Expression, purification and biochemical characterization of recombinant Ca-dependent protein kinase 2 of the malaria parasite Plasmodium falciparum.

Calcium-dependent protein kinases (CDPKs) are serine/threonine kinases that react in response to calcium which functions as a trigger for several mechanisms in plants and invertebrates, but not in mammals. Recent structural studies have defined the role of calcium in the activation of CDPKs and have elucidated the important structural changes caused by calcium in order to allow the kinase domain of CDPK to bind and phosphorylate the substrate. However, the role of autophosphorylation in CDPKs is still not fully understood. In Plasmodium falciparum, seven CDPKs have been identified by sequence comparison, and four of them have been characterized and assigned to play a role in parasite motility, gametogenesis and egress from red blood cells. Although PfCDPK2 was already discovered in 1997, little is known about this enzyme and its metabolic role. In this work, we have expressed and purified PfCDPK2 at high purity in its unphosphorylated form and characterized its biochemical properties. Moreover, propositions about putative substrates in P. falciparum are made based on the analysis of the phosphorylation sites on the artificial substrate myelin basic protein (MBP).

mitoKATP channel activation in the postanoxic developing heart protects E-C coupling via NO-, ROS-, and PKC-dependent pathways.

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH). Effects of the specific mitoK(ATP) channel opener diazoxide (Diazo, 50 microM) or the blocker 5-hydroxydecanoate (5-HD, 500 microM), the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 microM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 microM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or L-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, L-NAME, or Chel, whereas protection of the PR interval was abolished by L-NAME exclusively. Thus pharmacological opening of the mitoK(ATP) channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.

Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

TRADD protein is an essential component of the RIG-like helicase antiviral pathway

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses

INK4a/Arf is required for suppression of EGFR/DeltaEGFR(2-7)-dependent ERK activation in mouse astrocytes and glioma.

Amplification of the epidermal growth factor receptor (EGFR) or expression of its constitutively activated mutant, DeltaEGFR(2-7), in association with the inactivation of the INK4a/Arf gene locus is a frequent alteration in human glioblastoma. The notion of a cooperative effect between these two alterations has been demonstrated in respective mouse brain tumor models including our own. Here, we investigated underlying molecular mechanisms in early passage cortical astrocytes deficient for p16(INK4a)/p19(Arf) or p53, respectively, with or without ectopic expression of DeltaEGFR(2-7). Targeting these cells with the specific EGFR inhibitor tyrphostin AG1478 revealed that phosphorylation of ERK was only abrogated in the presence of an intact INK4a/Arf gene locus. The sensitivity to inhibit ERK phosphorylation was independent of ectopic expression of DeltaEGFR(2-7) and independent of the TP53 status. This resistance to downregulate the MAPK pathway in the absence of INK4a/Arf was confirmed in cell lines derived from our mouse glioma models with the respective initial genetic alterations. Thus, deletion of INK4a/Arf appears to keep ERK in its active, phosphorylated state insensitive to an upstream inhibitor specifically targeting EGFR/DeltaEGFR(2-7). This resistance may contribute to the cooperative tumorigenic effect selected for in human glioblastoma that may be of crucial clinical relevance for treatments specifically targeting EGFR/DeltaEGFR(2-7) in glioblastoma patients.

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

Improved Innate and Adaptive Immunostimulation by Genetically Modified HIV-1 Protein Expressing NYVAC Vectors.

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

Regulation of Placental Leptin Expression by Cyclic Adenosine 5 '-Monophosphate Involves Cross Talk between Protein Kinase A and Mitogen-Activated Protein Kinase Signaling Pathways

Leptin, a 16-kDa protein mainly produced by adipose tissue, has been involved in the control of energy balance through its hypothalamic receptor. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it was found to be expressed. In the current study, we examined the effect of cAMP in the regulation of leptin expression in trophoblastic cells. We found that dibutyryl cAMP [(Bu)(2)cAMP], a cAMP analog, showed an inducing effect on endogenous leptin expression in BeWo and JEG-3 cell lines when analyzed by Western blot analysis and quantitative RT-PCR. Maximal effect was achieved at 100 microM. Leptin promoter activity was also stimulated, evaluated by transient transfection with a reporter plasmid construction. Similar results were obtained with human term placental explants, thus indicating physiological relevance. Because cAMP usually exerts its actions through activation of protein kinase A (PKA) signaling, this pathway was analyzed. We found that cAMP response element-binding protein (CREB) phosphorylation was significantly increased with (Bu)(2)cAMP treatment. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor CREB caused a significant stimulation on leptin promoter activity. On the other hand, the cotransfection with a dominant negative mutant of the regulatory subunit of PKA inhibited leptin promoter activity. We determined that cAMP effect could be blocked by pharmacologic inhibition of PKA or adenylyl ciclase in BeWo cells and in human placental explants. Thereafter, we decided to investigate the involvement of the MAPK/ERK signaling pathway in the cAMP effect on leptin induction. We found that 50 microm PD98059, a MAPK kinase inhibitor, partially blocked leptin induction by cAMP, measured both by Western blot analysis and reporter transient transfection assay. Moreover, ERK 1/2 phosphorylation was significantly increased with (Bu)(2)cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 microm PD98059 inhibited cAMP-dependent phosphorylation of CREB in placental explants. In summary, we provide some evidence suggesting that cAMP induces leptin expression in placental cells and that this effect seems to be mediated by a cross talk between PKA and MAPK signaling pathways.

Demyelination induced by protein kinase C-activating tumor promoters in aggregating brain cell cultures.

The plasticity of mature oligodendrocytes was studied in aggregating brain cell cultures at the period of maximal expression of myelin marker proteins. The protein kinase C (PKC)-activating tumor promoters mezerein and phorbol 12-myristate 13-acetate (PMA), but not the inactive phorbol ester analog 4alpha-PMA, caused a pronounced decrease of myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity. In contrast, myelin/oligodendrocyte protein (MOG) content was affected relatively little. Northern blot analyses showed a rapid reduction of MBP and PLP gene expression induced by mezerein, and both morphological and biochemical findings indicate a drastic loss of compact myelin. During the acute phase of demyelination, only a relatively small increase in cell death was perceptible by in situ end labeling and in situ nick translation. Basic fibroblast growth factor (bFGF) also reduced the levels of the oligodendroglial differentiation markers and enhanced the demyelinating effects of the tumor promoters. The present results suggest that PKC activation resulted in severe demyelination and partial loss of the oligodendrocyte-differentiated phenotype.

Regulation of dendritic development by BDNF requires activation of CRTC1 by glutamate.

Dendritic growth is essential for the establishment of a functional nervous system. Among extrinsic signals that control dendritic development, substantial evidence indicates that BDNF regulates dendritic morphology. However, little is known about the underlying mechanisms by which BDNF controls dendritic growth. In this study, we show that the MAPK signaling pathway and the transcription factor cAMP response element-binding protein (CREB) mediate the effects of BDNF on dendritic length and complexity. However, phosphorylation of CREB alone is not sufficient for the stimulation of dendritic growth by BDNF. Thus, using a mutant form of CREB unable to bind CREB-regulated transcription coactivator (CRTC1), we demonstrate that this effect also requires a functional interaction between CREB and CRTC1. Moreover, inhibition of CRTC1 expression by shRNA-mediated knockdown abolished BDNF-induced dendritic growth of cortical neurons. Interestingly, we found that nuclear translocation of CRTC1 results from activation of NMDA receptors by glutamate, a process that is essential for the effects of BDNF on dendritic development. Together, these data identify a previously unrecognized mechanism by which CREB and the coactivator CRTC1 mediate the effects of BDNF on dendritic growth.

Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad.

Pseudomonas fluorescens CHA0 is a root-associated biocontrol agent that suppresses soil-borne fungal diseases of crops. Remarkably, the pseudomonad is also endowed with systemic and oral activity against pest insects which depends on the production of the insecticidal Fit toxin. The toxin gene (fitD) is part of a virulence cassette encoding three regulators (FitF, FitG, FitH) and a type I secretion system (FitABC-E). Immunoassays with a toxin-specific antibody and transcriptional analyses involving fitG and fitH deletion and overexpression mutants identified LysR family regulator FitG and response regulator FitH as activator and repressor, respectively, of Fit toxin and transporter expression. To visualize and quantify toxin expression in single live cells by fluorescence microscopy, we developed reporters which in lieu of the native toxin protein express a fusion of the Fit toxin with red fluorescent mCherry. In a wild-type background, expression of the mCherry-tagged Fit toxin was activated at high levels in insect hosts, i.e. when needed, yet not on plant roots or in batch culture. By contrast, a derepressed fitH mutant expressed the toxin in all conditions. P. fluorescens hence can actively induce insect toxin production in response to the host environment, and FitH and FitG are key regulators in this mechanism.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Redirecting NK cells mediated tumor cell lysis by a new recombinant bifunctional protein.

Natural killer (NK) cells are at the crossroad between innate and adaptive immunity and play a major role in cancer immunosurveillance. NK cell stimulation depends on a balance between inhibitory and activating receptors, such as the stimulatory lectin-like receptor NKG2D. To redirect NK cells against tumor cells, we designed bifunctional proteins able to specifically bind tumor cells and to induce their lysis by NK cells, after NKG2D engagement. To this aim, we used the 'knob into hole' heterodimerization strategy, in which 'knob' and 'hole' variants were generated by directed mutagenesis within the CH3 domain of human IgG1 Fc fragments fused to an anti-CEA or anti-HER2 scFv or to the H60 murine ligand of NKG2D, respectively. We demonstrated the capacity of the bifunctional proteins produced to specifically coat tumor cells surface with H60 ligand. Most importantly, we demonstrated that these bifunctional proteins were able to induce an NKG2D-dependent and antibody-specific tumor cell lysis by murine NK cells. Overall, the results show the possibility to redirect NK cytotoxicity to tumor cells by a new format of recombinant bispecific antibody, opening the way of potential NK cell-based cancer immunotherapies by specific activation of the NKG2D receptor at the tumor site.

Role of the host cell's unfolded protein response in arenavirus infection.

Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.

Perinatal Hypoxia Enhances Cyclic Adenosine Monophosphate-mediated BKCa Channel Activation in Adult Murine Pulmonary Artery.

Exposure to perinatal hypoxia results in alteration of the adult pulmonary circulation, which is linked among others to alterations in K channels in pulmonary artery (PA) smooth muscle cells. In particular, large conductance Ca-activated K (BKCa) channels protein expression and activity were increased in adult PA from mice born in hypoxia compared with controls. We evaluated long-term effects of perinatal hypoxia on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway-mediated activation of BKCa channels, using isoproterenol, forskolin, and dibutyryl-cAMP. Whole-cell outward current was higher in pulmonary artery smooth muscle cells from mice born in hypoxia compared with controls. Spontaneous transient outward currents, representative of BKCa activity, were present in a greater proportion in pulmonary artery smooth muscle cells of mice born in hypoxia than in controls. Agonists induced a greater relaxation in PA of mice born in hypoxia compared with controls, and BKCa channels contributed more to the cAMP/PKA-mediated relaxation in case of perinatal hypoxia. In summary, perinatal hypoxia enhanced cAMP-mediated BKCa channels activation in adult murine PA, suggesting that this pathway could be a potential target for modulating adult pulmonary vascular tone after perinatal hypoxia.