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Abstract: Endogenous danger signals released during cell damage contribute to alert the immune system. Typically, their release results in the activation and maturation of innate immune cells, and the production of pro-inflammatory cytokines. In addition, extracellular NAD+ stimulates immune responses by hindering regulatory T cells (Tregs), and could, therefore, represent the prototype of a new category of danger signals. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: NAD+ plays central roles in energy metabolism as redox carrier. Recent research has identified important signalling functions of NAD+ that involve its consumption. Although NAD+ is synthesized mainly in the cytosol, nucleus and mitochondria, it has been detected also in vesicular and extracellular compartments. Three protein families that consume NAD+ in signalling reactions have been characterized on a molecular level: ADP-ribosyltransferases (ARTs), Sirtuins (SIRTs), and NAD+ glycohydrolases (NADases). Members of these families serve important regulatory functions in various cellular compartments, e.g., by linking the cellular energy state to gene expression in the nucleus, by regulating nitrogen metabolism in mitochondria, and by sensing tissue damage in the extracellular compartment. Distinct NAD+ pools may be crucial for these processes. Here, we review the current knowledge about the compartmentation and biochemistry of NAD+-converting enzymes that control NAD+ signalling. [Copyright &y& Elsevier]

Inositol 5′-phosphatase SHIP-1 (SHIP) is a negative regulator of signal transduction in haematopoietic cells. SHIP inactivation may be involved in the pathogenesis of leukaemia. An inducible expression system was combined with microarray analysis to identify target genes regulated by SHIP in the human T-cell leukaemia cell line Jurkat. One gene identified was Krüppel-like factor 2 ( KLF2), which was up-regulated two to threefold at the RNA and protein level after the induced expression of SHIP. KLF2, a negative regulator of T cell proliferation, has been implicated in T cell quiescence. KLF2 or SHIP expression in Jurkat cells caused 45% or 60% reduction of proliferation, respectively. SHIP can up-regulate KLF2 expression, implicating KLF2 in the SHIP-mediated growth inhibition of a human leukaemic T-cell line. [ABSTRACT FROM AUTHOR]

Cytotoxicity induced by protein ADP-ribosylation is a common theme of certain bacterial toxins and of the mammalian ectoenzyme ART2. Exposure of T cells to NAD, the substrate for ART2-catalyzed ADP-ribosylation, induces exposure of phosphatidylserine, uptake of propidium iodide, and fragmentation of DNA. ART2-specific antibodies raised by gene gun immunization block NAD-induced apoptosis. ART2 catalyzed ADP-ribosylation of cell membrane proteins induces formation of cytolytic membrane pores by activating the P2X7 purinoceptor. This alternative pathway to T cell apoptosis could be triggered upon the release of NAD from intracellular stores, for example, during inflammatory tissue damage. [ABSTRACT FROM AUTHOR]

Abstract: Mono-ADP-ribosylation of arginine is a protein modification in eukaryotic cells regulating protein activity and thereby influencing signal transduction and metabolism. Due to the complexity of the modification and the fragmentation pattern in MS/MS CID experiments, the identification of ADP-ribosylation sites in complex mixtures is difficult. Here we describe a two-step strategy, in the first step enriching and identifying potentially ADP-ribosylated proteins and in the second step identifying the sites of modification by a combination of LC/MS-, LC/MSE (MS at elevated fragmentation energy)- and LC/MS/MS experiments. Using this technique we could identify two ADP-ribosylation sites in TNFα digested with trypsin, protease V8 and both proteases and thereby demonstrate the specific ADP-ribosylation of TNFα. In complex samples the detection of ADP-ribosylated peptides requires further enrichment of the modified peptides. We tested various materials routinely used for the isolation of phosphopeptides. IMAC as well as TiO2 chromatography were successfully applied for the selective enrichment of ADP-ribosylated model peptides. [Copyright &y& Elsevier]