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      Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349

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          Abstract

          TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca 2+-permeable non-selective cation channel activated by the binding of adenosine 5′-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5′-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.

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          Most cited references 34

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          Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches

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            Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

            Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism.
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              ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology.

              Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.
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                Author and article information

                Journal
                Biochem J
                Biochem. J
                ppbiochemj
                BCJ
                Biochemical Journal
                Portland Press Ltd.
                0264-6021
                1470-8728
                1 July 2017
                17 May 2017
                16 June 2017
                : 474
                : 13
                : 2159-2175
                Affiliations
                [1 ]The Calcium Signalling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
                [2 ]Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, U.K.
                [3 ]Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
                Author notes
                Correspondence: Andreas H. Guse ( guse@ 123456uke.de )
                Article
                BCJ-2017-0091
                10.1042/BCJ20170091
                5473349
                28515263
                © 2017 The Author(s)

                This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).

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