Abstract
Despite substantial progress in ADP-ribosylation research in recent years, the identification of ADP-ribosylated proteins, their ADP-ribose acceptors sites, and the respective writers and erasers remains challenging. The use of recently developed mass spectrometric methods helps to further characterize the ADP-ribosylome and its regulatory enzymes under different conditions and in different cell types. Validation of these findings may be achieved by in vitro assays for the respective enzymes. In the below method, we describe how recombinant ADP-ribosylated proteins are demodified in vitro with mono-ADP-ribosylhydrolases of choice to elucidate substrate and potentially also site specificity of these enzymes.
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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
Luo X, Kraus WL (2012) On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1. Genes Dev 26(5):417–432
Barkauskaite E, Jankevicius G, Ahel I (2015) Structures and mechanisms of enzymes employed in the synthesis and degradation of PARP-dependent protein ADP-ribosylation. Mol Cell 58(6):935–946
Caldecott KW (2014) Protein ADP-ribosylation and the cellular response to DNA strand breaks. DNA Repair 19:108–113
Butepage M et al (2015) Intracellular mono-ADP-ribosylation in signaling and disease. Cell 4(4):569–595
Laing S et al (2011) ADP-ribosylation of arginine. Amino Acids 41(2):257–269
Du J, Jiang H, Lin H (2009) Investigating the ADP-ribosyltransferase activity of sirtuins with NAD analogues and 32P-NAD. Biochemistry 48(13):2878–2890
Pan PW et al (2011) Structure and biochemical functions of SIRT6. J Biol Chem 286(16):14575–14587
Ueda K et al (1972) Poly ADP-ribose glycohydrolase from rat liver nuclei, a novel enzyme degrading the polymer. Biochem Biophys Res Commun 46(2):516–523
Oka S, Kato J, Moss J (2006) Identification and characterization of a mammalian 39-kDa poly(ADP-ribose) glycohydrolase. J Biol Chem 281(2):705–713
Moss J, Jacobson MK, Stanley SJ (1985) Reversibility of arginine-specific mono(ADP-ribosyl)ation: identification in erythrocytes of an ADP-ribose-L-arginine cleavage enzyme. Proc Natl Acad Sci U S A 82(17):5603–5607
Kato J et al (2011) ADP-ribosylarginine hydrolase regulates cell proliferation and tumorigenesis. Cancer Res 71(15):5327–5335
Jankevicius G et al (2013) A family of macrodomain proteins reverses cellular mono-ADP-ribosylation. Nat Struct Mol Biol 20(4):508
Rosenthal F et al (2013) Macrodomain-containing proteins are new mono-ADP-ribosylhydrolases. Nat Struct Mol Biol 20(4):502–507
Sharifi R et al (2013) Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease. EMBO J 32(9):1225–1237
Xi HQ, Zhao P, Han WD (2010) Clinicopathological significance and prognostic value of LRP16 expression in colorectal carcinoma. World J Gastroenterol 16(13):1644–1648
Mohseni M et al (2014) MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers. Proc Natl Acad Sci U S A 111(49):17606–17611
Zang L et al (2013) Identification of LRP16 as a negative regulator of insulin action and adipogenesis in 3T3-L1 adipocytes. Horm Metab Res 45(5):349–358
Han WD et al (2007) Estrogenically regulated LRP16 interacts with estrogen receptor alpha and enhances the receptor’s transcriptional activity. Endocr Relat Cancer 14(3):741–753
Yang J et al (2009) The single-macro domain protein LRP16 is an essential cofactor of androgen receptor. Endocr Relat Cancer 16(1):139–153
Wu Z et al (2011) LRP16 integrates into NF-kappaB transcriptional complex and is required for its functional activation. PLoS One 6(3):e18157
Abplanalp J et al (2017) Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase. Nat Commun 8(1):2055
Fontana P et al (2017) Serine ADP-ribosylation reversal by the hydrolase ARH3. elife 6:e28533
Martello R et al (2016) Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue. Nat Commun 7:12917
Bilan V et al (2017) Combining higher-energy collision dissociation and electron-transfer/higher-energy collision dissociation fragmentation in a product-dependent manner confidently assigns proteomewide ADP-ribose acceptor sites. Anal Chem 89(3):1523–1530
Leidecker O et al (2016) Serine is a new target residue for endogenous ADP-ribosylation on histones. Nat Chem Biol 12(12):998
Acknowledgments
The authors would like to thank Tobias Suter (University of Zurich) for providing editorial assistance and critical input during manuscript writing. Work on ADP-ribosyltransferases and hydrolases in the laboratory of MOH is supported by the Kanton of Zurich and the Swiss National Science Foundation (SNF 310030_157019 and 31003A_176177).
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Abplanalp, J., Hopp, AK., Hottiger, M.O. (2018). Mono-ADP-Ribosylhydrolase Assays. 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_13
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DOI: https://doi.org/10.1007/978-1-4939-8588-3_13
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