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2017

Friday, 12:30-13:30, June 9

Speaker: Michael O. Hottiger, Ph.D. 
Professor,
Head of Department
Department of Molecular Mechanisms of Disease
University of Zurich

Topic: Uncovering the functional role of protein ADP-ribosylation on the proteome-wide level

Host: Mengqiu Dong, Ph.D.

Abstract:

Protein ADP-ribosylation is a reversible posttranslational modification (PTM) that results from the transfer of the ADP-ribose (ADPr) moiety from NAD+ to specific amino acid residues or to ADPr itself.  In terms of complexity and functional consequences, ADP-ribosylation is on a par with other protein PTMs.  The covalent attachment of ADPr to amino acids or ADPr is governed by ADP-ribosyltransferases (ARTs), it is recognized by ADPr binding domains and removed by ADP-ribosylhydrolases (ARHs).  This PTM can affect the structure and the function of the modified proteins.  One of the greatest needs, and most significant challenges, within the field is to complete the ADP-ribosylation discovery phase (target protein identification and/or ADPr acceptor site localization).  This would allow us to transition towards defining the functional links between ARTs (writers) and ARHs (erasers) and their targets, and characterizing the molecular relevance of ADP-ribosylation on the modification site or protein level.

We recently further advanced the development our established ADPr-peptide affinity enrichment mass spectrometry (MS) workflow (Martello, Leutert, Jungmichel et al., Nat Commun. 2016).  This optimized MS method, is based on the enrichment of the ADP-ribosylated peptides with a newly engineered Af1521 macrodomain and a product preview MS scan step that triggers subsequent HCD and EThCD fragmentations only when marker ions are present.  This workflow allows us now to localize the ADP-ribose acceptor site with high accuracy and to define protein ADP-ribosylation motifs.  Using cells with perturbed ARTD1/2 expression, we identified ARTD1 as the main catalyzer of nuclear ADP-ribosylation during oxidative stress, while ARTD2 modified fewer and, importantly, different specific targets.  Moreover, we observed ARTD1-catalyzed ADP-ribosylation to be selective towards serine and to a lower extent tyrosine as ADPr acceptors.  Using a comparable MS approach, we could provide evidence that ARH3, unlike PARG, is capable of releasing ADP-ribose from peptides modified on serine, indicating that ARH3 has mono-ADP-ribosylhydrolase activity and is the first known eraser of serine ADP-ribosylation. Together these findings allow us to start resolving the functional relevance of protein ADP-ribosylation and shed light on how ADP-ribosylation contributes to different cellular processes.