Abstract
ADP-ribosylation is an important post-translational protein modification (PTM) that regulates diverse biological processes. ADP-ribosyltransferase diphtheria toxin-like 10 (ARTD10, also known as PARP10) mono-ADP-ribosylates acidic side chains and is one of eighteen ADP-ribosyltransferases that catalyze mono- or poly-ADP-ribosylation of target proteins. Currently, no enzyme is known that reverses ARTD10-catalyzed mono-ADP-ribosylation. Here we report that ARTD10-modified targets are substrates for the macrodomain proteins MacroD1, MacroD2 and C6orf130 from Homo sapiens as well as for the macrodomain protein Af1521 from archaebacteria. Structural modeling and mutagenesis of MacroD1 and MacroD2 revealed a common core structure with Asp102 and His106 of MacroD2 implicated in the hydrolytic reaction. Notably, MacroD2 reversed the ARTD10-catalyzed, mono-ADP-ribose–mediated inhibition of glycogen synthase kinase 3β (GSK3β) in vitro and in cells, thus underlining the physiological and regulatory importance of mono-ADP-ribosylhydrolase activity. Our results establish macrodomain-containing proteins as mono-ADP-ribosylhydrolases and define a class of enzymes that renders mono-ADP-ribosylation a reversible modification.
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Acknowledgements
We are grateful to I. Ahel (Paterson Institute for Cancer Research, Manchester, UK) and M. Neuvonen (Institute of Biotechnology, University of Helsinki, Helsinki, Finland) for MacroD1 and MacroD2 constructs and J. Moss (National Institutes of Health, Bethesda, Maryland, USA) for the ARH1 construct. F. Althaus (University of Zurich, Zurich, Switzerland) and K. Aktories (University of Freiburg, Freiburg, Germany) are acknowledged for generously providing a baculovirus expressing PARG and the CDTa and TccC3 enzymes together with purified actin, respectively. F. Freimoser (University of Zurich, Zurich, Switzerland) provided editorial assistance and critical input during the writing and M. Fey technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft DFG (LU 466/15-1) to B.L. and in part by the University of Zurich (Forschungskredit 54041205 to E.F.), the Swiss National Science Foundation (SNF-31003A_125190 (to P.O.H.) and 31-122421 (to M.O.H.)) and the Kanton of Zurich (to M.O.H.).
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F.R. and P.O.H. performed experiments with MacroD1, MacroD2 and other macrodomains, and MS analysis was done together with D.F.; K.L.H.F. analyzed the influence of MacroD2 on GSK3β; E.F. performed the computer modeling; E.F. and A.C. analyzed the modeling and simulation results; M.B. identified MacroD2 as an ADP-ribosylhydrolase; A.H.F. cloned and purified mMacro1 and mMacro2; R.I. performed the HPLC analysis; H.C.W. compared the macro sequences; A.C., P.O.H., B.L. and M.O.H. supervised the work. B.L. and M.O.H. wrote the manuscript.
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Rosenthal, F., Feijs, K., Frugier, E. et al. Macrodomain-containing proteins are new mono-ADP-ribosylhydrolases. Nat Struct Mol Biol 20, 502–507 (2013). https://doi.org/10.1038/nsmb.2521
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DOI: https://doi.org/10.1038/nsmb.2521
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