The Polycomb group protein EED couples TNF receptor 1 to neutral sphingomyelinase.

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer's, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1.FAN.RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the "missing link" that completes one of the last unresolved signaling pathways of TNF-R1.

Confluence induced threonine41/serine45 phospho-beta-catenin dephosphorylation via ceramide-mediated activation of PP1cgamma.

It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels [Marchesini N., Osta W., Bielawski J., Luberto C., Obeid L.M. and Hannun Y.A. (2004) J. Biol. Chem., 279, 25101-11]. In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-beta-catenin at threonine41/serine45. The effect of confluence on beta-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of beta-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented beta-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cgamma prevented dephosphorylation of beta-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cgamma to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced beta-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence.

The extended family of neutral sphingomyelinases.

The neutral sphingomyelinases (N-SMases) are considered major candidates for mediating the stress-induced production of ceramide, and N-SMase activity has been identified, characterized, and cloned from bacteria, yeast, and mammalian cells. Although the level of identity between these enzymes is low, a number of key residues thought to be involved in metal binding and catalysis are conserved. This has led to the suggestion of a common catalytic mechanism, and thus, these enzymes are considered to form an extended family of N-SMases. Despite considerable research into N-SMase activity in cell culture and various tissues, the lack, until recently, of molecular identification of specific N-SMase enzymes had precluded specific insights into the regulation, physiological, and pathological roles of these proteins. In this review, we summarize, for the first time, current knowledge of the N-SMase family, focusing on cloned members from bacteria, yeast, and mammalian cells. We also briefly consider the major future directions for N-SMase research which promises highly significant and specific insight into sphingolipid-mediated functions.

A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol.

The Toxoplasma gondii phosphoinositide-specific phospholipase C gene (TgPI-PLC) was cloned, sequenced and expressed in Escherichia coli and its enzymatic characteristics were investigated. TgPI-PLC is present in the genome as a single-copy gene consisting of 22 exons interrupted by 21 introns, and encodes a polypeptide of 1097 amino acids with a predicted molecular mass of 121 kDa. In addition to the conserved catalytic X and Y domains, TgPI-PLC contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When compared with mammalian delta-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31-35% identity over the whole sequence. Recombinant TgPI-PLC, as well as the native enzyme obtained from crude membrane extracts of the parasite, was more active with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. Indirect immunofluorescence analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme localizes in the plasma membrane of the parasites.

A malaria parasite-encoded vacuolar H(+)-ATPase is targeted to the host erythrocyte.

The asexual development of malaria parasites inside the erythrocyte is accompanied by changes in the composition, structure, and function of the host cell membrane and cytoplasm. The parasite exports a membrane network into the host cytoplasm and several proteins that are inserted into the erythrocyte membrane, although none of these proteins has been shown to have enzymatic activity. We report here that a functional malaria parasite-encoded vacuolar (V)-H(+)-ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte. This localization was determined by separation of parasite and erythrocyte membranes and determination of enzyme marker activities and by immunofluorescence microscopy assays using antibodies against the B subunit of the malarial V-H(+)-ATPase and erythrocyte (spectrins) and parasite (merozoite surface protein 1) markers. Our results suggest that this pump has a role in the maintenance of the intracellular pH (pH(i)) of the infected erythrocyte. Our results also indicate that although the pH(i) maintained by the V-H(+)-ATPase is important for maximum uptake of small metabolites at equilibrium, it does not appear to affect transport across the erythrocyte membrane and is, therefore, not involved in the previously described phenomenon of increased permeability of infected erythrocytes that is sensitive to chloride channel inhibitors (new permeation pathway). This constitutes the first report of the presence of a functional enzyme of parasite origin in the plasma membrane of its host.

Role for mammalian neutral sphingomyelinase 2 in confluence-induced growth arrest of MCF7 cells.

Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth (Marchesini, N., Luberto, C., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 13775-13783). In this study, the role of endogenous nSMase2 in regulating growth arrest was investigated. The results show that endogenous nSMase2 mRNA was up-regulated approximately 5-fold when MCF7 cells became growth-arrested at confluence, and total neutral SMase activity was increased by 119 +/- 41% with respect to control. Cell cycle analysis showed that up-regulation of endogenous nSMase2 correlated with G(0)/G(1) cell cycle arrest and an increase in total ceramide levels (2.4-fold). Analysis of ceramide species showed that confluence caused selective increases in very long chain ceramide C(24:1) (370 +/- 54%) and C(24:0) (266 +/- 81%) during arrest. The role of endogenous nSMase2 in growth regulation and ceramide metabolism was investigated using short interfering RNA (siRNA)-mediated loss-of-function analysis. Down-regulation of nSMase2 with specific siRNA increased the cell population of cells in S phase of the cell cycle by 59 +/- 14% and selectively reverted the effects of growth arrest on the increase in levels of very long chain ceramides. Mechanistically, confluence arrest also induced hypophosphorylation of the retinoblastoma protein (6-fold) and induction of p21(WAF1) (3-fold). Down-regulation of nSMase2 with siRNA largely prevented the dephosphorylation of the retinoblastoma protein and the induction of p21(WAF1), providing a link between the action of nSMase2 and key regulators of cell cycle progression. Moreover, studies on nSMase2 localization in MCF7 cells showed that nSMase2 distributed throughout the cells in subconfluent, proliferating cultures. In contrast, nSMase2 became nearly exclusively located at the plasma membrane in confluent, contact-inhibited cells. Hence, we demonstrate for the first time that nSMase2 functions as a growth suppressor in MCF7 cells, linking confluence to the G(0)/G(1) cell cycle check point.

Acid and neutral sphingomyelinases: roles and mechanisms of regulation.

Ceramide, an emerging bioactive lipid and second messenger, is mainly generated by hydrolysis of sphingomyelin through the action of sphingomyelinases. At least two sphingomyelinases, neutral and acid sphingomyelinases, are activated in response to many extracellular stimuli. Despite extensive studies, the precise cellular function of each of these sphingomyelinases in sphingomyelin turnover and in the regulation of ceramide-mediated responses is not well understood. Therefore, it is essential to elucidate the factors and mechanisms that control the activation of acid and neutral sphingomyelinases to understand their the roles in cell regulation. This review will focus on the molecular mechanisms that regulate these enzymes in vivo and in vitro, especially the roles of oxidants (glutathione, peroxide, nitric oxide), proteins (saposin, caveolin 1, caspases), and lipids (diacylglycerol, arachidonic acid, and ceramide).

Purification, characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa. PlcH is a multifunctional enzyme.

Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of approximately 75 kDa and one of 30-35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.

Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism.

Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.

Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores.

Calcium-mediated microneme secretion in Toxoplasma gondii is stimulated by contact with host cells, resulting in the discharge of adhesins that mediate attachment. The intracellular source of calcium and the signaling pathway(s) triggering release have not been characterized, prompting our search for mediators of calcium signaling and microneme secretion in T. gondii. We identified two stimuli of microneme secretion, ryanodine and caffeine, which enhanced release of calcium from parasite intracellular stores. Ethanol, a previously characterized trigger of microneme secretion, stimulated an increase in parasite inositol 1,4,5-triphosphate, implying that this second messenger may mediate intracellular calcium release. Consistent with this observation, xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, inhibited microneme secretion and blocked parasite attachment and invasion of host cells. Collectively, these results suggest that T. gondii possess an intracellular calcium release channel with properties of the inositol 1,4,5-triphosphate/ryanodine receptor superfamily. Intracellular calcium channels, previously studied almost exclusively in multicellular animals, appear to also be critical to the control of parasite calcium during the initial steps of host cell entry.

Cellular signalling in Trypanosoma cruzi: biphasic behaviour of inositol phosphate cycle components evoked by carbachol.

Stimulation of epimastigote forms of Trypanosma cruzi with carbachol resulted in a long-lasting response. The earlier phase for inositol phosphates was rapid and transient, peaking at 1 min with a return to basal levels by 6 min. In a second phase, these metabolite levels reached maximal values at 10-12 min, with a later declination to basal values at about 20 min. The inositol phosphate response was quenched by parasite treatment with atropine. The elevation in intracellular free calcium ([Ca(2+)]i) was transient, reaching the resting level at 87+/-8 s (n=48) of agonist addition. Myo-inositol 1,4,5-triphosphate (InsP(3)) production and [Ca(2+)]i mobilisation were carbachol dose-dependent. The maximally effective concentrations of agonist ranged between 1x10(-6) and 1x10(-5) M. The increase in carbachol concentration resulted in an evident attenuation of [Ca(2+)]i mobilisation and in [3H]InsP(3) levels. Pretreatment of the cells with 10 microM U73122, a phospholipase C (PLC) inhibitor, showed that both InsP(3) peaks triggered by carbachol were completely abolished, whereas there was not substantial change on the maximum elevation in [Ca(2+)]i. The first peak of InsP(3) and InsP(s) was completely abolished when the cells were incubated with phorbol 12-myristate 13-acetate ester (TPA) for 30 min before carbachol stimulation. A biphasic behaviour for PtdIns 4-kinase activity was demonstrated by changes in [32P]PtdInsP levels. The time-course of PtdIns4P 5-kinase activity showed a rapid, significant and transient decrease of [32P]PtdInsP(2) from 0 time to the third min. At the end of this time the polyphosphoinositide began to return towards control levels but, interestingly, after 5-6 min of stimulation there was a subsequent more important increase over control levels which peaked at 10 min. There was also a detectable increment of DAG at 1 min with a maximum at 3 min, this level remaining elevated until at least 10 min. Subsequently, these levels returned to the base line or even below it.

A plasma membrane P-type H(+)-ATPase regulates intracellular pH in Leishmania mexicana amazonensis.

A recent report (Mukherjee et al., J. Biol. Chem. 276 (2001) 5563) has proposed that the plasma membrane Mg(+)-ATPase of promastigotes of Leishmania donovani, that is involved in its intracellular pH regulation, is an electroneutral H(+)/K(+) antiporter rather than an electrogenic H(+) pump. Since this proposition has important implications for the use of the pump as a target for chemotherapy, we investigated its nature in the mammalian stage (amastigote) of L. mexicana amazonensis and compared it with that present in promastigotes. Intracellular pH and H(+) efflux were measured using the acetotoxymethyl ester and the free form of 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein, respectively. Intracellular pH in amastigotes (at an external pH of 5.5) and promastigotes (at an external pH of 7.4) was 6.36+/-0.02 and 6.83+/-0.07, respectively. Differences in the mechanisms for regulation of intracellular pH were noted between amastigote and promastigote forms. Amastigotes maintained their intracellular pH neutral over a wide range of external pHs in the absence of K(+) or Na(+). The H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodi-imide, diethylstilbestrol and N-ethylmaleimide, substantially decreased their steady-state intracellular pH, inhibited proton efflux, and their recovery from acidification. The data support the presence of an H(+)-ATPase as the major regulator of intracellular pH in amastigotes. In contrast, promastigotes were unable to maintain a neutral pH under acidic conditions and although their steady-state intracellular pH and recovery from acidification were affected by H(+)-ATPase inhibitors, bicarbonate was able to overcome intracellular acidification. Bicarbonate was also able to raise the steady-state intracellular pH from 6.80+/-0.03 to 7.25+/-0.09 and induce membrane hyperpolarization. No evidence was found of the possible involvement of a K(+)/H(+)-ATPase in intracellular pH regulation in both developmental stages of L. m. amazonensis.

Acidocalcisomes are functionally linked to the contractile vacuole of Dictyostelium discoideum.

The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H(+)-ATPase, an H(+)-pyrophosphatase, and a Ca(2+)-ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H(+)-pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylenediphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dose-dependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4' -diisothiocyanatodihydrostilbene-2, 2' -disulfonic acid and the vacuolar H(+)-ATPase inhibitor bafilomycin A(1). Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.