Autophagy, a major clearance route for many long-lived proteins and organelles, has long been implicated in cancer development. Myc is a proto-oncogene often found to be deregulated in many cancers, and thus is an attractive target for design of cancer therapy. Therefore, understanding the relationship between anti-Myc strategies and autophagy will be important for development of effective therapy. Here, we show that Myc depletion inhibits autophagosome formation and impairs clearance of autophagy substrates. Myc suppression has an inhibitory effect on autophagy via reduction of c-Jun N-terminal kinase 1 (JNK1) and B-cell lymphoma 2 (Bcl2) phosphorylation. Additionally, the decrease in JNK1 phosphorylation observed with Myc knockdown is associated with a reduction in ROS production. Our data suggest that targeting Myc in cancer therapy might have the additional benefit of inhibiting autophagy in the case of therapy resistance associated with chemotherapy-induced autophagy. It involves the sequestration of portions of cytoplasm through the formation of double-membraned autophagosomes, which are subsequently targeted for degradation after fusion with lysosomes. Under normal conditions, autophagy is maintained at a basal level , where i demands . TherefoAutophagy can be regulated via diverse pathways. For instance, amino acid depletion can induce autophagy by inhibiting the mammalian target of rapamycin (mTOR) . There aBif-1), ultraviolet radiation resistance-associated gene (UVRAG) and Atg4, has been shown to promote the development of spontaneous tumours in mice  is one of the most common lesions occurring in human cancers of diverse origin ,15, as hgression . These dgression .In situations where Myc levels were elevated, autophagy appeared to be decreased ,23. ThisTo investigate whether Myc plays a role in autophagy, we first performed siRNA knockdown of the gene and examined its effects on microtubule associated protein 1 light chain 3 (LC3-II) protein levels. LC3-II is the only known protein to specifically associate with autophagosomes, and thus, it is widely accepted as a marker for monitoring autophagy . LC3 is Supplementary Material, Fig. S1A) and Myc knockout (−/−) rat fibroblasts , in which Myc depletion decreased LC3-II levels in both the absence and the presence of Baf A1.Knockdown of Myc in HeLa cells decreased LC3-II levels, and this correlated with a reduction in endogenous Myc expression Fig. A. To verSupplementary Material, Fig. S1C).We went on to test a small molecule inhibitor of Myc, 10058-F4 , hereafter). This compound appears to inhibit Myc both by disrupting its dimerization with its obligate partner, Max, which is required for transactivation of target genes and by reducing Myc mRNA expression in different cell lines . ConsistTo exclude the possibility that the decrease in LC3-II levels observed is due to suppression in the transcriptional activity of Myc on LC3, Atg5 knockout (−/−) murine embryonic fibroblast (MEFs) were used. Atg5 −/− MEFs lack the Atg5 protein that is required for the conversion of LC3-I to LC3-II, and thus, only LC3-I can be detected in Atg5−/− MEFs. If there was less transcription of LC3 itself, then lower LC3-I levels would be detected in Atg5−/− MEFs upon Myc inhibition. We observed no change in LC3-I levels in Atg5−/− MEFs treated with an Myc inhibitor, suggesting that Myc inhibition does not result in reduced transcription of LC3 Fig. E.In addition to immunoblotting, an alternative approach to assess autophagy is by immunofluorescence. For this, we used HeLa cells stably expressing GFP-LC3, in which GFP-LC3 associated with autophagosomes appears as green puncta. We observed that Myc knockdown decreased the number of GFP-LC3 puncta Fig. F.Fab 1 (yeast orthologue of PIKfyve), YOTB, Vac 1 (vesicle transport protein), and EEA1) domain-containing protein (DFCP1) vesicles. DFCP1 labels phosphatidylinositol-3-phosphate (PI3P) on autophagosome precursors, called omegasomes . To validate that this was not predominantly a consequence of Myc on p62 transcription, U2OS cells stably expressing Halotag-p62 were used. The cells were labelled with a Halotag fluorescent ligand before they were treated with Myc-I. The Halotag ligand forms a covalent bond and binds irreversibly to the Halotag protein. This enables a fixed pool of Halotag-p62 protein to be labelled and its levels can be monitored over time after labelling in a chase period which is independent of any changes in protein synthesis (t = 0), and 24 h after switching-off (t = 24). Myc knockdown caused a reduction in the degradation rate of GFP-p62 over time, as evident by the ratio of GFP-p62 remaining at t = 24 relative to t = 0  is a widely characterized endogenous autophagy substrate ,30. Consynthesis . InhibitAs p62 expression might be affected by various factors that are unrelated to autophagy , we alsoTo investigate the molecular mechanism by which Myc modulates autophagy, we first examined the effect of Myc knockdown on mTOR activity, which negatively regulates autophagy. The activity of mTOR can be assayed by measuring the phosphorylation its downstream targets, such as p70S6 kinase or S6 ribosomal protein . Myc knoSupplementary Material, Fig. S3).To understand the link between Myc-modulated autophagy and cancer, we tested whether the compromise of autophagy resulting from Myc inhibition was mediated through changes in Bcl2, since the unphosphorylated form of this protein inhibits Beclin 1 to decrease the production of PI3P, which would manifest in decreased DFCP1 vesicles that was observed earlier Fig. A and B. The effect of Myc knockdown on LC3-II was abrogated when Bcl2 was inhibited with HA14-1, a BH3 mimetic that interferes with the inhibitory Bcl2–Beclin 1 interaction, and thus, would be expected to mimic the functional effects of Bcl2 phosphorylation in this context . This reSupplementary Material, Fig. S4A). JNK-Inh also decreased LC3-II levels in Myc WT rat fibroblasts . These data supported the notion that Myc depletion led to autophagy impairment through decreased JNK activity and phosphorylation of its downstream target, Bcl2.To further investigate the molecular mechanism involved in Myc-modulated autophagy, we examined potential molecular players upstream of Bcl2. Activation of JNK1 has been reported to phosphorylate Bcl2, thereby inducing autophagy in cells under starvation conditions . Hence, sts Fig. C. JNK-Insts Fig. D. In addPhosphorylation of Bcl2 by JNK1 has previously been shown to result in dissociation of Bcl2 from Beclin 1, thereby relieving Beclin 1 from its inhibitory interaction with Bcl2 and allowing Beclin 1 to activate autophagy via Vps34 . We therSupplementary Material, Fig. S4C). The extent of autophagy inhibition caused by Myc-I treatment in starvation conditions appears to be greater than the magnitude of autophagy inhibition in full medium condition because starvation is capable of increasing autophagy. Similar to Myc-I, treatment with JNK-Inh failed to trigger starvation-induced autophagy, consistent with the notion that the effects of Myc inhibition on autophagy are mediated through JNK activation.In addition, as activation of JNK1 has been shown to induce autophagy under starvation conditions, we went on to investigate if Myc inhibition has the same effect on autophagy during starvation. While starvation clearly induced autophagy compared to nutrient-replete full medium, it did not increase the number of GFP-LC3 dots in cells treated with Myc-I .JNK1 is a member of the MAPK family which is activated by various stress signals, including oxidative stress, UV irradiation and injury stress associated with pro-inflammatory cytokines . Thus, w2DCFDA) or MitoSOX™ red .In addition, recent work has demonstrated that the superoxide-inducing agent, menadione, strongly enhanced autophagy . As oxidred Fig. A and B. m. Our data indicate that autophagy-lysosome attenuation with Baf A1 decreased cell viability itself and delayed the onset of response towards Myc-I in terms of inhibition of cell viability. This also implies that the effects of Myc inhibition on cell viability were at least partially dependent on the autophagy-lysosome pathway.Inhibition of Myc has been shown to trigger cell growth arrest and apoptosis in transformed cells as well as tumour xenografts –20. ThusCollectively, our data suggest that Myc knockdown, through decreased generation of ROS, resulted in less phosphorylation of JNK1 and Bcl2, which consequently led to a reduction in autophagy Fig. D.Myc plays important roles in many fundamental aspects of cell biology, such as cell proliferation, differentiation and sensitivity to apoptosis . Our stuPreviously, the anti-neoplastic effect of Myc inactivation has been associated with mechanisms such as proliferative arrest, cellular senescence, terminal differentiation and apoptosis induction ,19. NonePreviously, autophagy inhibition with chloroquine has been demonstrated to promote apoptosis and tumour regression in a Myc-induced lymphoma model . The stuElucidation of the molecular mechanisms involved in Myc regulation of autophagy is important for understanding of the feasibility and potential side effects that could arise from inactivation of Myc and its affected downstream targets. The closest observation was made by Tsuneoka and colleagues using a stable inducible expression of the MycER system in rat 3Y1 fibroblasts , wherebyIn addition to the plausible pathway that we have characterized in this study, we cannot exclude the possibility that there are other indirect pathways involved as well. Although decrease in ROS is expected to affect other members of the MAPK family, p38 MAPK and ERK were not significantly affected by Myc suppression, perhaps due to other compensatory mechanisms caused by changes in Myc activity . Nonetheml-glutamine and 100 U/ml penicillin/streptomycin at 37°C in 5% CO2. HeLa cells stably expressing the GFP-LC3 (kind gift of A.M. Tolkovsky)  and c-Myc null HO15.19 (Myc −/−) rat fibroblast cell lines (generous gift of J.M. Sedivy) , Atg5 −/lkovsky)  and HEK2lkovsky)  were cullkovsky)  were culCells were seeded 24 h before transfection such that they reached 70–80% confluency the next day. cDNA constructs or siRNA were transfected using Lipofectamine 2000 reagent (Invitrogen) in OptiMEM (Invitrogen) by following the manufacturer's protocols. pcDNA3.1 empty vector was used as a control in overexpression studies, whereas nontargeting siRNA was used in siRNA experiments .The cDNA constructs used in transfection were obtained from the following sources: pcDNA3-c-Myc from W. El-Deiry (Addgene plasmid 16011) , and Flam, Millipore), 10058-F4 , 10074-G5 , HA14-1 , JNK inhibitor VIII , Carboxy-H2DCFDA  and MitoSOX™ red .All chemicals used in this study were dissolved in dimethyl sulfoxide (DMSO) unless otherwise stated. The following chemical compounds were used for treatment at the following concentrations: bafilomycin A1 , rabbit anti-LC3 , rabbit anti-actin , mouse anti-α-tubulin , mouse anti-GFP , rabbit anti-phospho-Bcl2 , rabbit anti-Bcl2 , rabbit anti-phospho-JNK , mouse anti-JNK , rabbit anti-Beclin1 , mouse anti-p62 , rabbit anti-phospho-S6K , rabbit anti-S6K , rabbit anti-phospho-S6  and rabbit anti-S6 .m sodium chloride, 1% (w/v) sodium deoxycholate, 50 mm Tris (pH 7.4), 1% (v/v) Triton X-100, 0.25 mm EDTA, 5 mm sodium orthovanadate, 50 mm sodium fluoride, phosphatase inhibitor and protease inhibitor ] and the supernatant was collected for analysis by immunoblotting. To ensure equal loading of samples, protein concentration was determined by using a DC protein assay (Biorad) before being subjected to a SDS–PAGE.Assessment of protein levels was carried out by standard sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting techniques. In brief, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [150 mBlots were probed with primary antibodies overnight followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Roche) at 1:5000. Protein bands were detected using an enhanced chemiluminescence (ECL) western blotting kit  and densitometry quantified using ImageJ software .HeLa cells stably expressing GFP-LC3 were cultured on coverslips and subjected to siRNA knockdown or drug treatment. They were then rinsed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde, followed by mounting of coverslips on slides using Prolong Gold antifade reagent with 4’, 6-diamidino-2-phenylindole (Invitrogen). The number of GFP puncta per cell was quantified with the Spot detector V3 Cellomics Bioapplication in a Thermo Scientific Cellomics ArrayScan High Content Screening Reader. At least 300 cells were scored for each sample and the average number of dots per cell was computed by the ArrayScan software.ml-glutamine, 100 U/ml streptomycin/penicillin and 100 µg/ml hygromycin B at 37 °C, 5% CO2. To assay for p62 clearance, cells were seeded in 12-well plates and the next day, they were labelled with a diAcFam-conjugated fluorescent HaloTag ligand (Promega) according to manufacturer's instructions. Labelled cells were subsequently treated with Myc-I (60 µm) or DMSO as a control for 16 h before being harvested as described above and subjected to SDS–PAGE. Gels were scanned and fluorescence was measured with a Typhoon 8600 imager. For quantification of total protein loading, Krypton Flourescent Protein Stain (Pierce) was used following the manufacturer's instructions. Fluorescence levels were quantified by densitometric analysis using Image J software.U2OS cells stably expressing HaloTag-p62 (construct was obtained from Kazusa DNA Research Institute collection) (previously described in ) were cuDendra-tau was transfected into HeLa cells with Lipofectamine 2000. After 24 h, Dendra-tau was either photoconverted from green to red by exposure of cells to a 405 nm LED light source (constructed in-house) for 2 min or left unconverted (as controls). Both converted and unconverted cells were subjected to control or Myc siRNA knockdown for 48 h before being fixed and analysed by fluorescence-activated cell sorting (FACs) for Dendra-tau levels. The red fluorescent signal was assessed at excitation and emission wavelengths of 561/586 using a FACS analysis BD LSR Fortessa cell analyser (BD Biosciences). For compound treatment, cells were similarly photoconverted prior to treatment with the various drugs for 16 h before being analysed by FACS.m Tris–HCl, pH 7.4, 2 mm MgCl2, 150 mm NaCl, 5 mm NaF, 1 mm Na3VO4, 0.5% NP-40, protease inhibitor cocktail (Roche)) for 20 min on ice, followed by centrifugation at 13 000g for 15 min. Cell lysates were incubated with an anti-Beclin 1 antibody  overnight at 4°C, and subsequently with Dynabeads Protein G (Invitrogen) for 3 h at 4°C. Products of IP were boiled in Laemmli buffer and analysed by western blotting.Cells collected for immunoprecipitation (IP) study were lysed in Buffer A (20 m2DCFDA); or mitochondrial superoxide sensitive dye, MitoSOX™. After 72 h of siRNA knockdown, cells were washed once with PBS and incubated with phenol red-free complete medium (Gibco) containing 20 µm carboxy-H2DCFDA for 20 min or 5 µm MitoSOX™ red (Invitrogen) for 10 min at 37°C. Subsequently, cells were harvested with trypsin and washed twice with phenol red-free medium. Following this, they were resuspended in phenol red-free medium. The fluorescent signal was assessed at excitation and emission wavelengths of 488/530 using a FACS Calibur flow cytometer  for carboxy-H2DCFDA and using a BD LSR Fortessa cell analyser (BD Biosciences) for MitoSOX™ red. At least 10 000 cells were measured for each sample. Data were analysed using the FlowJo software  and presented as the geometric mean of green fluorescence for carboxy-H2DCFDA or red signal for MitoSOX™ red.HeLa cells were monitored for intracellular ROS generation using a peroxide-sensitive fluorescent probe, 2′, 7′-dichlorofluorescin diacetate (Carboxy-HCell viability was determined by using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) following the manufacturer's instructions. Briefly, 100 µl of Cell Titer-Glo reagent was added to cells grown in 100 µl of culture medium in a 96-well plate. The contents were mixed on a shaker for 5 min and incubated at room temperature for 10 min. Luminescence was then measured with a GloMax luminometer (Promega).http://rsb.info.nih.gov/ij/, 1997–2005). Protein signals were normalized to actin as loading control. The P-values were determined by two-tailed Student's t-test in at least three independent experiments. Statistical significance for comparisons between more than two groups was analyzed by one-way ANOVA with a Dunnett or Bonferroni multiple comparisons post hoc test in PRISM software.Immunoblots were quantified by densitometric analysis with the Image J program (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, USA, HMG onlineSupplementary Material is available at .Conflict of Interest statement. None declared.


SOCS3 gene expression and identified the flavanoids naringenin and flavone as effective inducers of SOCS3 protein, mRNA and promoter activity. This was in contrast with the action of traditional JAK/STAT3 inhibitors and the polyphenol resveratrol, which effectively suppress SOCS3 gene expression. Both naringenin and flavone also effectively suppressed IL-6-stimulated phosphorylation of STAT3 (Tyr705) which led to suppression of IL-6-induction of the atherogenic STAT3 target gene MCP1 (monocyte chemotactic protein-1), suggesting that their ability to induce SOCS3 gene expression is STAT3-independent. Supporting this idea was the observation that the general kinase inhibitor compound C inhibits flavone- and cAMP-dependent, but not JAK-dependent, SOCS3 induction in VECs. Indeed, the ability of flavanoids to induce SOCS3 expression requires activation of the ERK -dependent transcription factor SP3, and not STAT3. In the present paper we therefore describe novel molecular actions of flavanoids, which control SOCS3 gene induction and suppression of STAT3 signalling in VECs. These mechanisms could potentially be exploited to develop novel anti-atherogenic therapies.The atherogenic cytokine IL-6 (interleukin-6) induces pro-inflammatory gene expression in VECs  by activating the JAK (Janus kinase)/STAT3  signalling pathway, which is normally down-regulated by the STAT3-dependent induction of the E3 ubiquitin ligase component SOCS3 . Novel treatments based on the regulation of SOCS3 protein levels could therefore have value in the treatment of diseases with an inflammatory component, such as atherosclerosis. To this end we carried out a screen of 1031 existing medicinal compounds to identify inducers of  The NINDS-II [NINDS  Custom Collection II] was obtained from MRCT .ECL reagents and secondary antibodies (horseradish peroxidase-conjugated anti-rabbit-IgG and horseradish peroxidase-conjugated anti-mouse-IgG) were bought from GE Healthcare. HUVECs  and endothelial cell growth medium 2 were obtained from PromoCell. Dulbecco's PBS was from Sigma–Aldrich. Forskolin, rolipram, PMA, compound C and MG132 were obtained from Merck. 5,7-dihydroxy-2-(4-hydroxyphenyl) chroman-4-one (naringenin) and 5-[(E)-2-(4-hydroxyphenyl)-vinyl]-1,3-benzenediol (Socs3 promoter construct (pGL3-SOCS3-107Luc) was a gift from Professor J.G. Bode  with permission from Professor Shlomo Melmed . This plasmid contains the promoter region −107 to +929 of the murine Socs3 gene fused to the coding region of firefly luciferase as described previously . Protein samples were then separated by SDS/PAGE (10% gels), transferred on to nitrocellulose, blocked for 1 h at room temperature in 5% (w/v) BSA, and then immunoblotted with antibodies specific for SOCS3 (Santa Cruz Biotechnology), phospho-STAT3  according to the manufacturer's instructions. RT–PCRs were carried out in a 25 μl reaction mixture containing 5–10 ng of RNA, 0.4 mM dNTPs and 0.6 μM of each primer using the OneStep RT-PCR Kit (Qiagen). The primer sequences used were: hSOCS3 , hActin  and hMCP-1 . Reactions were carried out in a thermocycler with the following settings: 30 min at 50°C, 15 min at 95°C, 30 cycles of 30 s at 94°C, 30 s at 50°C and 1 min at 72°C, followed by 10 min at 72°C. PCR products were visualised on 2% (w/v) agarose gels following ethidium bromide staining.2], washed with PBS (1×1 ml) and then incubated with 300 μl/dish of sample diluent from the DetectX High Sensitivity Direct Cyclic AMP Chemiluminescent Immunoassay kit from Arbor Assays. Cells were incubated with occasional mixing for a further 10 min at room temperature, scraped into Eppendorf tubes and then centrifuged at 1000 g for 15 min at 4°C. Supernatants were transferred to fresh tubes and frozen at −70°C. Detection of cAMP levels in standards and experimental samples was performed according to the manufacturer's protocols and chemiluminescent signals were captured using a BMG Labtech luminometer set to a 0.1 s read time per well of a 96-well plate.HUVECs were incubated with pharmacological stimuli in 30 mm dishes for 5 h [37°C, 5% (v/v) COData were analysed using one-way ANOVA with a Tukey–Kramer post-test.SOCS3 gene expression we carried out a high-throughput screen of small molecules capable of inducing the expression of the minimal human SOCS3 gene promoter [SOCS3 promoter-expressing COS-1 cells were stimulated for 16 h with each compound and then luciferase activities were determined using a luminometer. The details of individual compounds and their respective abilities to regulate SOCS3 promoter activity are detailed in the Supplementary Data File (at http://www.biochemj.org/bj/454/bj4540283add.htm). Of particular note from the screen was the observation that two structurally related flavanoid compounds, 5,7-dihydroxy-2-(4-hydroxyphenyl) chroman-4-one (naringenin) and 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (apigenin), both produced a robust (approximately 4-fold) induction of SOCS3 promoter activity ; 705 phosphorylation demonstrated that naringenin began to exert a significant inhibitory effect after approximately 3 h stimulation . From these results it is clear that 100 μM naringenin effectively inhibits tyrosine phosphorylation of STAT3 and it is therefore unlikely that the effects of naringenin on SOCS3 induction are related to activation of STAT3 signalling. Indeed, results suggest that the ability of naringenin to inhibit tyrosine phosphorylation of STAT3, and yet induce SOCS3 expression, may occur through distinct signalling mechanisms.We first examined the effects of naringenin on IL-6-regulated STAT3 activation, since STAT3 is a principle regulator of n HUVECs . We founmulation b. Anotheactivity . HoweverSOCS3 promoter activity and inhibit STAT3 Tyr705 phosphorylation , since previous work implicates a role for flavanoids in modulating AMPK activity [SOCS3 gene induction. Stimulation of HUVECs with flavone was found to elicit a degree of AMPK activation, as determined by immunoblotting cell extracts with phospho-specific antibodies that detect the AMPK substrate ACC (Ser79); however, direct activation of AMPK with the specific AMPK agonist 10 μM A769622 failed to induce SOCS3 expression  by both 10 μM compound C and the inhibitor of ERK activation 1 μM PD184352 [SOCS3 gene expression. We therefore investigated the roles of cAMP and ERK signalling in mediating the actions of flavone on SOCS3 gene induction.To this end we tested the ability of compound C to inhibit SOCS3 induction promoted by activation of the PKC (protein kinase C) pathway, the JAK/STAT3 signalling pathway and the cAMP cascade, since all of these pathways have previously been implicated in controlling n HUVECs ,21,24,29n HUVECs . AccordiPD184352 . That cASOCS3 gene expression by cAMP is dependent on the combined actions of cAMP on EPAC1 activation and ERK signalling in HUVECs [http://www.biochemj.org/bj/454/bj4540283add.htm), indicating that both flavone and resveratrol could potentially enhance cAMP production either through inhibition of CK2 or PDE4. However, direct measurement of cAMP levels following treatment of HUVECs with combinations of flavone, resveratrol, rolipram and forksolin demonstrated that rolipram, and to a minor extent resveratrol, but not flavone, were able to potentiate forskolin-induced cAMP synthesis in HUVECs , indicating that flavone induces SOCS3 gene induction independently of cAMP. To determine whether the combined actions of flavanoid treatment and IL-6 stimulation effected cAMP levels, we examined whether co-stimulation of HUVECs with IL-6 and forskolin, and either flavone, reseveratrol or rolipram, effects phosphorylation of the PKA (protein kinase A) substrate VASP on Ser157 . Results demonstrated that IL-6 did not promote VASP Ser157 phosphorylation, after either 30 min or 5 h stimulation . In contrast, a combination of forskolin and IL-6 promoted a robust phosphorylation of VASP after 30 min stimulation, which was enhanced by 5 h co-treatment with rolipram and, to a lesser extent, resveratrol, but not by flavone . Taken together these results indicate that the ability of flavone to induce SOCS3 expression and inhibit IL-6 promoted STAT3 activation occurs independently of elevations in intracellular cAMP in HUVECs.We have previously demonstrated that induction of n HUVECs ,32. We tn HUVECs , an enzyn HUVECs , and than HUVECs , we deciSOCS3 gene expression [SOCS3 promoter activity in transfected COS-1 cells and SOCS3 protein synthesis in HUVECs . Indeed, IL-6-promoted phosphorylation of STAT3 on Tyr705 appears to be enhanced, rather than inhibited, in the presence of PD184352 and AZD6244 (SOCS3 gene induction in response to flavone treatment. Indeed, our previous work has demonstrated that the activity of the minimal SOCS3 promoter is dependent on basal activity of ERK and the ERK-targeted transcription factors AP1 and SP3, in addition to STAT3 [SOCS3 promoter in luciferase reporter assays as described previously [Socs3 promoter spanning −49 to −2757 nt from the initiating ATG (SOCS3 gene induction by flavanoids. This idea is supported by further experiments where we ablated individual transcription factor-binding sites within the SOCS3 minimal promoter (SOCS3 promoter activity (SOCS3 gene activity. This idea is supported by observations that treatment of COS-1 cells with naringenin led to an increase in both AP1 and SP3 transcription factor activity (trans-resveratrol, which generally suppressed AP1, STAT and SP3 transcription factor activity (SOCS3 gene activity, the ERK inhibitor AZD6244 suppressed both basal and naringenin-stimulated SP3 transcription factor activity (SOCS3 gene activity by flavanoids.We also noted that neither PD184352 nor AZD6244 affected the ability of resveratrol and flavone to block IL-6-promoted STAT3 activation b, indica AZD6244 b. These eviously ,24. We fting ATG a. We fouting ATG a. Furtheting ATG a. Howeveting ATG a, suggespromoter b. We fouactivity b. Howeveactivity b, indicaactivity c, as detactivity c. In addactivity d. MoreovSOCS3 gene expression. The ability of these compounds to induce SOCS3 gene expression appears to rely on ERK-dependent SP3 transcription factor activation (in vivo measurements have demonstrated that flavone acetate exerts anti-neoplastic actions at a plasma concentration of around 360 μM and is acutely lethal at 2.1 mM [In summary, in the present paper we describe a new class of chemical probes based on the flavone core structure that have the novel combined actions of STAT3 inhibitor and inducer of tivation . Our objtivation c. Since t 2.1 mM . Plasma t 2.1 mM . Thus th


IgG4 is believed to act as a blocking antibody preventing IgE binding to Bet v 1, thus alleviating allergic symptoms. Only little information on the location and relationship of IgE and IgG4 binding sites of Bet v 1 is available. In this study we seek to identify epitopes of IgE and IgG4 antibodies on Bet v 1.Millions of patients with allergy to birch pollen develop clinically cross-reactive IgE against Bet v 1-like proteins in plant foods. Specific immunotherapy (SIT) with birch pollen extracts induces the biosynthesis of Bet v 1-specific immunoglobulin (Ig)G4 antibodies by bioinformatic means. Predicted epitope residues potentially critical for antibody binding were substituted by site-directed mutagenesis. Recombinant Bet v 1 (rBet v 1) and rBet v 1 variants were purified from Escherichia coli. The proteins were physicochemically characterized using circular dichroism (CD) and dynamic light scattering. To test the IgE and IgG4 interactions with rBet v 1 variants, western blot analyses, ELISA, and cellular mediator release assays were performed.A competitive immunoscreening of phage-displayed peptides was applied to predict Bet v 1 epitopes of allergen-specific IgE and IgGE. coli. Circular dichroism and structural modeling of the variants revealed Bet v 1-like theoretical secondary structure topology. The rBet v 1 variants showed reduced IgE and IgG4 binding with sera of birch pollen allergic subjects in western blot analyses and competitive ELISAs. The rBet v 1 variants showed decreased IgE-mediated mediator release in humanized rat basophil leukemia cells sensitized with sera of birch pollen allergic subjects.Several rBet v 1 variants were expressed in 4epitopes of Bet v 1. Although patient-specific variability was observed, the antibody interactions of the respective rBet v 1 variants were compromised for both IgE and IgG4, respectively. We conclude that epitopes for IgE and IgG4 share common residues critical for antibody interaction, suggesting an overlap of IgE and IgG4-binding sites on the molecular surface of Bet v 1. The knowledge of clinically relevant immunoglobulin-allergen interactions on the molecular level enables new strategies in the diagnosis, prognosis, and therapy of both birch pollen allergies and birch pollen-related food allergies.We identified critical residues in IgE and IgGNone declared.


Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL  resembles a contractile phage tail structure and functions to deliver effectors to eukaryotic or prokaryotic target cells for the survival of many pathogenic bacteria. T6SS is highly regulated by various regulatory systems at multiple levels in response to environmental cues. Post-translational regulation via threonine (Thr) phosphorylation is an emerging theme in regulating prokaryotic signaling, including T6SS; the knowledge is mainly contributed by studies of Hcp secretion island 1-encoded T6SS (H1-T6SS) of  The type VI secretion system (T6SS) is the most recently described protein secretion system encoded as one or multiple copies in ∼25% of all sequenced Gram-negative bacteria Escherichia coli Sci-1 T6SS Francisella novicida cytoplasmic TssL revealed a surface-exposed groove that may represent a functional site for T6SS function Bioinformatics and mutagenesis analyses revealed that T6SS consists of approximately 13–14 conserved components of type VI secretion  P. aeruginosa, in which the Hcp secretion island 1-encoded T6SS (H1-T6SS) is regulated positively by PpkA and negatively by the cognate phosphatase PppA via Thr phosphorylation on an FHA domain-containing protein, Fha1 Serratia marcescenstype VI secretion associated genes Q, R, S, T (TagQRST) function upstream of PpkA to promote kinase activity and subsequent type VI secretion in P. aeruginosaP. aeruginosa surface growth P. aeruginosa T6SS dueling activity when encountering the T6SS attack via contact with Vibrio cholerae cells Interestingly, a subset of T6SS gene clusters also encode orthologs of Ser/Thr protein kinase (PpkA), phosphatase (PppA), and forkhead-associated (FHA) domain-containing proteins that are involved in regulating the Thr phosphorylation event Mycobacterium tuberculosis encoding 11 Ser/Thr protein kinases and 7 FHA domains in 6 proteins P. aeruginosaAlthough quite common in eukaryotes, this Ser/Thr phosphorylation regulatory mechanism is an emerging theme in prokaryotic signaling Agrobacterium tumefaciens, a soil phytopathogen capable of causing crown gall disease in wide range of plants. We discovered that TssL, a T6SS core component of the inner-membrane complex A. tumefaciens is transcriptionally activated by the ExoR-ChvG/ChvI signaling cascade upon sensing the acidic signal secreted small protein (Ssp) in S. marcescensp-TssL) and this specific p-TssL-Fha interaction is required for efficient binding of Hcp and Atu4347 to p-TssL and activation of type VI secretion. We proposed that PpkA kinase initiates the complex assembly by phosphorylating TssL to increase the ATP binding ability of TssM ATPase for energy production and leads to the recruitment of Fha for activation of type VI secretion.In this study, we investigated the involvement of Thr phosphorylation in regulating type VI secretion in P. aeruginosaS. marcescensA. tumefaciens strain C58  Thr phosphorylation pathway (TPP) may play a role to regulate type VI secretion in A. tumefaciens, but the molecular mechanisms underlying this potential Thr phosphorylation regulation remain unknown.The presence of genes encoding the Fha-family protein, PpkA kinase, and PppA phosphatase in a subset of T6SSs suggested the involvement of a Thr phosphorylation regulatory pathway in these bacteria rain C58  [11], . Tos 1–255)  [20]. Bes 1–255) , Thr 14 ssLT14A) . TherefoppkA mutant encoding PpkAD161AN166A with alanine substitutions in 2 conserved Asp 161 (D161) and Asn 166 (N166) residues located within the kinase magnesium binding loop . Not surprisingly, purified TssL-His remained with the wild-type phosphorylation pattern in fhaR30AS46A by Phos-tag gel  or glutamic acid (E) substitutions (TssLT14D and TssLT14E). We observed complete loss of Hcp secretion from ΔtssL and fhaR30AS46A and low levels of secreted Hcp proteins from ΔppkA and TssL phosphorylation site mutants   . ConsistE. coli (data not shown), we purified truncated Fha proteins with different lengths for the ITC experiments , although with relatively low affinity (dK 1.58±0.35 mM) (dK 0.54±0.07 mM) with a longer phosphorylated TssL peptide   or the 6 mer phosphorylated CHK2 peptide  as controls R30AS46A) completely lost the ability to interact with the 8 mer p-TssL peptide (p-TssL peptide but not unphosphorylated TssL peptide with a longer version of the truncated Fha protein (Fha7-309WT)  . The binL20 mer) . These d peptide . We show7-309WT) . These dp-TssL-Fha complex formation in vivo by pulldown assays using functional Strep-tagged TssL  as compared with the wild type and fhaR30AS46A strains before protease treatments  phosphorylation site of TssL is highly conserved in all analyzed Agrobacterium and Rhizobium species that also encode Thr phosphorylation components  and no phosphorylation of TssL could be detected in ΔppkA . The formation of this TssM-p-TssL-Fha complex combined with TssM ATP hydrolysis activates the efficient recruitment of Hcp and Atu4347 into this T6SS inner-membrane subassembly for type VI tube assembly and effector secretion across outer membrane (step V). The direct evidence for formation of this inner-membrane subassembly remains lacking and other alternative or multiple pathways may exist. Future work to investigate whether and how TssM ATP binding/hydrolysis influence Fha recruitment to activate type VI complex formation and secretion shall provide further mechanistic insights in type VI assembly and secretion.Taken together, we proposed a hypothetical model for the TssL phosphorylation-induced T6SS assembly pathway . The forS. marcescens identified Fha but not TssL as PpkA-phosphorylated substrate P. aeruginosa, Fha1 focally co-localizes with ClpV1 and is required for ClpV1 focal recruitment tssM (icmF) for ClpV1 foci recruitment in P. aeruginosap-TssL inner-membrane complex. Our findings now extend the knowledge that phosphorylation of TssL may function to initiate the inner-membrane subassembly formation, for which Fha may come into play at a later stage for recruiting ClpV1 for TssB-TssC tubule disassembly. Future work to determine the conservation or uniqueness of Fha and TssL phosphorylation in different bacteria and to investigate their hierarchy and roles for type VI complex assembly will elucidate the mechanistic role of Thr phosphorylation in T6SS. Only a subset of T6SS-containing bacterial species encodes the Thr phosphorylation components Recent phosphoproteomic analysis in A. tumefaciens and Escherichia coli strains were grown at 28°C in 523 A. tumefaciens and 100 µg/ml ampicillin (Ap), and 50 µg/ml Gm for E. coli.Strains, plasmids, and primer sequences used in this study are in A. tumefaciens cells grown in liquid AB-MES medium (pH 5.5) A. tumefaciens C58 via double crossover using the suicide plasmid pJQ200KS All in-frame deletion and amino acid substitution(s) mutants were generated in A. tumefaciens strains were resuspended in 1× CIAP buffer containing 100 mM NaCl, 50 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT, and 1× protease inhibitor cocktail (EDTA-free) with CIAP at 1 unit per µg of protein. The protein samples treated with or without CIAP were incubated at 37°C for 90 min. An equal volume of 2× SDS loading buffer was added and incubated at 96°C for 20 min and analyzed by Phos-tag SDS-PAGE.Dephosphorylation analysis by calf intestinal alkaline phosphatase (CIAP) was performed according to the user manual  with minor modifications. Equal amounts of Ni-NTA resins with purified TssL-His or total protein extracts isolated from various 2, with electrophoresis conducted at 40 mA/gel under a maximum voltage of 90V in a cold room. After electrophoresis, Phos-tag gels were washed with transfer buffer  containing 1 mM EDTA for 15 min with gentle shaking followed by a second wash in transfer buffer without EDTA for 15 min. The gels were washed with transfer buffer containing 1% SDS for 15 min before transfer to PVDF membranes with a submarine blotting apparatus.The Phos-tag SDS-PAGE analysis was performed according to the user manual for Phos-tag Acrylamide AAL-107  with minor modifications. Protein samples were separated on 7% polyacrylamide gels containing 0.35 M Bis-Tris-HCl (pH 6.8), 35 µM Phos-tag Acrylamide AAL-107, and 100 µM ZnClA. tumefaciens were purified by use of Ni-NTA resins and separated by 12% SDS-PAGE followed by Coomassie blue staining. TssL-His protein bands were cut out for in-gel trypsin or trypsin/chymotrypsin digestion as described Phosphorylation site mapping was determined by in-gel trypsin or trypsin/chymotrypsin digestion of purified TssL-His followed by nanoLC/nanospray/tandem mass spectrometry (LC-ESI/MS/MS) analysis. TssL-His proteins expressed in A. tumefaciens. The pulldown assay with Strep-Tag was performed according to the user manual for StrepTactin Sepharose . In total, 500 ml A. tumefaciens cell cultures were centrifuged and washed 3 times with 12 ml phosphate buffer  and resuspended in the same buffer adjusted to OD600 about 4. Membrane-permeable cross-linker dimethyl 3,3′-dithiobispropionimidate (DTBP) was added at a final concentration of 5 mM, and the mixture was incubated at room temperature for 45 min. The reaction was stopped by adding Tris-HCl (pH 7.6) to a final concentration of 20 mM for 15 min. The cells were collected by centrifugation and washed twice with 12 ml of 50 mM Tris-HCl (pH 7.6) before pulldown assay. The harvested cells were resuspended in binding buffer  containing 0.05% Triton X-100 and underwent sonication on ice. The cell lysate was centrifuged twice at 10,000× g for 15 min at 4°C. The soluble fraction was passed through Strep-Tactin resins  and washed 6 times with binding buffer  containing 0.05% Triton X-100. The bound proteins were eluted by use of elution buffer . The fractions were examined by western blot analysis.To avoid the non-specific protein–protein interactions occurring when proteins are released into solution after cell breakage, we used the cleavable and membrane permeable cross-linker dimethyl 3,3′-dithiobispropionimidate (DTBP) to cross-link interacting proteins before cell lysis 200 system  and analyzed with ORIGIN software. Truncated His-tagged Fha7–267 and Fha7–309 proteins were overexpressed and purified from E. coli BL21(DE3) as described p-TssL8 mer; DNPSSWQDLPpTVVEITEESR, p-TssL20 mer), unphosphorylated TssL , and phosphorylated CHK2  7–267 or Fha7–309 proteins at 25°C with a total 25 times of injections with 200 µM protein in the sample cell and 6 mM peptide in the injection syringe. The titration heat was calculated to eliminate the effect of heat generated from titrating the ligand into buffer. Thermal data were fitted to the One Set of Sites binding model with the N value fixed at 1 to yield the value of the equilibrium dissociation constant (dK).ITC analysis was used to determine the binding affinity and stoichiometry between Fha and TssL T-14 peptide with use of the MicroCal iTCA. tumefaciens cells were grown in liquid AB-MES medium (pH 5.5) Streptomyces griseus protease (Sigma) at a final concentration of 12.5 or 25 µg/ml for 10 min on ice in the presence of 10 mM MgSO4. The reaction was stopped by adding an equal amount of 2× SDS loading buffer and incubated at 96°C for 20 min before SDS-PAGE.Spheroplast preparation and protease susceptibility assay were performed as described 41, TssB, TssA, ClpV, Atu4347, VgrGs, RpoA, AopB) Western blot analysis was performed as described Figure S1Western blot analysis of T6SS components in various strains. Western blot analysis of total proteins from various Agrobacterium tumefaciens strains resolved by 10% or 12% Glycine-SDS-PAGE and examined with specific antibodies. The soluble protein ActC and RNA polymerase α subunit (RpoA) were internal controls. The proteins analyzed are indicated on the left, the molecular weight standards are on the right, and with arrows when necessary. The TssL protein band with faster migration in ΔppkA mutant is marked with an asterisk.(TIF)Click here for additional data file.Figure S2Alignment of the amino acid sequences of Fha orthologs. Amino acid sequences of Fha-family proteins from Pseudomonas aeruginosa , A. tumefaciens , Rhizobium leguminosarum bv. viciae 3841 , R. leguminosarum bv. trifolii WSM2297 , Rhodobacter sphaeroides WS8N , Bradyrhizobium japonicum , Pseudomonas fluorescens , Shewanella frigidimarina , Vibrio parahaemolyticus , Xanthomonas axonopodis pv. citri , and Xanthomonas campestris pv. vesicatoria . Identical amino acid residues are highlighted in black. Conserved amino acid residues in FHA domain used for mutagenesis are indicated with red asterisks, and Thr 362 phosphorylation site of Fha1 of P. aeruginosa is indicated with an arrowhead. Sequences were aligned and highlighted by use of ClustalW2 .(TIF)Click here for additional data file.Figure S3Protein secretion assay and Phos-tag SDS-PAGE analysis. (A) His-tagged or Strep-tagged TssL has full function in mediating Hcp and Atu4347 secretion. Western blot analysis of total (T) and secreted (S) protein isolated from various A. tumefaciens strains grown in AB-MES (pH 5.5) for 6 h at 25°C and separated by 12% Glycine-SDS-PAGE. The secreted proteins were collected from 1 ml of culture medium after removal of bacterial cells by centrifugation and were concentrated by TCA precipitation B) Phos-tag SDS-PAGE analysis of TssL-His. Total proteins isolated from ΔtssL(pTssL-His) grown in AB-MES (pH 5.5) for 6 h at 25°C were diluted (1× served as 0.5 µg/µl) and treated with (+) or without (−) calf intestinal alkaline phosphatase (CIAP). Western blot analysis of protein samples separated by 7% Phos-tag SDS-PAGE and examined with specific antibody against 6×His. Total protein isolated from ΔtssL mutant was a negative control. Phos-tag SDS-PAGE revealed the upper band indicating the phosphorylated TssL-His (p-TssL-His) and lower band indicating unphosphorylated TssL-His. (C) Phos-tag SDS-PAGE analysis for Fha. Western blot analysis of total protein isolated from wild-type C58 grown in AB-MES (pH 5.5) for 6 h at 25°C that was diluted (1× served as 0.5 µg/µl), treated with (+) or without (−) CIAP and separated by 7% Phos-tag SDS-PAGE and examined with specific antibodies against 6×His and Fha. TssL-His was pulled down by Ni-NTA binding beads and treated with (+) or without (−) CIAP as a positive control. Total protein isolated from Δt6 was a negative control. No phosphorylated Fha protein could be detected. The upper band detected in both C58 and Δt6 mutant was a non-specific cross-reacted protein as indicated.(TIF)Click here for additional data file.Figure S4Sequence coverage and phosphorylated Thr 14 of TssL-His by Mass spectrometry identification. Sequence coverage of TssL-His by MS/MS analysis is marked in red and the phosphorylated Thr 14 is indicated by a blue box. Thr 14 is the only phosphorylation site of TssL-His detected by MS from samples prepared by trypsin only or trypsin/chymotrypsin double digestion. Similar results were obtained from 3 independent experiments.(TIF)Click here for additional data file.Figure S5Amino acid sequence alignment of PpkA orthologs. Partial amino acid sequences of PpkA orthologs from A. tumefaciens C58 , P. aeruginosa PAO1 , Mycobacterium tuberculosis UT205 , and Yersinia enterocolitica . Identical amino acid residues are highlighted in black. Amino acid residues used for mutagenesis are indicated with red asterisks. Sequences were aligned and highlighted by use of ClustalW2 .(TIF)Click here for additional data file.Figure S6SDS-PAGE analysis of purified Fha proteins and isothermal titration calorimetry (ITC) analysis. (A) Truncated Fha proteins tagged with 6×His were purified with Ni-NTA resins followed by HiLoad 16/600 Superdex75 pg column. Five µg of purified Fha proteins  was analyzed by 12% SDS-PAGE and stained by Coomassie blue to ensure protein purity. Molecular markers are indicated on the left (kDa). (B) ITC of specific interaction and binding kinetics between the Fha7-309WT protein and phosphorylated TssL peptide . The equilibrium dissociation constant (dK) between Fha7-309WT protein and DLPpTVVEI (p-TssL8 mer) is 2.15±0.01 mM. The binding affinity of Fha7-309WT protein and DLPTVVEI unphosphopeptide (TssL8 mer) is undetectable. Top panel shows the raw calorimetric data for the interaction and bottom panel the integrated heat changes, corrected for heat of dilution, and fitted to a single-site binding model.(TIF)Click here for additional data file.Figure S7N-terminal amino acid sequence alignment of A. tumefaciens TssL with TssL orthologs encoded by closely related bacterial species. Alignment of first 50 amino acid sequences of TssL orthologs identified from various Agrobacterium and Rhizobia strains were performed. The identical amino acid residues are highlighted in black and the conserved T14 residue is indicated with a red arrowhead. The analyzed proteins are TssL/Atu4333/OmpA-like porin [Agrobacterium tumefaciens C58]/(GI:159186121), OmpA-like porin [A. tumefaciens 5A]/(GI:418409003), OmpA-like porin [A. tumefaciens CCNWGS0286]/(GI:418297583), OmpA-like porin [Agrobacterium sp. ATCC 31749]/(GI:335037634), OmpA-like porin [Agrobacterium sp. H13-3]/(GI:332715427), OmpA-type porin [Agrobacterium vitis S4]/(GI:222106979), Type VI secretion system OmpA/MotB family protein [Rhizobium leguminosarum bv. trifolii WSM597]/(GI:424918217), Type VI secretion system OmpA/MotB family protein [R. leguminosarum bv. trifolii WSM2297]/(GI:424892095), Nitrogen fixation outer membrane porin [R. leguminosarum bv. viciae 3841]/(GI:116249130). Sequences were aligned and highlighted by use of ClustalW2 .(TIF)Click here for additional data file.Figure S8Amino acid sequence alignment of A. tumefaciens TssL with TssL orthologs encoded by distantly related bacterial species. Amino acid sequence of TssL orthologs from A. tumefaciens , Pseudomonas aeruginosa PAO1 , P. tolaasii , Pseudomonas fluorescens , Pseudomonas mandelii , Vibrio cholerae O1 biovar EI Tor str. N16961 , Edwardsiella tarda , Burkholderia mallei , Salmonella typhimurium , Burkholderia sp. WSM4176 , Acidovorax avenae subsp. avenae ATCC 19860 , Thiorhodococcus drewsii , Shewanella baltica BA175 , enteroaggregative Escherichia coli Sci-1 T6SS , and Francisella novicida U112 . Part of the aligned result is shown here. Identical amino acid residues are highlighted in black. The T14 residue of TssL of A. tumefaciens is indicated with a red arrowhead. Sequences were aligned and highlighted by use of ClustalW2 .(TIF)Click here for additional data file.Figure S9Secretion and western blot analyses assay on agar plate. Western blot analysis of total and secreted (Sup) proteins isolated from various A. tumefaciens strains grown on agar plates were resolved by 10% or 12% Glycine-SDS-PAGE and examined with specific antibodies. ActC and RpoA were used as internal controls. The proteins analyzed are indicated on the left, and the molecular weight standards are indicated on the right and with arrows when necessary.(TIF)Click here for additional data file.Figure S10Overexpression of TssL with T14A substitution in ΔtssL is unable to complement type VI secretion. The ΔtssL mutant harboring the vector pRL662 (V) or complemented plasmid pTssL or pTssLT14A was analyzed for Hcp and Atu4347 secretion. Western blot analysis of total (T) and secreted (S) proteins isolated from various strains grown in AB-MES (pH 5.5) for 6 h at 25°C and separated by 12% Glycine-SDS-PAGE and examined with specific antibodies. The non-secreted protein ActC and RpoA were internal controls for secretion and protein accumulation analyses, respectively. The proteins analyzed and the molecular weight standards are indicated on the left and right, respectively, and with arrows when necessary. The TssL with different migrations are marked with black asterisks.(TIF)Click here for additional data file.Figure S11TssM is not required for TssL phosphorylation. (A) Western blot analysis with regular SDS-PAGE of total proteins isolated from various A. tumefaciens strains examined with specific antibodies. RNA polymerase α subunit RpoA was an internal control. The TssL protein band with faster migration in ΔppkA mutant is marked with an asterisk. (B) Phos-tag SDS-PAGE analysis. Western blot analysis of TssL-His proteins purified by Ni-NTA resins were separated by 7% Phos-tag SDS-PAGE and examined by specific antibody against 6×His. Total proteins from ΔtssL mutant were a negative control. Phos-tag SDS-PAGE revealed the upper band indicating phosphorylated TssL-His (p-TssL-His) and lower band indicating unphosphorylated TssL-His. The proteins analyzed and the molecular weight standards are indicated on the left and right, respectively.(TIF)Click here for additional data file.Table S1Bacterial strains and plasmids.(PDF)Click here for additional data file.Table S2Primer information.(PDF)Click here for additional data file.Information S1Supporting Information.(PDF)Click here for additional data file.


People can inhibit an action because of an instruction by an external stimulus, or because of their own internal decision. The similarities and differences between these two forms of inhibition are not well understood. Therefore, in the present study the neural correlates of intentional and stimulus-driven inhibition were tested in the same subjects. Participants performed two inhibition tasks while lying in the scanner: the marble task in which they had to choose for themselves between intentionally acting on, or inhibiting a prepotent response to measure intentional inhibition, and the classical stop signal task in which an external signal triggered the inhibition process. Results showed that intentional inhibition decision processes rely on a neural network that has been documented extensively for stimulus-driven inhibition, including bilateral parietal and lateral prefrontal cortex and pre-supplementary motor area. We also found activation in dorsal frontomedian cortex and left inferior frontal gyrus during intentional inhibition that depended on the history of previous choices. Together, these results indicate that intentional inhibition and stimulus-driven inhibition engage a common inhibition network, but intentional inhibition is also characterized by additional context-dependent neural activation in medial prefrontal cortex. In daily life, most people experience and exercise a degree of voluntary control over their actions. The concept of intentional action is well recognized in the neuroscience literature. Several studies have focused on the voluntary choice between alternative actions , and the voluntary choice of when to initiate action (the “when–component”) . Within neuroscience research stimulus-driven inhibition has been extensively studied using different paradigms, such as go/nogo tasks  also involved in intentional inhibition, or is a different neural network involved in intentional inhibition (including dFMC)? Additionally, is intentional inhibition dependent on preceding context, as has been previously observed for stimulus-driven inhibition  participated in the experiment. All participants had normal or corrected-to-normal vision, and no neurological or psychiatric impairments according to self-report. Before participating in the experiment, all participants signed informed consent. In accordance with guidelines of the Leiden University Medical Center, all anatomical scans were reviewed by a radiologist. No anomalous findings were reported. To obtain an estimate of cognitive functioning participants completed two subtests of the Wechsler Adult Intelligence Scale (WAIS) .Twenty-four healthy right-handed adults between 18 and 26 years of age  and instructed (green) trials discouraged participants from strategically pre-deciding a pattern of intentional action, such as act-inhibit-act-inhibit etc.The stop-signal task  a stop-signal was presented. In this case the arrow suddenly changed color to red. This color change indicated that participants had to inhibit responding to the direction of the arrow. Stop-signal delay (SSD) was adjusted using a staircase-tracking procedure to guarantee a 50% inhibition rate . The first 2 volumes of each run were discarded in order to allow for equilibration of T1 saturation effects . After the functional runs a high-resolution 3D T1-FFE scan for anatomical reference was obtained . Head motion was restricted by using foam inserts between the head and the head coil. Visual stimuli were projected onto a screen in the magnet bore that could be viewed through a mirror attached to the head coil.Scanning was performed with a standard whole-head coil on a 3.0 Tesla Philips scanner at the Leiden University Medical Center. The marble task consisted of 3 event-related runs, each lasting approximately 6 min, and the stop-signal task consisted of 2 event-related runs, both lasting approximately 5 min. Functional data were acquired using T2For the marble task, repeated measures analyses of variance were performed to examine the effect of preceding context on intentional inhibition. Planned comparisons were performed between the different numbers of preceding green trials, to examine which conditions differed from each other.The use of response selection strategies on the marble task was evaluated by computing the Random Number Generation 2 (RNG2) index using the program RgCalc  was calculated according to the horse-race model of stopping . Images were corrected for rigid-body motion. Structural and functional volumes were spatially normalized to T1 templates. The normalization algorithm used a 12-parameter affine nonlinear transformation involving cosine basis functions, and then resampled the volumes to 3-mm cubic voxels. Translational movement parameters never exceeded 1 voxel (<3 mm) in any direction for any subject or scan. Templates were based on the MNI305 stereotaxic space .Region of interest (ROI) analyses were performed to further characterize the involvement of brain regions in intentional inhibition. ROI analyses were performed with the MarsBaR toolbox in SPM8  = 18.09, p < 0.001  and when there was 1 compared to 3 preceding green trials (p < 0.05). The level of inhibition was comparable for the conditions where there were 1 or 2, and 2 or 3 preceding green trials .Participants successfully responded to the green marble on 63.22% of the trials. Participants intentionally inhibited responding to the white marble on 53.17% of the trials. Participants more often decided to inhibit responding to the white marble when there were fewer preceding green trials, SD = 0.012) was observed. To examine the randomness the participants' RNG2 index was compared with a RNG2 index computed over a set of randomly generated sequences of go- and nogo-responses. For the randomly generated set of go- and nogo-response sequences a mean RNG2 index of 0.801 (SD = 0.002) was observed. Although the RNG2 index for the randomly generated sequences was marginally but significantly smaller compared to the participants' RNG2 index, F = 5.71, p < 0.05, these results indicate that the participants' behavior was close to being random and not driven by simple alternation strategies.To examine the randomness of response selection the RNG2 index was computed. A mean RNG2 index of 0.807  compared to the white marble trials , F = 32.55, p < 0.001, indicating that the decision process in the white marble trials took more time. However, longer reaction times on the white marble trials might also be partially related to the slower marble speed on those trials. Reaction times on the white marble trials were faster when there were more preceding green trials, F = 5.52, p < 0.01 . Reaction times did not differ between the other conditions of preceding green trials .Reaction times were shorter for the green marble trials (SD = 133). Participants successfully inhibited responding to the direction of the arrow on 46.03% of the stop-trials. SSRT was 281 ms (SD = 45).Participants successfully responded to the direction of the arrow on 96.46% of the go-trials. The average reaction time on the successful go-trials was 519 ms  was not correlated with stimulus-driven inhibition as measured by the stop-signal task (SSRT), First, to identify the brain regions underlying the intentional inhibition decision process the contrast intentional inhibition > stimulus-driven action (White NoGo > Green Go) was computed. This analysis revealed activation in a widespread neural network . This analysis revealed activation in bilateral IPL, left IFG, left MFG, right medial temporal gyrus (MTG), and occipital lobe  . For left IFG pars orbitalis and pars triangularis contrast values were significantly different from zero when there were 0 preceding green trials .In order to examine the effect of the number of preceding green trials on intentional inhibition decision outcomes, a parametric analysis of the number of preceding green trials was performed on the contrast intentional inhibition > intentional action (White NoGo > White Go). This analysis revealed stronger activation in dFMC, left IFG pars orbitalis, left IFG pars triangularis, and right SFG when there were fewer preceding green trials  both intentional action and intentional inhibition decisions resulted in a large network of activation including the lateral prefrontal cortex, parietal cortex, and preSMA, regions previously referred to as the intentionality network  prefrontal cortex. Indeed, the contrast intentional action vs. stimulus-driven action (white vs. green marble Go responses) confirmed that this network was largely engaged, consistent with prior studies in the literature on intention action (Lau et al., per se (Karch et al., Secondly, we tested whether there were brain regions uniquely related to the intentional inhibition decision outcome by contrasting intentional inhibition with intentional action. A network of brain regions was active for intentional inhibition compared to intentional action including bilateral IPL and left IFG, suggesting that the inhibition process cannot be reduced to intentionality Contrary to prior findings by Kühn et al. , we obseoptions (Cisek, The results of the parametric analysis shed important light on the role of dFMC in intentional inhibition, and on the significance of intentional inhibition more generally. Briefly, we found dFMC activation was reduced when previous trials had created a prepotent urge to act. Our design differs from the original free-choice whether decision of Brass and Haggard , by incls Cisek, .This reciprocal antagonism corresponds to the common intuition that inhibition of action is harder when the drive to act is strong—for example in cases such as craving and addiction. Interestingly, these are exactly the circumstances when intentional inhibition may also be most necessary. It may also explain why we did not find dFMC activation in our main contrast, while previous studies that did not use instructed action trials to enforce a prepotent urge to act did (Brass and Haggard, side-by-side comparison between the intentional and the stimulus driven tasks was made. Stimulus driven inhibition resulted in the expected network of activation, including the right IFG and pre-SMA (Aron et al., Third, a Some limitations of the present study deserve mention. First, the fact that two different tasks were used to measure intentional and stimulus-driven inhibition pre-empted the possibility to compute a direct contrast between intentional and stimulus-driven inhibition. Future research might benefit from using one single task to measure both forms of inhibition, to allow for such a direct contrast. Second, for the marble task we cannot completely rule out the possibility that participants have pre-decided not to initiate an action on the intentional inhibition trials, instead of deciding in the instant to inhibit an already initiated action. The observed pattern of results showing that intentional inhibition was less likely following a run of instructed action trials, suggests that this was not the case. However, future research could shed more light on this issue, for instance by including electromyography measures to ascertain that the initial action initiation is also present in intentional inhibition trials.per se (Karch et al., Taken together, this study was the first to test the neural correlates of intentional and stimulus-driven inhibition within the same subjects. The results confirmed the hypothesis that these two types of inhibition rely on the same neural network including lateral PFC and preSMA, regions previously associated with intentionality (Lau et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trypanosoma cruzi. It is transmitted to humans by triatomine bugs where T. cruzi multiplies and differentiates in the digestive tract. The differentiation of proliferative and non-infective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) can be correlated to nutrient exhaustion in the gut of the insect vector. In vitro, metacyclic-trypomastigotes can be obtained when epimastigotes are submitted to nutritional stress suggesting that metacyclogenesis is triggered by nutrient starvation. The molecular mechanism underlying such event is not understood. Here, we investigated the role of one of the key signaling responses elicited by nutritional stress in all other eukaryotes, the inhibition of translation initiation by the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), during the in vitro differentiation of T. cruzi. Monospecific antibodies that recognize the phosphorylated Tc-eIF2α form were generated and used to demonstrate that parasites subjected to nutritional stress show increased levels of Tc-eIF2α phosphorylation. This was accompanied by a drastic inhibition of global translation initiation, as determined by polysomal profiles. A strain of T. cruzi overexpressing a mutant Tc-eIF2α, incapable of being phosphorylated, showed a block on translation initiation, indicating that such a nutritional stress in trypanosomatids induces the conserved translation inhibition response. In addition, Tc-eIF2α phosphorylation is critical for parasite differentiation since the overexpression of the mutant eIF2α in epimastigotes abolished metacyclogenesis. This work defines the role of eIF2α phosphorylation as a key step in T. cruzi differentiation.Chagas' disease is a potentially life-threatening illness caused by the unicellular protozoan parasite  In this process, eIF2 plays an important role in delivering Met-tRNAiMet to the translational machinery. This is achieved when the ternary complex formed by eIF2 bound to GTP and Met-tRNAiMet is transferred to the 40S subunit. For the assembly of the 80S complex on the AUG initiator codon, eIF2 is released in the form of the binary inactive eIF2-GDP complex. For another round of translation initiation, eIF2 must be converted to its eIF2-GTP active form. This recycling event is mediated by eIF2B, a nucleotide guanine exchange factor (GEF) 51, eIF2-GDP becomes a competitive inhibitor of eIF2B The reversible phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α) is a major survival response, conserved from yeast to mammals, and plays a significant role when cells must respond to environmental stresses. The process of translation initiation in eukaryotes consists of binding of methionyl-initiator tRNA and mRNA to the 40S ribosomal subunit, pairing the Met-tRNAPhosphorylation of eIF2α is mediated by different kinases that respond to specific environmental stresses Toxoplasma gondii, the agent of toxoplasmosis, into quiescent stages, which are responsible for microbial persistence and latent infection . Also, phosphorylation of eIF2α of Plasmodium falciparum is required in the formation of sporozoytes in the salivary gland of the insect vector Leishmania was demonstrated. In this case, mutant promastigotes that have impaired eIF2α phosphorylation during ER stress, showed delayed differentiation into amastigotes grown axenically, or within macrophages The response to stress via phosphorylation of eIF2α is conserved from yeast to mammals and has been shown to underlie cell differentiation in protozoan parasites. For example, phosphorylation of eIF2α by different forms of stresses results in the differentiation of T. cruzi, a protozoan parasite from the order Kinetoplastida, involves stress responses. A variety of conditions can initiate the process of metacyclogenesis, including changes in pH and osmolarity, presence of mammalian serum in vitro when epimastigotes are transferred to a poor medium containing no proteins, amino acids or sugars , and then incubated in TAU medium supplemented with amino acids and glucose The developmental transition in Trypanosoma brucei TbeIF2K2, showing that this kinase is able to phosphorylate yeast and mammalian eIF2α at Ser51. It also phosphorylates the highly unusual form of eIF2α found in trypanosomes specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes Our previous work indicated that trypanosomes encode three potential eIF2 kinases (eIF2K1-K3), and we characterized the 169 residue of T. cruzi eIF2α, we show that this phosphorylation mediates translational control in response to nutritional stress. Importantly, we also found that phosphorylation of Tc-eIF2α at Thr169 residue controls metacyclogenesis, since a mutation of Thr169 to Ala residue impaired differentiation. Interestingly, epimastigotes overexpressing wild type Tc-eIF2α differentiate at higher frequency compared to epimastigotes overexpressing the Tc-eIF2αThr169Ala mutant. The eIF2α pathway is then proposed to participate in the adaptive response of T. cruzi to nutritional stress, contributing to parasite differentiation to disease-causing metacyclic-trypomastigotes.In this study, using specific antibodies that recognize the phosphorylated ThrT. cruzi Dm28c and Y epimastigotes were cultured in liver infusion tryptose (LIT) medium at 28°C. Metacyclogenesis was performed as described 7 parasites ml− 1) were collected by centrifugation at 2,000 g for 10 min at room temperature, and were subjected to nutritional stress for 2 h at 28°C in TAU medium  at a density of 5× parasites ml−1. The parasites were diluted to 5 × 106 parasites ml− 1 in TAU supplemented with 2.5% sodium bicarbonate, 500 U ml− 1 penicillin, 10 mM L-proline, 50 mM L-glutamic acid, 2 mM L-aspartic acid, 10 mM glucose and incubated at 28°C for the designated length of time according to the experimental protocol.T. cruzi eIF2α motif PYTEITPR, containing the phosphorylated threonine at position 169, and a peptide carrying the corresponding non-phosphorylated threonine were synthesized by Dr. Maria Aparecida Juliano  as described T. brucei eIF2α recombinant protein purified from E. coliT. cruzi metacyclic trypomastigote mucins (10D8) was kindly provided by Dr. Nobuko Yoshida  A peptide corresponding to the T. cruzi wild-type eIF2α coding sequence was amplified from genomic DNA of DM28c strain using oligonucleotides eIF2aF (5′-CCTGTATTTTCAGGGCATGGCGTCGCATGGCCCAGTGAA-3′) and eIF2aR (5′-GTACAAGAAAGCTGGGTATTTCAATCTGTCTCATCATCATC-3′) based on the CL-Brener sequence with recombination sites appended to the 5′ end for insertion into the pDONR221 vector (GatewayTM Platform). The amplified fragment was inserted into pDONR221 (entry vector) and transferred to pDEST17 according to the manufacturer's protocol (Invitrogen). The insert was removed from by XbaI and HindIII digestion and transferred to the p33 vector 169A substitution in Tc-eIF2α was performed by overlapping PCR, of two PCR fragments, using the oligonucleotides: Tc-eIF2 FX (5′-TCTAGAATGTCGTACTACCATCACC-3′) and Tc-eIF2 Rbg (5′-GCAACGTGACATACAATGGATCTCACC-3′) for a 788 base pair 5′ fragment, and Tc-eIF2 TA169 (5′-CCGTACACGGAAATTGCGAGGATCCGCATTCG-3′) and Tc-eIF2 RH (5′-AAGCTTTCAATCTGTCTCATCATC-3′) for a 765 base pair 3′ fragment. The products were cloned into the pGEM®-T easy vector systems (Promega) and the constructions were confirmed by sequencing. The sequences encoding the wild type and the mutant Tc-eIF2α were transferred as XbaI-HindIII fragments into the p33 vector The T. cruzi epimastigote cells of Y strain were transfected with the plasmids using a Gene Pulser Electroporator (Bio-Rad Laboratories) under the following conditions: 2.5×108 parasites grown in LIT medium at 28°C were harvested by centrifugation , washed with PBS and resuspended in 400 μl of electroporation buffer . The cell suspension was mixed with 50 μg of plasmid DNA and electroporated in 0.4 cm gap cuvettes (Bio-Rad Laboratories) with a single discharge of 450 V and 500 μF generating a time constant of about 5 ms. After electroporation, parasites were left to recover for 48 h at 28°C in LIT medium, followed by selection in the same medium containing 500 μg ml−1 geneticin G418 (Invitrogen). A p33-GFP (green fluorescent protein) construct was used as a control for transfection efficiency T. cruzi extracts (106 parasites μl−1) were separated by electrophoresis using a 10% polyacrylamide gel in the presence of 0.1% SDS (SDS-PAGE). Proteins were transferred to nitrocellulose membranes, incubated with 5% non-fat milk in TBS (pH 7.8) containing 0.1% Tween-20 (TBS-T) for 2 h and probed with either rabbit anti-Tc-eIF2α antibody  or anti-phospho-specific (T169) Tc-eIF2αP antibody (diluted 1:200), followed by Protein A-peroxidase conjugate . Bound antibodies were visualized using the Immobillon Western blotting substrate (Millipore).Phosphorylation of Tc-eIF2α was monitored by Western blotting. 9) were incubated in 5 ml of RPMI, TAU or TAU3AAG supplemented with 200 μCi of Express [35S]-methionine (PerkinElmer) for 2 h at 28°C. Then, 5×108 cells were harvested and lysed in 50 μl of SDS-PAGE sample buffer, boiled for 5 min and 10 µl were fractionated on 10% SDS-PAGE. After Coomassie staining, the gels were incubated in Amersham AmplifyTM Fluorographic Reagent  for 30 min, dried and exposed to Kodak X-ray film at −70°C for detection of [35S]-labeled proteins. For the determination of [35S]- methionine incorporation into proteins parasites incubated in RPMI, TAU or TAU3AAG in the presence of radiolabelled methionine for different times were washed in PBS, resuspended in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl2 in the presence of a protease inhibitor cocktail  and lysed by freezing and thawing. The insoluble fraction obtained from a trichloroacetic acid (TCA) extraction was recovered on a membrane disc filter by vacuum filtration. After rinsing with a 10% TCA solution and then with 95% ethanol the membrane disc filters were air-dried and placed in a scintillation tube for radioactivity count.For radiolabeling experiments, epimastigote cells (10T. cruzi epimastigotes extract was prepared as described T. cruzi epimastigotes were washed in PBS and resuspended in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl2, 2 mM EGTA, 2 mM benzamidine, 1 µg ml−1 of leupeptin, 4 µg of aprotinin ml−1 and 10 µg ml−1 of pepstatin A in the absence of protein phosphatase inhibitor cocktail to an equivalent of 3×106 parasites per microliter. After freezing and thawing, the suspension was centrifuged at 10,000 g for 15 min and the supernatant was kept as soluble material. A portion of the lysate containing 20 µg of protein was incubated with 100 U of lambda protein phosphatase (New England Biolabs) in 30 µL of lambda-PPase buffer  at 30°C for 1 h. Negative controls were performed in the same experimental conditions without protein phosphatase, or with heat inactivated protein phosphatase (65°C for 1 h).For dephosphorylation assays, a soluble fraction of T. cruzi polysomes were isolated and separated on sucrose gradients as described 9) in 20 ml of the indicated medium were incubated with 100 µg ml−1 cycloheximide for 5 min at 28°C. They were then kept on ice for additional 10 min, pelleted by centrifugation and washed with cold PBS buffer supplemented with 100 µg ml−1 cycloheximide. After centrifugation at 3,000 g for 10 min at 4°C the pellet was resuspended in 500 µl buffer A  containing 100 µg ml−1 cycloheximide, 1% (v/v) Triton X-100 and 0.2 M sucrose −1 heparin was added. After spectrophotometric quantification, 10 U Abs260nm were layered onto linear 7% to 47% sucrose density gradients prepared in buffer A and centrifuged at 4°C for 2:30 h at 39,000 RPM in a Beckman SW41 rotor. The gradients were collected from the top and the absorbance at 254 nm was monitored in a continuous flow.169) that corresponds to the serine 51 (Ser51) that is phosphorylated in all other eukaryotes  and the KGYID201 motif, known to play an important role in both translation regulation and Ser51 phosphorylation 169 in Tb-eIF2α was specifically phosphorylated by Tb-eIF2K2 in vitro169 might be the actual target of stress activated eIF2 kinases that mediate translational inhibition. To study this pathway in trypanosomatids, we then raised antibodies against the T. cruzi eIF2α sequence PYTEITp169R in which the Tp169 represents the phosphorylated threonine. In ELISA assays the affinity-purified antibody showed reactivity to the phosphorylated peptide but not to the unphosphorylated one, or to an unrelated control peptide corresponding to the T. cruzi histone H1 T. cruzi epimastigotes these antibodies recognized a protein with an apparent molecular mass of 50 kDa that migrated at the same position as the protein recognized by the antibodies against total Tc-eIF2α . Both proteins were expressed as fusions to a His-tag. The expression of the fusion proteins before and after purification with a nickel chelating resin was confirmed by Western blot with anti-His antibodies  allows the transformation of epimastigotes into infective metacyclic trypomastigotes within 72 h T. cruzi and that addition of the three amino acids and glucose allows for a fast partial dephosphorylation of eIF2α.Having shown that the antibodies recognized specifically the phosphorylated form of 35S] methionine during the 2 h period of stress treatment (or mock treatment). The autoradiography of SDS-PAGE showed that the overall protein synthesis was greatly reduced in parasites maintained in TAU medium compared to parasites maintained in LIT medium  according to their densities on a sucrose gradient. Efficient translation results in mRNAs bound to many ribosomes, called polysomes, whereas poor translation initiation causes a decrease in size and abundance of polysomes and a concomitant increase in 80S ribosomes (monosomes). We observed that incubation of exponential epimastigotes for 2 h in TAU medium completely abolished the polysomal fractions and enhanced the amount of monosomes, indicating a very strong inhibition of translation initiation  and as a control, the parasites overexpressing the wild type protein (OE-WT). Parasites were incubated in LIT, or subjected to nutritional stress in TAU or in TAU followed by TAU3AAG media. The profile of the OE-T169A parasites grown in LIT was similar to that of the OE-WT kept under the same experimental condition . Parasites overexpressing Tc-eIF2α or Tc-eIF2αT169A grown in LIT medium were stressed for 2 h at 28°C in TAU and then submitted to differentiation in TAU3AAG medium for 72 h. Under this condition, the parasites attach to the substrate and metacyclics are progressively released 169A mutants  involved in both kinase and eIF2B recognition in typical eIF2α 51 residue in all other eukaryotes is also found at residues 42 to 46 in the N-terminal extension of Trypanosoma eIF2α  are subjected in vitro to heat shock, alkaline pH, or other forms of stress, they phosphorylate eIF2α and start expressing cyst wall antigens of bradyzoites, the parasite's latent forms Plasmodium berghei is also dependent on the phosphorylation status of eIF2α, involving this signaling cascade in parasite's survival and recrudescence of the disease T. gondii, two eIF2α kinases were characterized  and described as involved in the phosphorylation of Tg-eIF2α, in P. berghei three eIF2 kinases were described  T. brucei (TbeIF2K1-K3) being one of them, TbeIF2K1, a possible GCN2 orthologue Leishmania encodes a homologue of the ER stress inducible PERK eIF2α kinase but while in T. brucei eIF2K2-PERK kinase is localized in the flagellar pocket Leishmania spp. it is associated with the ER T. brucei are also present in the genomes of T. cruzi and bioinformatics search revealed two potential homologues of TbeIF2K3 in this parasite T. cruzi. The eIF2α kinase, or kinases, activated under the conditions used here has yet to be determined. The GCN2 ortholog might be a likely candidate. Whether this signaling mechanism influences other T. cruzi differentiation events, i.e., the transformation of infective stages to intracellular amastigotes after cell invasion, probably driven by acidification Cell differentiation in response to stress via phosphorylation of eIF2α has been described in others parasites. In Figure S1Nutritional stress leads to translation repression in epimastigotes. Kinetic of [35S]-methionine incorporation in growing epimastigotes , epimastigotes maintained for 2 h in TAU medium  and epimastigotes incubated in the differentiating TAU3AAG medium . These data were obtained in one experiment performed in triplicate.(TIF)Click here for additional data file.


We here characterize a novel soluble version of theIgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI), the high affinityreceptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa andcontains an intact IgE-binding site. In human serum, sFcεRI is found as asoluble free IgE receptor as well as a complex with IgE. Using a newlyestablished ELISA, we show that serum sFcεRI levels correlate with serum IgEin patients with elevated IgE. We also show that serum of individuals withnormal IgE levels can be found to contain high levels of sFcεRI. AfterIgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI inthe exosome-depleted, soluble fraction of cell culture supernatants. We furthershow that sFcεRI can block binding of IgE to FcεRI expressed at the cellsurface. In summary, we here describe the alpha-chain of FcεRI as acirculating soluble IgE receptor isoform in human serum.Soluble IgE receptors are potential  Allergic patients are commonly characterized by high serum IgE and high IgE-receptorexpression on effector cells of the innate and adaptive immune system in vivo and is generated by cleavage of membrane CD23 from thesurface of B-cells in vivo role of sCD23 and soluble galectin-3highlights the need for continued research on soluble factors that modulate serumIgE responses in the context of an allergic response.In addition to the transmembrane forms, CD23 and galectin-3 are found as solubleproteins in human serum. Soluble CD23 (sCD23) is a modulator of IgE responsesFcεRI is an activating immune receptor of the immunoglobulin superfamily, whichincludes the Fc receptors CD16, CD32, CD64 and CD89 invitro and that the soluble form of the receptor can inhibit binding ofIgE to FcεRI at the cell surface.Here we describe a soluble form of the FcεRI alpha-chain (sFcεRI). In humanserum, this sFcεRI is found as both a free form and bound to its ligand IgE. Weshow that IgE-mediated cell activation induces the release of sFcεRI To give a definitive answer whether a soluble form of the alpha chain ofFcεRI exists in humans, we performed immunoprecipitation experiments toisolate this protein from serum. Sera from patients with normal IgE levels andelevated IgE were run over IgE-columns. Eluates from these columns were analyzedwith the FcεRI alpha-chain specific mAb 19-1 by Western blot To perform a more detailed molecular characterization of sFcεRI, we nextcompared sFcεRI-alpha precipitated from serum to FcεRI-alphaprecipitated from the cell surface of MelJuso-αγ cells. As expected fora soluble form, sFcεRI has a lower molecular weight than the surfaceexpressed protein. Unlike transmembrane FcεRI-alpha, which forms amultimeric complex with the common FcR-gamma chain In summary, this set of results show that the Fc-portion of human IgE caninteract with a soluble alpha-chain protein in serum and that this serumsFcεRI does not have the molecular characteristics of the multimericmembrane-associated FcεRI.To allow for semi-quantitative analysis of sFcεRI levels in human serum, wenext established a sandwich-ELISA system  with a wide range of normal and elevated serum IgE levels. Patients werecategorized based on the specifications given in the in vitroIgE incubation step from the signal with the IgE incubation step allowed us todetermine how much sFcεRI was complexed to IgE in vivo. Werandomly selected 14 sera that were positive for sFcεRI and found that inhuman serum, sFcεRI is present as both a free and an IgE-bound protein chain thatcirculates in human serum as a free protein or bound to its natural ligand, IgE. Weshow that sFcεRI is released upon IgE-antigen-mediated activation of cellsurface FcεRI Commonly, the reagents used to detect transmembrane forms of FcεRI-alpha aredirected against the IgE-binding epitope of the protein. Thus, the identification ofsFcεRI could easily have been missed if the detection reagents were not selectedcarefully. We here established an ELISA system that uses a monoclonal antibodydirected against the stalk region of the protein et al. most likely described a version of the receptor that wasreleased in an exosomal fraction rather than a bona fide soluble protein.Several studies with recombinant versions of sFcεRI are found in the literatureetal.Based on our current understanding of the mechanism of sFcεRI generation, it isfair to assume that serum sFcεRI is a reflection of FcεRI activation. In anindependent study, we were able to confirm the observation of Liang in vivomodulator of IgE-mediated responses. While sCD23 has to trimerize to developconsiderable affinity for its ligand in vivo.sFcεRI is also an excellent candidate for an efficient in vitro studies suggest that sFcεRI may also preventIgE-mediated activation of the immune system by clearing the serum of IgE in amanner comparable to omalizumab. Omalizumab is a recombinant humanized monoclonalantibody directed against serum IgE and currently approved for the treatment ofsevere allergic asthma Our in vivo.In summary we here describe a new soluble form of human FcεRI, the high affinityreceptor for IgE (s FcεRI), in human serum. Establishing an improvedquantitative ELISA with a standard protein is now highly important, because such aquantitative method will allow for an extensive comparative analysis of serum levelsin various patient groups. Such studies will be of outmost importance to drawconclusions about the clinical relevance of this new serum IgE receptor. It will bealso essential to gain a better understanding of how endogenous levels of sFcεRIare regulated and how sFcεRI is linked to IgE-mediated immune activationPatient sera used for this study were obtained from an ongoing prospective cohortstudy on the role of FcεRI in the gastrointestinal tract at Children'sHospital Boston or had been previously obtained as part of routine clinical careat the Medical University of Vienna. The prospective cohort study was approvedby the Investigational Review Board of Children's Hospital Boston  and patients or their legal guardians providedwritten informed consent. The retrospective study of patient sera was approvedby the Ethics Committee of Medical University of Vienna, Vienna, Austria.in vitro cell cultureexperiments. Phycoerythrin (PE)-conjugated was purchased from BiosearchTechnologies, Novato, CA, and used for flow cytometry analysis. Anti-mouse IgG wasused for coating of the ELISA plates. High-IgE human serum  was purchased from Bioreclamation, Hicksville, NY to assure the qualitycontrol of IgE used for detection of sFcεRI by ELISA. Goat anti-human IgEHRP conjugated antibody  was used as asecondary antibody.Anti-human FcεRI alpha mAb 19-1 was kindly provided by Dr. J.P. Kinet and used as previously described http://www.stanford.edu/group/nolan/) following the guidelinesof Children's Hospital Boston. MelJuso cells transfected with empty vector(MelJusoØØ) and MelJuso-αγ cells were maintained inDulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum , 2 mMglutamine (Cellgro), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were reselected using hygromycin (1 mg/ml)and puromycin . Jw 8/5/13 cells The MelJuso cell line was provided by Hidde L. Ploegh  and is well-established for studies on MHC class I and MHC classII trafficking Sera from adult individuals were tested for the presence of sFcεRI byimmunoblot and ELISA. Sera from eight poly-sensitized, highly atopic childrenwere analyzed for sFcεRI by ELISA. Total serum IgE and allergen-specific IgEwere measured by solid phase immunoassay . Total serum IgE levels are given in kU/l,specific IgE is given in kUA/l and CAP RAST classes.Sera from 119 children were obtained from an ongoing prospective cohort study onthe role of FcεRI in the gastrointestinal tract. Patients between 1 and 19years of age scheduled for an elective esophago-gastro-duodenoscopy at theDivision of Gastroenterology at Children's Hospital Boston were randomlyinvited to participate. Subjects who used steroids in any form, immunomodulatorydrugs, mast cell stabilizer, or leukotriene inhibitor within the last 3 months,as well as patients with an established diagnosis of autoimmune, inflammatory,or immunodeficiency disease were not enrolled. Total serum IgE levels wereassessed according to standard procedures at Children's Hospital Bostonusing the Elecsys IgE II kit . IgE levelsare given in kU/l. Expected normal ranges for this assay are 30 kU/l for age0–3 years, 200 kU/l for 3–10 years, 500 kU/l for 10–14 years,and 200 kU/l for >14 years.6 cells per ml; 0.5%Brij 96, 20 mM Tris, pH 8.2, 20 mM NaCl, 2 mM EDTA, 0.1% NaN3)containing protease inhibitors  for 30 min on ice. Immunoprecipitation was next performed with NIP-beads(Sigma) as previously described To target the fully mature form of FcεRI alpha as expressed on the cellsurface, we loaded MelJuso-αγ cells with cIgE (10 mg/ml in PBS) beforesolubilization in lysis buffer .total sFcεRI−ODIgE-sFcεRI complexes = ODfreesFcεRI.To improve sensitivity, wells were first incubated with a goat-anti-mouse coatingantibody , then with anti-alpha chain mAb . After a blocking step with 10% FCS in PBS, wellswere incubated with sera (1∶2 dilution) overnight. After repetitivewashing, plate-bound sFcεRI was loaded with IgE (Bioreclamation) anddetected with a goat anti-human IgE-HRP conjugated second step . Ratherthan using IgE purified from different patient sera, IgE was purchased to ensurethe quality and consistency of this reagent in our ELISA. Conversion of3,3′,5,5′-tetramethyl-benzidine liquid substrate  wasmeasured at 450 nm. Results are given as optical density (OD). To control forintra-assay variability, we included on each plate a positive and a negativecontrol sample consisting of a pool of three positive or three negative patientsrespectively. The levels of circulating IgE-sFcεRI complexes were determinedby omitting the IgE-loading step of the protocol. Levels of free sFcεRI werethen calculated as follows: ODIn a subset of patients, sFcεRI was measured in plasma and serum in parallel.For conversion of plasma samples into serum, BD Serum Separation Tubes (BectonDickinson) were used according to the manufacturer's guidelines.MelJuso-αγ cells were grown to confluence and incubated with cIgEovernight. Excess cIgE was washed away and ligand-bound receptor was activatedwith haptenized antigen . Cell culture supernatants were collected after theindicated time periods and analyzed for the presence of soluble alpha chain byELISA or by immunoprecipitation.et al.To remove exosomes from cell culture supernatants, MelJuso-αγsupernatants were treated with a sequence of ultracentrifugation steps followingthe protocol published by Thery Surface expression of FcεRI on MelJuso-αγ cells was determined bystaining with the anti-human FcεRI alpha mAb CRA1. IgE binding was tested byculturing MelJuso-αγ cells in the presence or absence of NP-specificcIgE and staining with phycoerythrin (PE)-conjugated NP . For the detection of sFcεRI, cells were loaded with either amix of cIgE (100 ng/ml) in culture supernatants that contained sFcεRI orsupernatants from unstimulated cell cultures for 30 min on ice. A number ofdifferent ratios of cIgE to culture supernatants was analyzed. FcεRI-boundcIgE was stained with NP-PE. Analysis was performed on a BD FACScan™ flowcytometer using CellQuest software for acquisition and data analysis (both fromBecton Dickinson).For FcεRI alpha internalization, MelJuso-αγ cells were grown oncoverslips (No 1.5), stained first with purified mouse anti-FcεRI alpha CRA1antibody for 20 min at 37°C and subsequently with an anti-mouse Alexa Fluor568 for 45 min at 37°C to induce receptor crosslinking. Cells were fixedwith 4% paraformaldehyde for 20 min and mounted using Prolong Antifadereagent (Invitrogen). Both antibodies were diluted in HBSS supplemented with 10mM HEPES (Invitrogen) and 5% NuSerum (Invitrogen). CRA1 was diluted at1∶100 and anti-mouse Alexa Fluor 568 was diluted at 1∶400. Plasmamembranes of fixed cells were stained with Alexa Fluor 647-conjugated wheat germagglutinin  for 10 min. For time pointt = 0 cells were fixed before incubation with anti-mouseAlexa Fluor 568 and WGA. All incubation steps were carried out in a humidifiedchamber. Confocal images were acquired on a Nikon TE2000 inverted microscopecoupled to a Yokogawa spinning disk confocal unit (Perkin-Elmer Inc.) and anOrca AG scientific-grade cooled CCD camera (Hamamatsu Photonics K.K.). Slidebooksoftware (Intelligent Imaging Innovations Inc.) was used for image capture,processing, and analysis.Correlations between serum IgE and serum sFcεRI were calculated bySpearman's rank correlation test using SPSS for Windows . Spearman's rank correlation coefficients are displayedas ‘rho’, a p-value of >0.05 was considered significant.Table S1Serum levels of sFcεRI in atopic patients.(DOC)Click here for additional data file.