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2′-Deoxyadenosine 5′-diphosphoribose is an endogenous TRPM2 superagonist

Nature Chemical Biology volume 13, pages 1036–1044 (2017)Cite this article

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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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Figure 1: ADPR analogs activated TRPM2 in whole-cell patch clamp experiments.
Figure 2: Modifications of adenosine ribose affect the ability of ADPR to activate TRPM2.
Figure 3: The effect of 2′-deoxy-ADPR on hTRPM2 in excised inside-out patches.
Figure 4: NMNAT-2 and CD38 synthesize 2′-deoxy-ADPR in vitro.
Figure 5: The increase in 2′-deoxy-ADPR in Jurkat cells exposed to hydrogen peroxide depends on CD38.
Figure 6: The concentration of 2′-deoxy ADPR in Jurkat cells increased after exposure to hydrogen peroxide.

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References

  1. Perraud, A.L. et al. ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology. Nature 411, 595–599 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Knowles, H., Li, Y. & Perraud, A.L. The TRPM2 ion channel, an oxidative stress and metabolic sensor regulating innate immunity and inflammation. Immunol. Res. 55, 241–248 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Faouzi, M. & Penner, R. in Mammalian Transient Receptor Potential (TRP) Cation Channels Vol. 222 (eds. Nilius, B. & Flockerzi, V.) 403–426 (Springer, 2014).

  4. Gasser, A. et al. Activation of T cell calcium influx by the second messenger ADP-ribose. J. Biol. Chem. 281, 2489–2496 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Buelow, B., Song, Y. & Scharenberg, A.M. The Poly(ADP-ribose) polymerase PARP-1 is required for oxidative stress-induced TRPM2 activation in lymphocytes. J. Biol. Chem. 283, 24571–24583 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Partida-Sanchez, S. et al. Chemotaxis of mouse bone marrow neutrophils and dendritic cells is controlled by ADP-ribose, the major product generated by the CD38 enzyme reaction. J. Immunol. 179, 7827–7839 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Uchida, K. et al. Lack of TRPM2 impaired insulin secretion and glucose metabolisms in mice. Diabetes 60, 119–126 (2011).

    Article  CAS  PubMed  Google Scholar 

  8. Song, K. et al. The TRPM2 channel is a hypothalamic heat sensor that limits fever and can drive hypothermia. Science 353, 1393–1398 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tan, C.-H. & McNaughton, P.A. The TRPM2 ion channel is required for sensitivity to warmth. Nature 536, 460–463 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wehage, E. et al. Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose. J. Biol. Chem. 277, 23150–23156 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Du, J., Xie, J. & Yue, L. Intracellular calcium activates TRPM2 and its alternative spliced isoforms. Proc. Natl. Acad. Sci. USA 106, 7239–7244 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Grubisha, O. et al. Metabolite of SIR2 reaction modulates TRPM2 ion channel. J. Biol. Chem. 281, 14057–14065 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Moreau, C. et al. Structure-activity relationship of adenosine 5′-diphosphoribose at the transient receptor potential melastatin 2 (TRPM2) channel: rational design of antagonists. J. Med. Chem. 56, 10079–10102 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tóth, B., Iordanov, I. & Csanády, L. Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2′-phosphate. J. Gen. Physiol. 145, 419–430 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Sano, Y. et al. Immunocyte Ca2+ influx system mediated by LTRPC2. Science 293, 1327–1330 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Beck, A., Kolisek, M., Bagley, L.A., Fleig, A. & Penner, R. Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes. FASEB J. 20, 962–964 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Klumpp, D. et al. Targeting TRPM2 channels impairs radiation-induced cell cycle arrest and fosters cell death of T cell leukemia cells in a Bcl-2-dependent manner. Oxid. Med. Cell. Longev. 2016, 8026702 (2016).

    Article  PubMed  CAS  Google Scholar 

  18. Brown, L.A., Ihara, M., Buckingham, S.D., Matsuda, K. & Sattelle, D.B. Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors. J. Neurochem. 99, 608–615 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Mortensen, M., Ebert, B., Wafford, K. & Smart, T.G. Distinct activities of GABA agonists at synaptic- and extrasynaptic-type GABAA receptors. J. Physiol. (Lond.) 588, 1251–1268 (2010).

    Article  CAS  Google Scholar 

  20. Tóth, B. & Csanády, L. Pore collapse underlies irreversible inactivation of TRPM2 cation channel currents. Proc. Natl. Acad. Sci. USA 109, 13440–13445 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Csanády, L. & Törocsik, B. Four Ca2+ ions activate TRPM2 channels by binding in deep crevices near the pore but intracellularly of the gate. J. Gen. Physiol. 133, 189–203 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Klenow, H. & Andersen, B. Some enzyme reactions with adenine deoxyriboside polyphosphates. Biochim. Biophys. Acta 23, 92–97 (1957).

    Article  CAS  PubMed  Google Scholar 

  23. Emanuelli, M. et al. Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase. J. Biol. Chem. 276, 406–412 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Di Stefano, M. & Conforti, L. Diversification of NAD biological role: the importance of location. FEBS J. 280, 4711–4728 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Suhadolnik, R.J. et al. ADP-ribosylation of isolated nuclei from HeLa cells, rat liver, fetal rat liver, and Novikoff rat hepatoma: effect of nicotinamide adenine dinucleotide analogs on template activity for DNA synthesis, incorporation into nuclear proteins, and a new 1″→3′ osidic linkage. J. Biol. Chem. 252, 4134–4144 (1977).

    CAS  PubMed  Google Scholar 

  26. Bakondi, E. et al. Detection of poly(ADP-ribose) polymerase activation in oxidatively stressed cells and tissues using biotinylated NAD substrate. J. Histochem. Cytochem. 50, 91–98 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Zhao, Y.J., Lam, C.M.C. & Lee, H.C. The membrane-bound enzyme CD38 exists in two opposing orientations. Sci. Signal. 5, ra67 (2012).

    Article  PubMed  CAS  Google Scholar 

  28. Muller-Steffner, H.M., Malver, O., Hosie, L., Oppenheimer, N.J. & Schuber, F. Slow-binding inhibition of NAD+ glycohydrolase by arabino analogues of β-NAD. J. Biol. Chem. 267, 9606–9611 (1992).

    CAS  PubMed  Google Scholar 

  29. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hara, Y. et al. LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol. Cell 9, 163–173 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Heiner, I. et al. Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD. Biochem. J. 371, 1045–1053 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fonfria, E. et al. TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase. Br. J. Pharmacol. 143, 186–192 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Rosenbluth, M.J., Lam, W.A. & Fletcher, D.A. Force microscopy of nonadherent cells: a comparison of leukemia cell deformability. Biophys. J. 90, 2994–3003 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ashamu, G.A., Sethi, J.K., Galione, A. & Potter, B.V.L. Roles for adenosine ribose hydroxyl groups in cyclic adenosine 5′-diphosphate ribose-mediated Ca2+ release. Biochemistry 36, 9509–9517 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Moreau, C. et al. Synthesis of cyclic adenosine 5′-diphosphate ribose analogues: a C2′ endo/syn “southern” ribose conformation underlies activity at the sea urchin cADPR receptor. Org. Biomol. Chem. 9, 278–290 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Del Nagro, C., Xiao, Y., Rangell, L., Reichelt, M. & O'Brien, T. Depletion of the central metabolite NAD leads to oncosis-mediated cell death. J. Biol. Chem. 289, 35182–35192 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dahmen, W., Webb, B. & Preiss, J. The deamido-diphosphopyridine nucleotide and diphosphopyridine nucleotide pyrophosphorylases of Escherichia coli and yeast. Arch. Biochem. Biophys. 120, 440–450 (1967).

    Article  CAS  PubMed  Google Scholar 

  39. Suhadolnik, R.J., Lennon, M.B., Uematsu, T., Monahan, J.E. & Baur, R. Role of adenine ring and adenine ribose of nicotinamide adenine dinucleotide in binding and catalysis with alcohol, lactate, and glyceraldehyde-3-phosphate dehydrogenases. J. Biol. Chem. 252, 4125–4133 (1977).

    CAS  PubMed  Google Scholar 

  40. Schuber, F., Pascal, M. & Travo, P. Calf-spleen nicotinamide-adenine dinucleotide glycohydrolase. Properties of the active site. Eur. J. Biochem. 83, 205–214 (1978).

    Article  CAS  PubMed  Google Scholar 

  41. Xie, Y.F., Macdonald, J.F. & Jackson, M.F. TRPM2, calcium and neurodegenerative diseases. Int. J. Physiol. Pathophysiol. Pharmacol. 2, 95–103 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Fonfria, E. et al. TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia. J. Recept. Signal Transduct. Res. 26, 179–198 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Yang, K.-T. et al. Activation of the transient receptor potential M2 channel and poly(ADP-ribose) polymerase is involved in oxidative stress-induced cardiomyocyte death. Cell Death Differ. 13, 1815–1826 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Zhang, B. et al. 2′-deoxy cyclic adenosine 5′-diphosphate ribose derivatives: importance of the 2′-hydroxyl motif for the antagonistic activity of 8-substituted cADPR derivatives. J. Med. Chem. 51, 1623–1636 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Kirchberger, T. et al. 8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist. Biochem. J. 422, 139–149 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. He, B. & Soderlund, D.M. Human embryonic kidney (HEK293) cells express endogenous voltage-gated sodium currents and Na v 1.7 sodium channels. Neurosci. Lett. 469, 268–272 (2010).

    Article  CAS  PubMed  Google Scholar 

  47. Gagné, J.P. et al. Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress. Nucleic Acids Res. 40, 7788–7805 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Bobalova, J., Bobal, P. & Mutafova-Yambolieva, V.N. High-performance liquid chromatographic technique for detection of a fluorescent analogue of ADP-ribose in isolated blood vessel preparations. Anal. Biochem. 305, 269–276 (2002).

    Article  CAS  PubMed  Google Scholar 

  49. Schmid, F., Fliegert, R., Westphal, T., Bauche, A. & Guse, A.H. Nicotinic acid adenine dinucleotide phosphate (NAADP) degradation by alkaline phosphatase. J. Biol. Chem. 287, 32525–32534 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Schmid, F., Bruhn, S., Weber, K., Mittrücker, H.W. & Guse, A.H. CD38: a NAADP degrading enzyme. FEBS Lett. 585, 3544–3548 (2011).

    Article  CAS  PubMed  Google Scholar 

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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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  1. Barry V L Potter and Andreas H Guse: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Cell Biology, The Calcium Signalling Group, University Medical Centre Hamburg–Eppendorf, Hamburg, Germany

    Ralf Fliegert, Andreas Bauche, Adriana-Michelle Wolf Pérez, Monika D Rozewitz, Riekje Winzer, Mareike Janus, Feng Gu, Annette Rosche, Angelika Harneit, Marianne Flato, Tanja Kirchberger, Valerie Wolters & Andreas H Guse

  2. Department of Pharmacy and Pharmacology, Wolfson Laboratory of Medicinal Chemistry, University of Bath, Bath, UK

    Joanna M Watt, Christelle Moreau & Barry V L Potter

  3. Department of Pharmacology, Medicinal Chemistry & Drug Discovery, University of Oxford, Oxford, UK

    Joanna M Watt & Barry V L Potter

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Contributions

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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Correspondence to Andreas H Guse.

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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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