Alternative splicing of the n-terminal cytosolic and transmembrane domains of P2X7 controls gating of the ion channel by ADP-ribosylation

P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP

ADP-ribosylation of arginine

Arginine adenosine-5′-diphosphoribosylation (ADP-ribosylation) is an enzyme-catalyzed, potentially reversible posttranslational modification, in which the ADP-ribose moiety is transferred from NAD+ to the guanidino moiety of arginine. At 540 Da, ADP-ribose has the size of approximately five amino acid residues. In contrast to arginine, which, at neutral pH, is positively charged, ADP-ribose carries two negatively charged phosphate moieties. Arginine ADP-ribosylation, thus, causes a notable change in size and chemical property at the ADP-ribosylation site of the target protein. Often, this causes steric interference of the interaction of the target protein with binding partners, e.g. toxin-catalyzed ADP-ribosylation of actin at R177 sterically blocks actin polymerization. In case of the nucleotide-gated P2X7 ion channel, ADP-ribosylation at R125 in the vicinity of the ligand-binding site causes channel gating. Arginine-specific ADP-ribosyltransferases (ARTs) carry a characteristic R-S-EXE motif that distinguishes these enzymes from structurally related enzymes which catalyze ADP-ribosylation of other amino acid side chains, DNA, or small molecules. Arginine-specific ADP-ribosylation can be inhibited by small molecule arginine analogues such as agmatine or meta-iodobenzylguanidine (MIBG), which themselves can serve as targets for arginine-specific ARTs. ADP-ribosylarginine specific hydrolases (ARHs) can restore target protein function by hydrolytic removal of the entire ADP-ribose moiety. In some cases, ADP-ribosylarginine is processed into secondary posttranslational modifications, e.g. phosphoribosylarginine or ornithine. This review summarizes current knowledge on arginine-specific ADP-ribosylation, focussing on the methods available for its detection, its biological consequences, and the enzymes responsible for this modification and its reversal, and discusses future perspectives for research in this field

Immuno-targeting the multifunctional CD38 using nanobody

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Extracellular NAD and ATP: Partners in immune cell modulation

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as ‘danger signals–to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response

Activation of the P2X7 ion channel by soluble and covalently bound ligands

The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7

Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation

International audienceIon channels are desirable therapeutic targets, yet ion channel-directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies-small, single-domain antibody fragments-may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5′-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1b (IL-1b). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1b release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeu-tics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7

Surrogate/spent fuel sabotage : aerosol ratio test program and Phase 2 test results.

A multinational test program is in progress to quantify the aerosol particulates produced when a high energy density device, HEDD, impacts surrogate material and actual spent fuel test rodlets. This program provides needed data that are relevant to some sabotage scenarios in relation to spent fuel transport and storage casks, and associated risk assessments; the program also provides significant political benefits in international cooperation. We are quantifying the spent fuel ratio, SFR, the ratio of the aerosol particles released from HEDD-impacted actual spent fuel to the aerosol particles produced from surrogate materials, measured under closely matched test conditions. In addition, we are measuring the amounts, nuclide content, size distribution of the released aerosol materials, and enhanced sorption of volatile fission product nuclides onto specific aerosol particle size fractions. These data are crucial for predicting radiological impacts. This document includes a thorough description of the test program, including the current, detailed test plan, concept and design, plus a description of all test components, and requirements for future components and related nuclear facility needs. It also serves as a program status report as of the end of FY 2003. All available test results, observations, and analyses - primarily for surrogate material Phase 2 tests using cerium oxide sintered ceramic pellets are included. This spent fuel sabotage - aerosol test program is coordinated with the international Working Group for Sabotage Concerns of Transport and Storage Casks, WGSTSC, and supported by both the U.S. Department of Energy and Nuclear Regulatory Commission

DNA immunization as a technology platform for monoclonal antibody induction

To combat the threat of many emerging infectious diseases, DNA immunization offers a unique and powerful approach to the production of high-quality monoclonal antibodies (mAbs) against various pathogens. Compared with traditional protein-based immunization approaches, DNA immunization is efficient for testing novel immunogen designs, does not require the production or purification of proteins from a pathogen or the use of recombinant protein technology and is effective at generating mAbs against conformation-sensitive targets. Although significant progress in the use of DNA immunization to generate mAbs has been made over the last two decades, the literature does not contain an updated summary of this experience. The current review provides a comprehensive analysis of the literature, including our own work, describing the use of DNA immunization to produce highly functional mAbs, in particular, those against emerging infectious diseases. Critical factors such as immunogen design, delivery approach, immunization schedule, use of immune modulators and the role of final boost immunization are discussed in detail

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis

Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line- or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects

CD38 Exacerbates Focal Cytokine Production, Postischemic Inflammation and Brain Injury after Focal Cerebral Ischemia

BACKGROUND: Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion. METHODOLOGY/PRINCIPAL FINDINGS: We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8(+) cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model. CONCLUSION/SIGNIFICANCE: CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke