Abstract
Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ entry and, consequently, apoptosis. To date, adenosine 5′-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2′-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2′-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2′-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2′-deoxy-ATP in vitro. Thus, 2′-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.
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Acknowledgements
This study was supported by the Deutsche Forschungsgemeinschaft (GU 360/16-1 to A.H.G.), the Wellcome Trust (Project Grant 084068 to B.V.L.P. and A.H.G.; Programme Grant 082837 to B.V.L.P.) and Landesforschungsförderung Hamburg (Research Group ReAd Me to A.H.G.). B.V.L.P. is a Wellcome Trust Senior Investigator (grant 101010). Wild-type HEK293 cells were kindly provided by M. Jücker (Department of Biochemistry and Signal Transduction, University Medical Centre Hamburg–Eppendorf, Hamburg, Germany). Expression plasmids pX330-Puro-T2A-hCas9 and pCAG-EGxxFP were kindly provided by A. Flügel (Department of Neuroimmunology, University Medical Center Göttingen, Göttingen, Germany).
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A.H.G., B.V.L.P. and R.F. designed the study and individual experiments. C.M. and J.M.W. synthesized and purified the ADPR analogs. T.K., R.F. and M.D.R. performed electrophysiological characterization of ADPR analogs. M.D.R. carried out the single-channel recordings. A.-M.W.P. and A.B. performed the enzyme assays with NMNAT and CD38. R.W. analyzed nucleotide products from poly-ADP ribosylated proteins. R.F. and A.H. prepared TRPM2 T5L and CD38 expression vectors and generated the TRPM2 T5L cell line. A.B., A.-M.W.P. and M.J. established the HPLC method for the determination of endogenous 2′-deoxy-ADPR. A.B. and M.J. quantitatively analyzed endogenous nucleotides. A.B. determined substrate saturation plots for NMNAT-2 and sCD38. J.M.W. carried out high-resolution mass spectrometry analysis of 2′-deoxy-ADPR and 2′-deoxy-NAD. V.W. generated the CD38−/− Jurkat cell line and produced and purified soluble recombinant CD38. M.F. characterized the CD38−/− Jurkat cell line. A.R., A.B. and F.G. determined the activity of CD38 in type III orientation. All authors wrote the manuscript.
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Fliegert, R., Bauche, A., Wolf Pérez, AM. et al. 2′-Deoxyadenosine 5′-diphosphoribose is an endogenous TRPM2 superagonist. Nat Chem Biol 13, 1036–1044 (2017). https://doi.org/10.1038/nchembio.2415
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DOI: https://doi.org/10.1038/nchembio.2415
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