Abstract
Mono-ADP-ribosylation is a posttranslational modification, which is catalyzed in cells by certain members of the ADP-ribosyltransferase diphtheria toxin-like family (ARTD) of ADP-ribosyltransferases (aka PARP enzymes). It involves the transfer of a single residue of ADP-ribose (ADPr) from the cofactor NAD+ onto substrate proteins. Although 12 of the 17 members of the ARTD family have been defined as mono-ARTDs in in vitro assays, relatively little is known about their exact cellular functions. A major challenge is the detection of mono-ADP-ribosylated (MARylated) proteins in cells as no antibodies are available that detect exclusively MARylated proteins. As an alternative to classical antibodies, the MAR-specific binding domains macro2 and macro3 of Artd8 can be utilized alone or in combination, to demonstrate intracellular auto-modification levels of ARTD10 in cells in both co-immunoprecipitation and co-localization experiments. Here we demonstrate that different macrodomain constructs of human ARTD8 and murine Artd8, alone or in combination, exert differences with regard to their interaction with ARTD10 in cells. Precisely, while the macrodomains of murine Artd8 interacted with ARTD10 in cells in a MARylation-dependent manner, the macrodomains of human ARTD8 interacted with ARTD10 independent of its catalytic activity. Moreover, we show that a combination of macro2 and macro3 of murine Artd8 was recruited more efficiently to ARTD10 during co-localization experiments compared to the single domains. Therefore, murine Artd8 macrodomain constructs can serve as a tool to evaluate intracellular ARTD10 auto-modification levels using the described methods, while the human ARTD8 macrodomains are less suited because of ADPr-independent binding to ARTD10. Protocols for co-immunoprecipitation and co-localization experiments are described in detail.
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References
Hottiger MO (2015) Nuclear ADP-ribosylation and its role in chromatin plasticity, cell differentiation, and epigenetics. Annu Rev Biochem 84:227–263. https://doi.org/10.1146/annurev-biochem-060614-034506
Hottiger MO, Hassa PO, Luscher B, Schuler H, Koch-Nolte F (2010) Toward a unified nomenclature for mammalian ADP-ribosyltransferases. Trends Biochem Sci 35(4):208–219. https://doi.org/10.1016/j.tibs.2009.12.003
Vyas S, Matic I, Uchima L, Rood J, Zaja R, Hay RT, Ahel I, Chang P (2014) Family-wide analysis of poly(ADP-ribose) polymerase activity. Nat Commun 5:4426. https://doi.org/10.1038/ncomms5426
Loseva O, Jemth AS, Bryant HE, Schuler H, Lehtio L, Karlberg T, Helleday T (2010) PARP-3 is a mono-ADP-ribosylase that activates PARP-1 in the absence of DNA. J Biol Chem 285(11):8054–8060. https://doi.org/10.1074/jbc.M109.077834
Kickhoefer VA, Siva AC, Kedersha NL, Inman EM, Ruland C, Streuli M, Rome LH (1999) The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase. J Cell Biol 146(5):917–928
Yang CS, Jividen K, Spencer A, Dworak N, Ni L, Oostdyk LT, Chatterjee M, Kusmider B, Reon B, Parlak M, Gorbunova V, Abbas T, Jeffery E, Sherman NE, Paschal BM (2017) Ubiquitin modification by the E3 Ligase/ADP-ribosyltransferase Dtx3L/Parp9. Mol Cell 66(4):503–516.e505. https://doi.org/10.1016/j.molcel.2017.04.028
Kleine H, Poreba E, Lesniewicz K, Hassa PO, Hottiger MO, Litchfield DW, Shilton BH, Luscher B (2008) Substrate-assisted catalysis by PARP10 limits its activity to mono-ADP-ribosylation. Mol Cell 32(1):57–69. https://doi.org/10.1016/j.molcel.2008.08.009
Yu M, Schreek S, Cerni C, Schamberger C, Lesniewicz K, Poreba E, Vervoorts J, Walsemann G, Grotzinger J, Kremmer E, Mehraein Y, Mertsching J, Kraft R, Austen M, Luscher-Firzlaff J, Luscher B (2005) PARP-10, a novel Myc-interacting protein with poly(ADP-ribose) polymerase activity, inhibits transformation. Oncogene 24(12):1982–1993. https://doi.org/10.1038/sj.onc.1208410
Feijs KL, Kleine H, Braczynski A, Forst AH, Herzog N, Verheugd P, Linzen U, Kremmer E, Luscher B (2013) ARTD10 substrate identification on protein microarrays: regulation of GSK3beta by mono-ADP-ribosylation. Cell Commun Signal 11(1):5. https://doi.org/10.1186/1478-811x-11-5
Verheugd P, Forst AH, Milke L, Herzog N, Feijs KL, Kremmer E, Kleine H, Luscher B (2013) Regulation of NF-kappaB signalling by the mono-ADP-ribosyltransferase ARTD10. Nat Commun 4:1683. https://doi.org/10.1038/ncomms2672
Eckei L, Krieg S, Butepage M, Lehmann A, Gross A, Lippok B, Grimm AR, Kummerer BM, Rossetti G, Luscher B, Verheugd P (2017) The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases. Sci Rep 7:41746. https://doi.org/10.1038/srep41746
Atasheva S, Frolova EI, Frolov I (2014) Interferon-stimulated poly(ADP-ribose) polymerases are potent inhibitors of cellular translation and virus replication. J Virol 88(4):2116–2130. https://doi.org/10.1128/JVI.03443-13
Herzog N, Hartkamp JD, Verheugd P, Treude F, Forst AH, Feijs KL, Lippok BE, Kremmer E, Kleine H, Luscher B (2013) Caspase-dependent cleavage of the mono-ADP-ribosyltransferase ARTD10 interferes with its pro-apoptotic function. FEBS J 280(5):1330–1343. https://doi.org/10.1111/febs.12124
Kaufmann M, Feijs KL, Luscher B (2015) Function and regulation of the mono-ADP-ribosyltransferase ARTD10. Curr Top Microbiol Immunol 384:167–188. https://doi.org/10.1007/82_2014_379
Feijs KL, Forst AH, Verheugd P, Luscher B (2013) Macrodomain-containing proteins: regulating new intracellular functions of mono(ADP-ribosyl)ation. Nat Rev Mol Cell Biol 14(7):443–451. https://doi.org/10.1038/nrm3601
Verheugd P, Butepage M, Eckei L, Luscher B (2016) Players in ADP-ribosylation: readers and erasers. Curr Protein Pept Sci 17(7):654–667
Forst AH, Karlberg T, Herzog N, Thorsell AG, Gross A, Feijs KL, Verheugd P, Kursula P, Nijmeijer B, Kremmer E, Kleine H, Ladurner AG, Schuler H, Luscher B (2013) Recognition of mono-ADP-ribosylated ARTD10 substrates by ARTD8 macrodomains. Structure 21(3):462–475. https://doi.org/10.1016/j.str.2012.12.019
Kleine H, Herrmann A, Lamark T, Forst AH, Verheugd P, Luscher-Firzlaff J, Lippok B, Feijs KL, Herzog N, Kremmer E, Johansen T, Muller-Newen G, Luscher B (2012) Dynamic subcellular localization of the mono-ADP-ribosyltransferase ARTD10 and interaction with the ubiquitin receptor p62. Cell Commun Signal 10(1):28. https://doi.org/10.1186/1478-811x-10-28
Caliandro R, Rossetti G, Carloni P (2012) Local fluctuations and conformational transitions in proteins. J Chem Theory Comput 8(11):4775–4785. https://doi.org/10.1021/ct300610y
Acknowledgments
We thank G. Müller-Newen and the core facility “Immunohistochemistry and Confocal Microscopy Facility” of the Medical School of the RWTH Aachen University for support. Our work was supported by grants from the German Science Foundation (DFG LU 466/16-1), the IZKF Aachen (O2-1-2014) of the Medical School of the RWTH Aachen University, the Start-Up program of the Excellence Initiative of the RWTH Aachen University (StUpPD_119_13), and by the START program of the Faculty of Medicine, RWTH Aachen University (117/15 and 121/17).
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Bütepage, M. et al. (2018). Assessment of Intracellular Auto-Modification Levels of ARTD10 Using Mono-ADP-Ribose-Specific Macrodomains 2 and 3 of Murine Artd8. In: Chang, P. (eds) ADP-ribosylation and NAD+ Utilizing Enzymes. Methods in Molecular Biology, vol 1813. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8588-3_4
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DOI: https://doi.org/10.1007/978-1-4939-8588-3_4
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