Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2'-phosphate.
ABSTRACT: Transient receptor potential melastatin 2 (TRPM2), a Ca(2+)-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid-adenine-dinucleotide (NAAD), and NAAD-2'-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified-specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2'-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest.
Project description:Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca(2+) and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H(2)O(2) or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H(2)O(2), cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca(2+) homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca(2+). To identify direct modulators of TRPM2, we have studied the effects of H(2)O(2), AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H(2)O(2) (1 mM) and enzymatically purified cADPR (10 ?M) failed to activate, whereas AMP (200 ?M) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ?50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K(0.5) = 104 and 35 ?M). H(2)O(2), cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.
Project description: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.
Project description:TRPM2 is a Ca2+-permeable cation channel that is specifically activated by adenosine diphosphoribose (ADPR). Channel activation in the plasma membrane leads to Ca2+ influx and has been linked to apoptotic mechanisms. The primary agonist, ADPR, is produced both extra- and intracellularly and causes increases in intracellular calcium concentration ([Ca2+]i), but the mechanisms involved are not understood. Using short interfering RNA and a knockout mouse, we report that TRPM2, in addition to its role as a plasma membrane channel, also functions as a Ca2+-release channel activated by intracellular ADPR in a lysosomal compartment. We show that both functions of TRPM2 are critically linked to hydrogen peroxide-induced beta cell death. Additionally, extracellular ADPR production by the ectoenzyme CD38 from its substrates NAD+ (nicotinamide adenine dinucleotide) or cADPR causes IP3-dependent Ca2+ release via P2Y and adenosine receptors. Thus, ADPR and TRPM2 represent multimodal signaling elements regulating Ca2+ mobilization in beta cells through membrane depolarization, Ca2+ influx, and release of Ca2+ from intracellular stores.
Project description:Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as an electron carrier in cellular metabolism and plays a crucial role in the maintenance of balanced redox homeostasis. Quantification of NAD+:NADH and NADP+:NADPH ratios are pivotal to a wide variety of cellular processes, including intracellular secondary messenger signaling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase (PARP), epigenetic regulation of gene expression by NAD-dependent histone deacetylase enzymes known as sirtuins, and regulation of the oxidative pentose phosphate pathway. We quantified changes in the NAD+ metabolome in plasma samples collected from consenting healthy human subjects across a wide age range (20-87 years) using liquid chromatography coupled to tandem mass spectrometry. Our data show a significant decline in the plasma levels of NAD+, NADP+, and other important metabolites such as nicotinic acid adenine dinucleotide (NAAD) with age. However, an age-related increase in the reduced form of NAD+ and NADP+-NADH and NADPH-and nicotinamide (NAM), N-methyl-nicotinamide (MeNAM), and the products of adenosine diphosphoribosylation, including adenosine diphosphate ribose (ADPR) was also reported. Whereas, plasma levels of nicotinic acid (NA), nicotinamide mononucleotide (NMN), and nicotinic acid mononucleotide (NAMN) showed no statistically significant changes across age groups. Taken together, our data cumulatively suggest that age-related impairments are associated with corresponding alterations in the extracellular plasma NAD+ metabolome. Our future research will seek to elucidate the role of modulating NAD+ metabolites in the treatment and prevention of age-related diseases.
Project description:Adenosine 5'-diphosphate ribose (ADPR) is an intracellular signalling molecule generated from nicotinamide adenine dinucleotide (NAD+). Synthetic ADPR analogues can shed light on the mechanism of activation of ADPR targets and their downstream effects. Such chemical biology studies, however, are often challenging due to the negatively charged pyrophosphate, also sensitive to cellular pyrophosphatases, and prior work on an initial ADPR target, the transient receptor potential cation channel TRPM2, showed complete pyrophosphate group replacement to be a step too far in maintaining biological activity. Thus, we designed ADPR analogues with just one of the negatively charged phosphate groups removed, by employing a phosphonoacetate linker. Synthesis of two novel phosphonoacetate ADPR analogues is described via tandem N,N'-dicyclohexylcarbodiimide coupling to phosphonoacetic acid. Neither analogue, however, showed significant agonist or antagonist activity towards TRPM2, underlining the importance of a complete pyrophosphate motif in activation of this particular receptor.
Project description:ADP-ribosyl cyclases are ubiquitous enzymes responsible for synthesis from NAD(+) of the intracellular calcium-releasing signal molecules cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP(+)). Here, we show that cyclases from lower and higher Metazoa also synthesize three adenylic dinucleotides from cADPR and adenine: diadenosine diphosphate and two isomers thereof. These dinucleotides are present and metabolized in mammalian cells and affect intracellular calcium and cell proliferation. The diadenosine diphosphate isomers are naturally occurring nucleotides containing an N-glycosidic bond different from the usual C1'-N9. The identification of these members of the family of NAD(+)-derived, calcium-active nucleotides opens new areas of investigation into their functional cooperation with cADPR and NAADP(+) and into their involvement in the physiology and pathology of calcium-controlled cell functions.
Project description:Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that ¹H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.
Project description:The Ca(2+)-permeable TRPM2 channel is a dual function protein that is activated by intracellular ADPR through its enzymatic pyrophosphatase domain with Ca(2+) acting as a co-factor. Other TRPM2 regulators include cADPR, NAADP and H(2)O(2), which synergize with ADPR to potentiate TRPM2 activation. Although TRPM2 has been thoroughly characterized in overexpression or cell-line systems, little is known about the features of TRPM2 in primary cells. We here characterize the regulation of TRPM2 activation in human neutrophils and report that ADPR activates TRPM2 with an effective half-maximal concentration (EC(50)) of 1microM. Potentiation by Ca(2+) is dose-dependent with an EC(50) of 300nM. Both cADPR and NAADP activate TRPM2, albeit with lower efficacy than in the presence of subthreshold levels of ADPR (100nM), which significantly shifts the EC(50) for cADPR from 44 to 3muM and for NAADP from 95 to 1microM. TRPM2 activation by ADPR can be suppressed by AMP with an IC(50) of 10microM and cADPR-induced activation can be blocked by 8-Bromo-cADPR. We further show that 100microM H(2)O(2) enables subthreshold concentrations of ADPR (100nM) to activate TRPM2. We conclude that agonistic and antagonistic characteristics of TRPM2 as seen in overexpression systems are largely compatible with the functional properties of TRPM2 currents measured in human neutrophils, but the potencies of agonists in primary cells are significantly higher.
Project description:Bacterial NadM-Nudix is a bifunctional enzyme containing a nicotinamide mononucleotide (NMN) adenylyltransferase and an ADP-ribose (ADPR) pyrophosphatase domain. While most members of this enzyme family, such as that from a model cyanobacterium Synechocystis sp., are involved primarily in nicotinamide adenine dinucleotide (NAD) salvage/recycling pathways, its close homolog in a category-A biodefense pathogen, Francisella tularensis, likely plays a central role in a recently discovered novel pathway of NAD de novo synthesis. The crystal structures of NadM-Nudix from both species, including their complexes with various ligands and catalytic metal ions, revealed detailed configurations of the substrate binding and catalytic sites in both domains. The structure of the N-terminal NadM domain may be exploited for designing new antitularemia therapeutics. The ADPR binding site in the C-terminal Nudix domain is substantially different from that of Escherichia coli ADPR pyrophosphatase, and is more similar to human NUDT9. The latter observation provided new insights into the ligand binding mode of ADPR-gated Ca2+ channel TRPM2.
Project description:Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a newly described Ca2+-mobilizing nucleotide that appears to target intracellular Ca2+-release channels distinct from those sensitive to inositol trisphosphate or ryanodine/cyclic ADP-ribose. Little, however, is known concerning the regulation of cellular NAADP levels. In the present study, we have characterized the metabolism of NAADP by brain membranes. From HPLC and MS analyses we show that loss of NAADP was associated with the appearance of a major product that is likely to be nicotinic acid-adenine dinucleotide (NAAD), the dephosphorylated form of NAADP. Dephosphorylation of NAADP, but not 3'-NAADP, was dramatically attenuated by Ca2+ chelators and stimulated by Ca2+ over a physiological range in a calmodulin-insensitive manner. In contrast, NADP was metabolized predominantly to ADP-ribose phosphate via glycohydrolase activity, although slower Ca2+-dependent dephosphorylation of both NADP and 2'-AMP could also be demonstrated. This is the first report describing a Ca2+-regulated 2'-specific phosphatase which is probably the major pathway for the inactivation of NAADP in brain. Our data provide a potential feedback mechanism for limiting NAADP-induced Ca2+ release within cells through stimulation of NAADP metabolism by Ca2+ and strongly support a signalling role for this novel nucleotide in the brain.