficking and (enzymatic) activity.

Among well-studied modifications, ADP-ribosylation is the least known in terms of its structural molecular details and regarding the proteins that are involved in its turnover and recognition. These shortcomings can be explained by the properties of ADP-ribose (ADPr)-based modifications such as the labile linkages with target amino acids in proteins, the complex polyanionic structure, and its dynamic nature. ADP-ribosylation plays a pivotal role in controlling cellular processes including the DNA damage response and deregulation of ADP-ribose signalling and has been linked to a number of diseases, including immune disorders and cancer. Hence, identification of components of the ADP-ribose signalling network is of great scientific importance.

Michiel Vermeulen and Kasia Kliza, theme cancer development and immune defense (from Faculty of Science of Radboud University and part of the interfaculty Radboud Institute for Molecular Life Sciences), collaborated with Qiang Liu and Dima Filippov (Leiden UMC) to synthesize biotinylated ADPr-based probes that would enable identification of ADPr readers and determination of their specificity towards mono- and polyADPr.

These probes were then used for affinity purifications in crude lysates combined with relative and absolute quantitative mass spectrometry to generate the first proteome-wide ADPr interactomes, including the determination of apparent binding affinities. Amongst the main findings, mono-ADPr and poly-ADPr interactors, also called ADPr ‘readers’, regulate various common and distinct processes, such as the DNA damage response, cellular metabolism, RNA trafficking and transcription.

The team also monitored the dynamics of polyADPr interactions upon induction of oxidative DNA damage and uncovered new mechanistic connections between ubiquitin signalling and ADP-ribosylation. Taken together, chemical biology enables the exploration of ADPr readers using interaction proteomics. Furthermore, the generated ADPr interaction maps significantly expand our current understanding of ADPr signalling. In the future, targeting certain ADPr – reader interactions may turn out to be beneficial from a therapeutic perspective. These results were published in the journal Molecular Cell.

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