Georg W. Mayr

Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.

Structural Analysis and Detection of Biological Inositol Pyrophosphates Reveal That the Family of VIP/Diphosphoinositol Pentakisphosphate Kinases Are 1/3-Kinases

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.

Ca2+ Entry Induced by Cyclic ADP-ribose in Intact T-Lymphocytes

Cyclic ADP-ribose (cADPr) is a potent Ca2+-mobilizing natural compound (Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615) which has been shown to release Ca2+ from an intracellular store of permeabilized T-lymphocytes (Guse, A. H., Silva, C. P., Emmrich, F., Ashamu, G., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Microinjection of cADPr into intact single T lymphocytes dose dependently induced repetitive but irregular Ca2+ spikes which were almost completely dependent on the presence of extracellular Ca2+. The Ca2+ spikes induced by cADPr could be blocked either by co-injection of cADPr with the specific antagonist 8-NH2-cADPr, by omission of Ca2+ from the medium, or by superfusion of the cells with Zn2+ or SK-F 96365. Ratiometric digital Ca2+ imaging revealed that single Ca2+ spikes were initiated at several sites (“hot spots”) close to the plasma membrane. These hot spots then rapidly formed a circular zone of high Ca2+ concentration below the plasma membrane which subsequently propagated like a closing optical diaphragm into the center of the cell. Taken together these data indicate a role for cADPr in Ca2+ entry in T-lymphocytes.

The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization.

The function of the transcription regulator ArgRIII in the expression of several genes involved in the metabolism of arginine in yeast has been well studied. It was previously reported that it is also an inositol phosphate multikinase and an important factor of the mRNA export pathway [reviewed by Shears (2000) Bioessays 22, 786-789]. In the present study we report the cloning of a full-length 1248-bp cDNA encoding a human inositol phosphate multikinase (IPMK). This protein has a calculated molecular mass of 47.219 kDa. Functionally important motifs [inositol phosphate-binding site, ATP-binding site, catalytically important SSLL (Ser-Ser-Leu-Leu) domain] are conserved between the human IPMK and yeast ArgRIII. Bacterially expressed protein demonstrated an inositol phosphate multikinase activity similar to that of yeast ArgRIII. Ins(1,4,5)P3 is phosphorylated at positions 3 and 6 up to Ins(1,3,4,5,6)P5. The human IPMK fused with a fluorescent protein tag is localized predominantly in the nucleus when transiently expressed in mammalian cells. A basic cluster in the proteins C-terminus is positively involved in nuclear targeting. These findings are consistent with the concept of a nuclear inositol phosphate signalling and phosphorylation pathway in mammalian cells.

Antiproliferative Plant and Synthetic Polyphenolics Are Specific Inhibitors of Vertebrate Inositol-1,4,5-trisphosphate 3-Kinases and Inositol Polyphosphate Multikinase

Inositol-1,4,5-trisphosphate 3-kinases (IP3K) A, B, and C as well as inositol polyphosphate multikinase (IPMK) catalyze the first step in the formation of the higher phosphorylated inositols InsP5 and InsP6 by metabolizing Ins(1,4,5)P3 to Ins(1,3,4,5)P4. In order to clarify the special role of these InsP3 phosphorylating enzymes and of subsequent anabolic inositol phosphate reactions, a search was conducted for potent enzyme inhibitors starting with a fully active IP3K-A catalytic domain. Seven polyphenolic compounds could be identified as potent inhibitors with IC50 < 200 nm (IC50 given): ellagic acid (36 nm), gossypol (58 nm), (–)-epicatechin-3-gallate (94 nm), (–)-epigallocatechin-3-gallate (EGCG, 120 nm), aurintricarboxylic acid (ATA, 150 nm), hypericin (170 nm), and quercetin (180 nm). All inhibitors displayed a mixed-type inhibition with respect to ATP and a non-competitive inhibition with respect to Ins(1,4,5)P3. Examination of these inhibitors toward IP3K-A, -B, and -C and IPMK from mammals revealed that ATA potently inhibits all kinases while the other inhibitors do not markedly affect IPMK but differentially inhibit IP3K isoforms. We identified chlorogenic acid as a specific IPMK inhibitor whereas the flavonoids myricetin, 3′,4′,7,8-tetrahydroxyflavone and EGCG inhibit preferentially IP3K-A and IP3K-C. Mutagenesis studies revealed that both the calmodulin binding and the InsP3 binding domain in IP3K are involved in inhibitor binding. Their absence in IPMK and the presence of a unique insertion in IPMK were found to be important for selectivity differences from IP3K. The fact that all identified IP3K and IPMK inhibitors have been reported as antiproliferative agents and that IP3Ks or IPMK often are the best binding targets deserves further investigation concerning their antitumor potential.

Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis.

Abstract Diphosphoinositol phosphates are a subclass of inositol phosphates possessing one or two high energy diphosphate groups instead of phosphoester substituents of the myo-inositol. Here we describe the enzymes responsible for their synthesis and degradation and how these may be regulated. Formation of diphosphoinositol phosphates in yeast and mammals is driven by an increase of the cellular energy charge, a lack of inorganic phosphate, and in mammals by osmotic or heat stress and in some cases by receptor mediated signaling. Known cellular actions are an improvement of the cell homeostasis by a reduction of the energy charge, increased phosphate uptake, improvement of mitochondrial performance, and an increase of insulin secretion in mammals. The underlying molecular mechanisms of action are far from being clarified but an increasing body of knowledge about molecular details has highlighted their complex participation in many cellular systems and metabolic processes.

Inositol hexakisphosphate increases L-type Ca2+ channel activity by stimulation of adenylyl cyclase

Inositol hexakisphosphate (InsP6) is a most abundant inositol polyphosphate that changes simultaneously with inositol 1,4,5‐trisphosphate in depolarized neurons. However, the role of InsP6 in neuronal signaling is unknown. Mass assay reveals that the basal levels of InsP6 in several brain regions tested are similar. InsP6 mass is significantly elevated in activated brain neurons and lowered by inhibition of neuronal activity. Furthermore, the hippocampus is most sensitive to electrical challenge with regard to percentage accumulation of InsP6. In hippocampal neurons, InsP6 stimulates adenylyl cyclase (AC) without influencing cAMP phosphodiesterases, resulting in activation of protein kinase A (PKA) and thereby selective enhancement of voltage‐gated L‐type Ca2+ channel activity. This enhancement was abolished by preincubation with PKA and AC inhibitors. These data suggest that InsP6 increases L‐type Ca2+ channel activity by facilitating phosphorylation of PKA phosphorylation sites. Thus, in hippocampal neurons, InsP6 serves as an important signal in modulation of voltage‐gated L‐type Ca2+ channel activity.—Yang, S.‐N., Yu, J., Mayr, G. W., Hofmann, F., Larsson, O., Berggren, P.‐O. Inositol hexakisphosphate increases L‐type Ca2+ channel activity by stimulation of adenylyl cyclase. FASEB J. 15, 1753–1763 (2001)

Human regulatory T cells rapidly suppress T cell receptor-induced Ca(2+), NF-κB, and NFAT signaling in conventional T cells.

Inhibition of calcium signaling is critical for the suppression of T cell responses by regulatory T cells. Suppressing Calcium Suppresses T Cells Regulatory T cells (Tregs) are required to keep conventional T cells in check, and disruption of the generation or function of Tregs leads to autoimmunity. Conversely, Tregs can have a deleterious effect by dampening antitumor responses of T cells. Thus, improved understanding of the mechanisms by which Tregs inhibit T cell receptor (TCR)–induced responses in conventional T cells would help to develop better therapies against autoimmune disorders and cancer. Schmidt et al. found that TCR-induced, Ca2+-dependent signaling in human conventional T cells that were incubated with Tregs was inhibited compared to that in nonsuppressed T cells, which led to defective activation of the transcription factors NFAT and NF-κB. In contrast, Ca2+-independent signaling was unaffected. Suppressed signaling persisted after the Tregs were removed from cocultures. Increasing the intracellular concentration of Ca2+ in conventional T cells reversed the inhibitory effects of Tregs. Together, these data suggest that inhibition of Ca2+ signaling is critical for the suppressive effects of Tregs. CD4+CD25hiFoxp3+ regulatory T cells (Tregs) are critical mediators of self-tolerance, which is crucial for the prevention of autoimmune disease, but Tregs can also inhibit antitumor immunity. Tregs inhibit the proliferation of CD4+CD25− conventional T cells (Tcons), as well as the ability of these cells to produce effector cytokines; however, the molecular mechanism of suppression remains unclear. Here, we showed that human Tregs rapidly suppressed the release of calcium ions (Ca2+) from intracellular stores in response to T cell receptor (TCR) activation in Tcons. The inhibition of Ca2+ signaling resulted in decreased dephosphorylation, and thus decreased activation, of the transcription factor nuclear factor of activated T cells 1 (NFAT1) and reduced the activation of nuclear factor κB (NF-κB). In contrast, Ca2+-independent events in Tcons, such as TCR-proximal signaling and activation of the transcription factor activator protein 1 (AP-1), were not affected during coculture with Tregs. Despite suppressing intracellular Ca2+ mobilization, coculture with Tregs did not block the generation of inositol 1,4,5-trisphosphate in TCR-stimulated Tcons. The Treg-induced suppression of the activity of NFAT and NF-κB and of the expression of the gene encoding the cytokine interleukin-2 was reversed in Tcons by increasing the concentration of intracellular Ca2+. Our results elucidate a previously unrecognized and rapid mechanism of Treg-mediated suppression. This increased understanding of Treg function may be exploited to generate possible therapies for the treatment of autoimmune diseases and cancer.

Non-radioactive, isomer-specific inositol phosphate mass determinations: high-performance liquid chromatography-micro-metal-dye detection strongly improves speed and sensitivity of analyses from cells and micro-enzyme assays

A microbore high-performance liquid chromatographic (HPLC) method is presented allowing rapid and sensitive mass analysis of inositol phosphates from cells and tissues. An analysis starting from inorganic phosphate up to inositol hexakisphosphate displaying a similar isomer selectivity as compared to the standard metal-dye detection system takes about 15 min. The detection sensitivity was about 15 pmol for inositol trisphosphate, about 10 pmol for inositol tetrakisphosphate, about 5 pmol for inositol pentakisphosphate and less than 5 pmol for inositol hexakisphosphate. The method was validated regarding day-to-day variations and variations at the same day of retention times and peak areas of standard inositol phosphates. Standard deviations of retention times ranged from 0.25 to 0.62% (same day) and from 0.64 to 1.61% (day-to-day variations). Ranges of standard deviations of peak areas were between 2.24% and 3.91% (same day) and 6.13% and 13.8% (day-to-day variations). Linearity of the post-column complexometric metal-dye detection system was demonstrated in the range of a few picomoles and at least 800 pmol. The method was applied to the analysis of inositol phosphates in Jurkat T-lymphocytes and assays from minute amounts of enzymes interconverting inositol phosphates. While measurements of inositol phosphates from cell extracts are now possible using significantly reduced cell numbers, micro-enzyme assays are feasible in reasonable repeated analysis times and with sufficient isomer selectivity. In conclusion, a substantial improvement towards speed of analysis and detection sensitivity of inositol phosphate mass analysis was achieved by microbore metal-dye detection HPLC.

Quantification of intracellular levels of cyclic ADP-ribose by high-performance liquid chromatography.

A combined two-step high-performance liquid chromatographic (HPLC) method was developed for the analysis of endogenous levels of cyclic adenosine diphosphoribose (cADPR) in cell extracts. The detection sensitivity for cADPR was about 10 pmol. Linearity of the HPLC detection system was demonstrated in the range of 10 pmol up to 2 nmol. The method was validated in terms of within-day and between-day reproducibility of retention times and peak areas of standard nucleotides. The method was applied to the analysis of endogenous cADPR in human T cell lines. Sequential separation of perchloric acid extracts from cells on strong anion-exchange and reversed-phase ion-pair HPLC resulted in a single symmetrical peak co-eluting with standard cADPR. The identity of this endogenous material was further confirmed by its ability to be converted to ADPR upon heating the cell samples at 80 degrees C for 2 h. Recoveries of the combined perchloric acid extraction-HPLC analysis procedures were 48.3 +/- 10.2%. The determined intracellular concentrations of cADPR in quiescent Jurkat and HPB. ALL human T cells were 198 +/- 41 and 28 +/- 9 pmol/10(8) cells, respectively. In conclusion, a non-radioactive HPLC method presenting a specificity and sensitivity suitable for precise quantification of cADPR in cell extracts was developed.