In these patients, the inhibition of such abnormal proteolysis rescues physiological amounts of active mutated CFTR in 90% of the patients (25 over 28). The recovery of functional F508del-CFTR at the physiological location, in cells treated with a synthetic calpain inhibitor, indicates that F508del-CFTR folding, maturation, and trafficking operate in CF-PBMC at significant rate. Thus, an increase in the basal calpain activity seems primarily involved in the CFTR defect observed in various CF cells. Furthermore, in CF-PBMC the recovery of the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF-1), occurring following inhibition of the aberrant calpain activity, can contribute to rescue CFTR-functional clusters.A basal calpain activity promotes the limited proteolysis of wild type (WT) cystic fibrosis conductance regulator (CFTR), inducing the internalization of the split channel. This process contributes to the regulation in the level of the active CFTR at the plasma membranes. In peripheral blood mononuclear cells (PBMC) from 16 healthy donors, the inhibition of calpain activity induces a 3-fold increase in the amount of active WT CFTR at the plasma membranes. Instead, in PBMC from cystic fibrosis (CF) patients, calpain activity is expressed at aberrant levels causing the massive removal of F These two fragments remain associated each other on the membranes, both having a transmembrane domain. The split CFTR is then immediately internalized in endocytic vesicles and slowly digested by lysosomes Our previous observations indicate that a third proteolytic system, represented by the Ca508del-CFTR is degraded by calpain, producing the same fragments obtained by the degradation of the wild type channel, and that the deletion of Phe508does not modify the sensitivity of the channel to the protease 508del-CFTR is almost undetectable and levels of the split channel form are higher than those detected in control cells 508del-CFTR could be further increased because activity of calpain is also sustained by a decreased level of its natural inhibitor calpastatin. The susceptibility of wild type CFTR to calpain digestion seems modulated by the association of the channel to its partner proteins in the CFTR-generated functional complexes. In this respect, of particular importance is the effect exerted by the chaperone HSP90 which protects almost completely membrane inserted mature CFTR from calpain digestion; this process is much less effective for F508del-CFTR Recently, it has been reported that the elastase released from activated neutrophils induces a limited proteolysis of CFTR in airway epithelial cells through activation of intracellular calpains + lymphocytes impairs cytokine secretion and hyperinflammatory adaptive immune responses In our studies, PBMC have been selected as cell model due to the observations indicating that a number of lymphocyte functional properties are altered in CF patients 508del-CFTR resulted functionally active at an extent comparable to that of the wild type channel. Our observations indicate that, in condition of calpain inhibition, F508del-CFTR could acquire the mature active form reaching its correct localization at the plasma membranes, although in an amount significantly lower as compared to the wild type channel form.In the present study, we are reporting that intracellular calpain inhibition promotes a large increase of CFTR associated to the plasma membranes in PBMC from healthy subjects. Rescue of the channel protein was obtained also in PBMC from CF patients following intracellular calpain inhibition. The recovered FThe present report not only provides new understanding of the mechanism involved in CFTR trafficking, but also suggests new approaches in the therapeutic strategies for cystic fibrosis.6,O2’-dibutyryl adenosine 3′:5′-cyclic monophpshoric acid (dibutyryl-cAMP) and genistein were purchased from Sigma-Aldrich. 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF) was obtained from Calbiochem. ECL ADVANCE® Detection System was obtained from GE Healthcare. t-BOC (t-butoxycarbonyl)-Leu-Met-CMAC (7-amino-4-chlorometylcoumarin), a fluorogenic calpain substrate was purchased fromMolecular Probes (Invitrogen). Ficoll-Pacque Plus was obtained from GE Healthcare. Anti -CFTR monoclonal antibody (clone M3A7) was purchased from Millipore. Anti-CFTR polyclonal antibody was purchased from Cell Signaling. Anti-NHERF-1 (H-100) polyclonal antibody was purchased from Santa Cruz biotechnology, Inc. CFTR inhibitor CFTR(inh)-172 Leupeptin, CI-2 , NAll participants and the authorized parents gave written informed consent prior to inclusion in the study, including permission to store the samples and to use them for research exclusively. The study protocol conforms to the provisions of the Declaration of Helsinki and of G. Gaslini Children Hospital, Genoa, Italy. The approval from the Ethics Committees is not required since our analysis were carried out on blood samples obtained from all participants during their routine clinical examinations at the hospital. The acquisition and analysis of the data were anonymous.508del-CFTR mutation and 16 healthy donors were enrolled in the study. CF patients  were regularly followed at the Cystic Fibrosis Center, G. Gaslini Hospital, Genoa, Italy and blood samples were collected under the supervision of Dr. L. Minicucci. For every patient and healthy donor, a sample of 6 ml of blood was collected in two 3 ml vacuette® PREMIUM tubes containing 5 mM EDTA. Samples were rapidly chilled in an ice bath and transferred to the DIMES, Section of Biochemistry, for the experimentation.28 CF patients homozygous for F6 cells/flask and incubated for 24 h at 37°C in culture medium supplemented with the vehicle  or the amounts of CI-2 indicated elsewhere in a humidified atmosphere containing 5% CO2. Viability as assessed by 0.2% trypan blue exclusion was higher than 80–90%. After incubation, cells were collected by centrifugation at 800×g for 10 min. PBMC were washed three times with ice-cold PBS solution and suspended in 50 mM sodium borate, pH 7.5 (500 µL), containing 1 mM EDTA, 2.5 µg leupeptin and 5 µg AEBSF. Cells were lysed by three cycles of freezing and thawing followed by sonication. The total particulate fraction was collected by centrifugation at 100,000 g for 10 min at 4°C. The pellet  was washed three times in 50 mM sodium borate, 0.1 mM EDTA, pH 7.5 and finally suspended in 0.1 mL of the washing buffer. The protein concentration was determined following the method of Lowry.PBMC, isolated as previously described Total membrane fraction (10 µg), prepared from PBMC as described above, was incubated at 37°C for 30 min in Laemmli loading buffer and submitted to 6% SDS-PAGE Total membrane fraction (100 µg in 0.5 ml) prepared from untreated and CI-2 treated PBMC was fractionated on a discontinuous sucrose density gradient . After centrifugation at 38000 rpm in a Beckman Coulter TLA-100.4 rotor for 30 min, membranes were recovered into two fractions layered at 10% and 30% sucrose interface. They were separately collected, suspended in 0.2 ml of sodium borate 50 mM, pH 7.5 containing 1 mM EDTA and 5′-nucleotidase (5′-NT) activity was assayed 7) isolated from control and CF patients were fixed and permeabilized by the Triton/paraformaldehyde method, as described in PBMC (1×106 cells/mL) were collected, washed three times in PBS solution, and incubated for 20 min at 37°C in buffer A  containing 50 µM t-Boc-Leu-Met-CMAC, the fluorogenic calpain substrate. Cells were washed with buffer A to remove substrate excess. The pellets were suspended to give 106 cells/mL in buffer A containing 0.1 mM CaCl2 and different concentrations (from 0 to 2 µM) of CI-2. Aliquots (2×105 cells) were transferred to 96-well plates and the fluorescence emission was continuously monitored at 37°C for 2 hours with a Mithras LB940 plate reader (Berthold Technologies). The excitation/emission wavelengths were 355/485 nm respectively.Calpain activity was detected in PBMC from healthy donors or CF patients following the procedure described in 508del-CFTR and halide-sensitive yellow fluorescent protein YFP-H148Q/I152L (HS-YFP) Fischer Rat Thyroid (FRT) cells, stably transfected with F6), washed twice with 0.25 M sucrose, containing 20 mM sodium borate, pH 7.5, and 0.2 mM Ca(NO3)2 (buffer CFTR), were suspended in 100 µL of buffer CFTR and exposed for 30 min at 37°C to vehicle or 100 µM dibutyryl-cAMP and 50 µM of the CFTR potentiator genistein - concentration measured by means of the increase in HS-YFP fluorescence, with respect to the fluorescence evaluated in the presence of 5 mM NaI.Measurements of CFTR activity were carried out on PBMC isolated from healthy donors (controls) and CF patients incubated for 24 hours in the absence or presence of 2 µM CI-2. Cells  in the presence of increasing concentrations (from 0 to 2 µM) of CI-2, a synthetic calpain inhibitor interacting with the essential Cys residue at the protease active site We have recently reported that degradation of CFTR in condition of basal calpain activity is detectable in growing cells as well as in PBMC. When calpain activity is evoked by a rise in [Ca508del-CFTR is nearly absent in PBMC from CF patients, suggesting an increased ER degradation of the misfolded channel by the proteasome machinery 508del-CFTR from the plasma membranes. Thus, PBMC from CF patients were exposed for 24 hours to increasing concentrations of CI-2 and the localization as well as the amount of mature F508del-CFTR were evaluated. As shown in 508del-CFTR was not detected, calpain activity was 5-fold higher than that observed in cells from healthy donors  is similar to that observed in untreated PBMC from healthy subjects (24 units), confirming the results shown in We have recently reported that mature Fnors see . When PBe images  revealede images , rescued CF-PBMC , was larEven though our observations clearly indicate that calpain inhibition promotes the accumulation of CFTR at its functional localization, the assay of CFTR activity is of great relevance, since it represents a direct indication that the recovered channel is present in its functional conformational state. To this purpose, the CFTR-mediated iodide flux across the plasma membranes was evaluated in untreated and in CI-2-treated PBMC from both healthy donors and CF patients. Our assay method is based on the activation of CFTR by cAMP-dependent phosphorylation and the measurement of iodide influx into the cells. The iodide concentration in the extracellular medium was determined by means of the fluorescent HS-YFP 508del-CFTR rescued in CI-2 treated CF-PBMC is active, the activity of the mutated channel was measured in the same conditions used in control-PBMC isolated from healthy donors. As shown in 508del-CFTR present in these cells. These data are in agreement with the report indicating that in the presence of cAMP and genistein the open probabilities of WT and F508del-CFTR are identical 508del-CFTR is able to reach the plasma membranes in a mature and active form.To establish if also the F508del-CFTR rescued in CF-PBMC exposed to CI-2 (508del-CFTR molecules can reach maturation and the functional localization Moreover, calculation of the ratio between the level of channel activity and the intensity of the 170 kD protein band revealed that the specific activity of WT CFTR is similar to that of the F to CI-2 . It is iTo establish if, in addition to CFTR, the CFTR partner protein NHERF-1, known to undergo digestion by calpain in CF-PBMC 508del-CFTR is absent from the plasma membranes being the split form largely accumulated inside the cells. Instead, when calpain activity was inhibited by CI-2, we observed the recovery of F508del-CFTR at the plasma membranes up to physiological amounts. In fact, following calpain inhibition, the level of the rescued mutated channel becomes similar to that of the WT CFTR expressed in PBMC from healthy donors. The effectiveness of such treatment indicates that calpain-mediated proteolysis of CFTR plays a crucial role in promoting the channel defect observed in CF cells. It is interesting to note that treatment with various effectors, operating in CFTR maturation steps 508del-CFTR has reached its appropriate localization it is biologically active. This fact indicates that the specific steps involved in processing, maturation, and trafficking of CFTR occur also in CF cells, although at lower rate. Thus, the limited efficiency of correctors could be due to their inability to reduce the aberrant calpain activity operating at the plasma membranes. In this respect, a recent paper 508del-CFTR was not recovered at significant levels. At present it is difficult to explain these negative results; it is possible that they may be due to the multiple therapeutic treatments at which these patients are exposed. Moreover, the level of NHERF-1, a CFTR partner protein known to be a calpain substrate, is increased in CF-PBMC following calpain inhibition. It has been reported 508del-CFTR at the plasma membranes. This observation is in agreement with our present results showing that the recovery of NHERF-1 can enhance the organization of CFTR functional clusters at the cell surface.We are herewith reporting that WT CFTR is 3-fold accumulated at the plasma membranes in an active form following intracellular inhibition of calpain activity. This CFTR rescue has been observed in PBMC from all the subjects analyzed, indicating that this process is of general significance. In fact, our findings indicate that a basal calpain activity, constitutively operating in PBMC, promotes the cleavage of WT CFTR and its removal from the plasma membranes. This process represents a physiological calpain function that is required to regulate the level of the active channel species, since calpain-mediated digestion of CFTR occurs only at the plasma membranes. In PBMC from CF patients, that are characterized by an aberrant calpain activity, FThese observations indicate that the calpain proteolytic system could be a promising target for CF therapy. However, since appropriate compounds are not available at present, experiments are in progress in our laboratory to obtain suitable inhibitors based on calpastatin structure.


Angiotensin II (Ang II) signaling occurs via two major receptors which activate non-receptor tyrosin kinases that then interact with protein tyrosin-phosphatases (PTPs) to regulate cell function. SHP-2 is one such important PTP that also functions as an adaptor to promote downstream signaling pathway. Its role in Ang II signaling remains to be clarified.Using cultured normal human fibroblasts, immunoprecipitation and western blots, we show for the first time that SHP-2 and PLCβ1 are present as a preformed complex. Complex PLCβ1 is tyr-phosphorylated basally and Ang II increased SHP-2-PLCβ1 complexes and caused complex associated PLCβ1 tyr-phosphorylation to decline while complex associated SHP-2's tyr-phosphorylation increased and did so via the Ang II type 1 receptors as shown by Ang II type 1 receptor blocker losartan's effects. Moreover, Ang II induced both increased complex phosphatase activity and decreased complex associated PLCβ1 tyr-phosphorylation, the latter response required regulator of G protein signaling (RGS)-2.Ang II signals are shown for the first time to involve a preformed SHP-2-PLCβ1 complex. Changes in the complex's PLCβ1 tyr-phosphorylation and SHP-2's tyr-phosphorylation as well as SHP-2-PLCβ1 complex formation are the result of Ang II type 1 receptor activation with changes in complex associated PLCβ1 tyr-phosphorylation requiring RGS-2. These findings might significantly expand the number and complexity of Ang II signaling pathways. Further studies are needed to delineate the role/s of this complex in the Ang II signaling system. Angiotensin II (Ang II) is a major regulator of a broad spectrum of important biological processes ranging from vasoconstriction to inflammatory processes including atherosclerosis and vascular ageing, which proceeds, in part, via phosphoinositide-specific phospholipase C (PLC) generated second messengers -4. Ang IThe previous report of a complex involving SHP-2 suggests that SHP-2 may function as part of a complex in other pathways. The concept of and the role(s) for complex formation has gained increasing attention as a means to direct signals toward a particular pathway along with reducing the likelihood of cross-talk by Golebiewska et al . For exaDuring the course of investigating Ang II signaling in our well characterized "in vivo" human model of altered Ang II long term signaling and vascular tone control, Bartter's and Gitelman's syndromes ,15-19, wThe effect of Ang II on PLCβ1 and SHP-2 in human skin fibroblasts was examined using cultured cells incubated with or without Ang II (100 nM) for 1 h. The effect of Ang II was examined by probing Western blots  of cell lysates immunoprecipitated with either anti PLCβ1 antibody or anti-SHP-2 antibody. The figures are the results of representative experiments. antibody. The figures are the results of representative experiments.Figure Figure Figure Figure Figure The phosphatase activity of the SHP-2 immunoprecipitates of cells significantly increased in normotensive healthy subject cells with Ang II compared to those without Ang II .The present study on Ang II signaling in normal human fibroblasts has produced the first description, to our knowledge, of the presence of a SHP-2-PLCβ1complex that responds to Ang II signaling associated events. The presence of a SHP-2-PLCβ1 complex in fibroblast from normotensive healthy subjects was demonstrated via immunoprecipitates obtained by incubating with either anti PLCβ1 or anti SHP-2 Figure  found waSHP-2, participates in multiple signal transduction cascades, including the Ras-Raf-MAP kinase, JAK/STAT, PI3K/Akt, NF-κB, and NFAT pathways -24 and aThe current study identifies a preformed SHP-2-PLC β1 complex as a part of Ang II signaling which strengthens the concept that preformed complexes are involved in cell signaling systems. These complexes have been suggested to function to direct signals toward a particular pathway along with reducing the likelihood of cross-talk . For exaThe identification of a SHP-2-PLCβ1preformed complex that responds to Ang II as shown in this study is an important first step but the role of this complex in the Ang II signaling remains to be delineated. We are aware that this is a limitation of the present study, however we think that the identification of this complex and its response to Ang II merits to be reported waiting for the results of further experiments specifically performed to clarify its role in the Ang II signaling. To this purpose, one approach to understanding the role of SHP-2-PLCβ1 complex is to assess its status in two systems with contrasting Ang II signaling. A comparison of the complex's levels and behavior in Bartter's and Gitelman's syndromes, a human model of blunted Ang II signaling system and RhoA-Rho kinase pathway ,15-18 anAnti-P-Tyr and anti-PLCβ1monoclonal antibodies were purchased from Biosource  and Upstate , respectively while rabbit anti-SHP-2 (C-18) polyclonal antibody was from Santa Cruz Biotechnology . Protease inhibitor cocktail was obtained from Roche Diagnostic . Anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) were from .Skin fibroblasts from 6 healthy subjects from the staff of the Department of Clinical and Experimental Medicine at the University of Padova, who gave their informed consent, were obtained via biopsy and individually cultured in F-10 HAM medium with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 4 mmol/l glutamine, as previously described ,27,28 anAnti-SHP-2 and Anti PLCβ1 immunoprecipitation was done using confluent cells. These were scraped, washed in buffer and extracted . After centrifugation, 200 μg of supernatant protein were diluted 1:1 in 20 mM Tris-HCl, pH 7.5, containing 1 mM sodium orthovanadate and protease inhibitor cocktail, precleared with protein A-Sepharose, and anti-SHP-2 or anti PLCβ1antibodies bound to protein A-Sepharose were added at 4°C. This was then incubated overnight, immunoprecipitates were washed 3× in buffer D , resuspended and then submitted to gel electrophoresis , transferred by blotting to nitrocellulose membranes and immunostained with the appropriate antibodies/second antibodies.Phosphatase activity was measured at 30°C using nitrophenyl phosphate (pNPP) (10 mM pNPP as substrate in 100 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and PTPase immunoprecipitates from 200 mg cell-protein content in Buffer D). After 10 min at 30°C, the reaction was quenched with 950 μl of 1 M NaOH. Absorbance at 405 nm was measured and in all cases the substrate-to-product conversion was less than 5%. All the reagents were from Sigma . Results are expressed as nanomoles per minute per 200 mg cell protein immunoprecipitate.5 cells) were plated the day before transfection in 6-well plates in growth medium without antibiotics containing 10% FBS. On the day of transfection, siRNA was incubated with Lipofectamine 2000 diluted in OPTI-MEM I  following manufacturer's instructions . We have chosen for our experimental protocol RGS-2 siRNA mapping for exon 5 at a concentration of 50 nmol/l and transfected the oligos with Lipofectamine 2000 (4 mg/ml) as previously reported [RGS-2 gene silencing was done using chemically synthesized siRNA that mapped to exon 5 of RGS-2 gene  as previously described . Fibroblreported . Followireported . HorseraData were evaluated statistically as normally distributed continuous variables and comparisons were performed using one-way ANOVA . Results with p < 0.05 were considered significant and data values are presented as mean±SD.The authors declare that they have no competing interests.LAC designed the experimental protocol and wrote the manuscript. LB contributed to design the experimental protocol, helped to drafting the manuscript and contributed to perform the experiments. PAD helped to design the experiments, contributed to drafting the manuscript and did the statistical analysis. EP and LDM performed the experiments. GPR, ACP and GC reviewed the manuscript. All authors read and approved the final version of the manuscript.


Influenza A viruses are a severe threat worldwide, causing large epidemics that kill thousands every year. Prevention of influenza infection is complicated by continuous viral antigenic changes. Newer anti-influenza agents include MEK/ERK and protein kinase C inhibitors; however, the downstream effectors of these pathways have not been determined. In this study, we identified a common mechanism for the inhibitory effects of a significant group of anti-influenza agents. Our studies showed that influenza infection activates a series of signaling pathways that converge to induce myosin light chain (MLC) phosphorylation and remodeling of the actin cytoskeleton. Inhibiting MLC phosphorylation by blocking RhoA/Rho kinase, phospholipase C/protein kinase C, and HRas/Raf/MEK/ERK pathways with the use of genetic or chemical manipulation leads to the inhibition of influenza proliferation. In contrast, the induction of MLC phosphorylation enhances influenza proliferation, as does activation of the HRas/Raf/MEK/ERK signaling pathway. This effect is attenuated by inhibiting MLC phosphorylation. Additionally, in intracellular trafficking studies, we found that the nuclear export of influenza ribonucleoprotein depends on MLC phosphorylation. Our studies provide evidence that modulation of MLC phosphorylation is an underlying mechanism for the inhibitory effects of many anti-influenza compounds. The complex of Ca+2 and calmodulin then activates MLCK, leading to increased MLC phosphorylation , and the Ras/Raf/MEK/ERK signaling cascade +2 levels and activating MLCP The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, is a serious threat to global human health. Two classes of anti-influenza agents are currently available for use during an influenza pandemic-M2 channel blockers and neurominidase inhibitors. However, continuous antigenic changes enhance the probability of increased resistance to these drugs. Moreover, a high frequency of resistance in clinical isolates in the United States has led to the conclusion that M2 inhibitors should not be used for the prevention or treatment of influenza until susceptibility to these drugs has been re-established among circulating influenza A isolates rylation . The Ca+Influenza-induced activation of Raf/MEK/ERK, PKC, and PLC has been reported 2+ chelator (BAPTA-AM), calcium channel blocker (verapamil), MLCP inhibitor , sodium nitroprosside, 8-Br-cGMP, and cytochalasin D were purchased from Calbiochem . LFL was purchased from EY Laboratories, Inc. Tyramide signal amplification kits  were obtained from PerkinElmer. Anti-influenza monoclonal antibody against nucleoprotein was purchased from Chemicon International, Inc. Premade recombinant adenoviruses of ERK2 (DN), Ras V12 (CA), Ras N17 (DN), Raf-1 (DN), RhoA N19 (DN), RhoA L63 (CA), null control, GFP, and ViraDuctin adenovirus transduction reagents were purchased from Cell Biolabs. A mutant construct for DN MLCK was a generous gift from Dr. Yasuo Mori . Small interfering RNA (siRNA) and TaqMan primers and probes for MYPT, Rho kinase1/2, Gα12/13, and Gαq11 were purchased from Applied Biosystems.Phospho-specific and nonphospho-specific antibodies against ERK1/2, nonmuscle myosin II, and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary and anti-FITC–HRP antibodies were obtained from Abcam. Monoclonal phospho-antibody against MLC and specific antibody against MLC were purchased from Sigma-Aldrich. Phospho-specific and nonphospho-specific antibodies against MLCK and the catalytic subunit of MLCP (MYPT) were obtained from Santa Cruz Biotechnology. Recombinant TNF-α, PKC activator , RhoA inhibitor (C3 exoenzyme), Rho kinase inhibitors , MLCK inhibitor (ML-7), MEK inhibitors , protein kinase C inhibitor , phospholipase C inhibitor (U73122), Raf-1 kinase inhibitor (GW 5074), calmodulin antagonist , intracellular CaHUVECs and MDCK cells were purchased from American Type Culture Collection. NHBE cells were purchased from Lonza . MDCK cells were grown in Dulbelcco's Modified Eagle Medium (DMEM)+10% FBS. HUVECs and NHBE were grown in Endothelial Cell Growth Medium-2  and Bronchial Epithelil Cell Growth Medium , respectively. HUVECs and NHBE cells from fewer than 4 generations were used for all experiments.The effects of chemicals on the viability of MDCK cells and HUVECs were determined by using the 3--2, 5-diphenyl tetrazoliumbromide (MTT) assay, as previously described Influenza virus A/Hong Kong/2/68; H3N2 [A/HK (H3N2)] was used as the primary influenza virus strain in all experiments. When grown to 90% confluency, HUVECs or MDCK cells were washed twice with PBS to remove residual fetal bovine serum (FBS) and were infected with virus at an MOI of 10 or 0.01 to allow single-cycle (8–10 h after infection) or multicycle (24 h after infection) replication, respectively. Viral stock was used in serum-free DMEM for 60 minutes at 37°C to inoculate the cells (adsorption phase). Cells were then washed with PBS and cultured in DMEM+2% FBS and tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK) trypsin , either with or without drug treatment, for 8 to 24 h (postinfection phase).These assays were performed in 24-well plates containing confluent MDCK monolayers (4 times for each experiment). The drugs were added to cells in 24-well plates, and the plates were incubated for 0.5 to 8 h at 37°C. The cells were then inoculated with virus, first for 1 h, and then again after washing for 24 h at 37°C in DMEM+2% FBS and TPCK trypsin. The medium was removed and centrifuged at 3,200× g for 5 min to remove the floating cells and then used for ribonucleic acid extraction and quantification of virus by using qPCR.5′ AAG ACC AAT CCT GTC ACC TCT GA 3′ and (reverse) 5′CAA AGC GTC TAC GCT GCA GTC C 3′ amplify a 104-base pair fragment in the M1 gene of influenza A. The influenza A–specific probe FAM (6-carboxyfluorescein)-5′ TTT GTG TTC ACG CTC ACC GT 3′ TAMRA (6-carboxytetramethylrhodamine) annealed to part of the sequence amplified by the 2 primers.TaqMan real-time PCR was used to quantify the presence of virus after infection with influenza virus, as described 2O2, 1 µg/ml anti-FITC–HRP (for 30 min) and FITC-Tyramide (Molecular Probes) were used to reveal FITC–anti-influenza staining. Hoechst 33342 (0.5 µg/ml for 5 minutes) was used as a nuclear counter-stain. Fluorescent images were acquired by MicroSuite FIVE software (Olympus Soft Imaging Solutions) with an Olympus BX61 motorized microscope (Olympus America).Cells were fixed in 4% paraformaldehyde for 15 min at 4°C, washed with PBS, and permeabilized for 5 min with 0.1% Triton-X-100. Viral nucleoprotein (NP) protein was detected by using mouse monoclonal antibody . The secondary antibody used was FITC-conjugated goat anti-mouse IgG. Tyramide signal amplification system was used in conjunction with anti-influenza antibody. After quenching the remaining horseradish peroxidase (HRP) activity with 1% HCell lysates were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred onto nitrocellulose membranes, and stained with primary antibodies, as indicated in figure legends. Secondary antibodies were conjugated to HRP (Sigma). Membranes were developed by use of an enhanced chemiluminescence system .All reagents and kits, including transduction reagents, adenovirus purification kit, and adenovirus titration kit were purchased from Cell Biolabs, Inc. After purification, the titration of each recombinant adenovirus was determined by an enzyme-linked immunosorbent assay (ELISA) titrating kit. Null control recombinant adenovirus (empty vector) served as a control for other recombinant adenovirus used. HUVECs, NHBE or MDCK cells were seeded into 6-well plates for 24 h until they reached 80% confluency. According to the manufacturer's protocol, adenovirus was transduced into cells by using ViraDuctin . HUVECs or MDCK cells were infected with adenoviral vectors with an MOI of 100 plaque-forming units per cell in the presence of ViraDuctin. After incubation with viral particles for 48 h, the cells were assessed for the expression of the transduced genes. Plasmids and siRNA were transfected into cells by using Lipofectamine 2000 (Invitrogen). Scrambled siRNA (a non-targeting siRNA pool) or pcDNA3.1 empty vector were transfected as the controls. Cells were collected 24 h after transfection with siRNA or plasmids.o-phenylenediamine) was added to each well. Stop solution (100 µl) was added after 3–5 min and the 96-well plate was read at 492 nm in a microplate reader.RhoA activity was determined by using a RhoA G-LISA Activation Assay kit according to the manufacturer's protocol . This assay is based on the principle that a Rho-GTP-binding protein is linked to the 96-well plates. The active GTP-bound Rho in the cell lysates binds to the wells, while the inactive GDP-bound Rho is removed during the washing steps. The bound active RhoA is detected with a RhoA specific antibody and quantified by absorbance. The degree of RhoA activation is determined by comparing readings from the infected cell lysates versus the non-infected cell lysates 2+]i in each sample as described Cells were loaded with 4 µg of fluo-3 acetoxymethyl (AM) and 10 µg of Fura-Red AM  as described Actin polymerization was measured by quantifying the ratio of F actin to G actin by using a G actin/F actin assay kit . Actin fibers were stained by using rhodamine phalloidin from the F-actin visualization Biochem Kit .Although the Madin Darby canine kidney (MDCK) cell line is most commonly used for influenza studies, we and others have shown that the influenza virus infects and replicates in human umbilical vein endothelial cells (HUVECs) The transduction conditions for the adenovirus recombinants were optimized by using adenovirus-encoding green fluorescent protein (GFP). The average transduction rate of HUVECs and MDCK cells with the recombinant GFP-adenovirus was 80% to 90% . To confInfection of permissive cells with influenza viruses induces activation of several intracellular signaling pathways that are then used by the virus for its own replication To facilitate infection, bacterial and viral pathogens induce remodeling of the actin cytoskeleton in the cytoplasm of host cells. The effects of ERK activation on phosphorylation of MLCK, MLC, and cell motility have been demonstrated Viruses use multiple mechanisms of movement within cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. Globular-actin (G-actin) readily polymerizes under physiologic conditions to form filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. To determine the effect of MLC phosphorylation on the actin cytoskeleton, we increased MLC phosphorylation by treating HUVECs with calyculin A (an inhibitor of MLC phosphatase) and then examined G-actin polymerization. Calyculin A treatment of HUVECs resulted in the induction of MLC phosphorylation and G-actin polymerization . InfectiThe viral surface glycoprotein hemagglutinin mediates cellular adhesion by recognizing and binding to sialic residues on the cell surface. The accumulation of influenza hemagglutinin on the surface of infected cells results in the activation of the Raf\MEK\ERK signaling cascade 2+ flux, cytoskeletal functions, and activation of Rho family of GTPases Signaling via the Ras/Raf/MEK/ERK pathway is commonly initiated by receptor tyrosine kinases or by G-protein–coupled receptors Several anti-influenza compounds have been described, including those that inhibit intracellular calcium, PKC, and Raf/MEK/ERK, as well as calmodulin antagonists and donors of nitric oxide (NO) Next, we tested whether increased MLC phosphorylation also increased proliferation of influenza virus. Thus, we induced MLC phosphorylation by treating MDCK cells with an MLC phosphatase inhibitor , a PKC activator (PMA), or TNF-α. Treatment of MDCK cells with either calyculin A, PMA or TNF-α increased phosphorylation of MLC . These fIt has been well established that inhibition of RhoA GTPase, calcium/calmodulin and PKC, or increase in NO or cGMP inhibit stimuli-induced MLC phosphorylation To investigate the signaling mechanism underlying the phosphorylation of MLC after influenza infection, we sought to examine the signaling pathways upstream of MLC phosphorylation. Previous studies demonstrated that activation of Raf/MEK/ERK is crucial for proliferation of the influenza virus The increase in ERK phosphorylation during the late phase of the influenza replication cycle has been shown to depend on accumulation of hemagglutinin on the cell surface of infected cells Replication and transcription of the influenza virus genome occurs exclusively within the nucleus of the infected cells. The viral RNA genome, polymerase subunits, and nucleoprotein form ribonucleoprotein (RNP) complexes, which are then exported from the nucleus to form budding virions at the cell membrane. This process requires viral activation of the cellular Raf/MEK/ERK signaling cascade. Accordingly, blocking this signaling pathway inhibits the export of RNP complexes and reduces titers of progeny virus The contractile function of the actin cytoskeleton depends on MLC phosphorylation and the presence of actin filaments. Impeding actin polymerization inhibits the internalization of influenza virus We sought to determine if MLC phosphorylation mediates the role of HRas\Raf\MEK\ERK in influenza proliferation. The reduction in influenza proliferation seen when MDCK cells were treated with the MEK/ERK inhibitor, U0126, or with the Raf kinase inhibitor, GW5074, was reversed when MDCK cells were also treated with the MLC phosphatase inhibitor, calyculin A . InductiTo assess the possible clinical applications of our findings in HUVECs and MDCK cells, we conducted experiments using the NHBE cells because lung epithelial cells are the primary target cell of influenza. In NHBE cells, knocking down Rho kinase with siRNA inhibited the nuclear translocation of influenza RNP complexes , as obseIn the present study, we have shown that influenza infection activates the signaling pathways that converge to induce MLC phosphorylation and actin cytoskeleton remodeling. Inhibiting MLC phosphorylation appears to be the underlying mechanism of action for a significant group of anti-influenza agents. In particular, we showed that MEK/ERK inhibitors suppressed the proliferation of influenza virus by blocking MLC phosphorylation. In addition, inhibition of MLC phosphorylation led to the nuclear retention of viral RNP complexes in late stages of the viral replication cycle, thus reducing proliferation of the influenza virus.To our knowledge, we are the first to report the influenza-induced activation of RhoA/Rho kinase and MLC phosphorylation. In addition, we observed that RhoA activation leads to ERK phosphorylation. RhoA had been shown to prevent apoptosis in zebrafish by activating ERK cascade 2+, and small GTPases, all of which have well-characterized effects on actin dynamics in the cytoplasm We found that MLC phosphorylation is critical for the export of influenza RNP complexes from the nucleus. However, it is not clear how MLC phosphorylation contributes to the nuclear translocation of influenza RNP complexes. Many bacterial and viral pathogens facilitate infection by inducing actin polymerization in the cytoplasm of host cells. The actin cytoskeleton plays a critical role in endocytosis and in intracellular trafficking of viruses In conclusion, our studies indicate that phosphorylation of MLC is critical for the proliferation of influenza virus, and inhibition of MLC phosphorylation is the mechanism underlying the effects of many anti-influenza agents.


Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage. Cohesin is a hetero-tetrameric protein complex consisting of SMC1, SMC3, Rad21 and either SA1 or SA2 in human cells Inverse fluorescence recovery after photo-bleaching (FRAP) was used to investigate how and when cohesin interacts with chromatin during the cell cycle The cohesin complex is known to accumulate at DNA double-strand breaks (DSB) in human cells SMC1 and SMC3 are phosphorylated by ATM Furthermore, cells defective in SMC1 and SMC3 phosphorylation show a defect in the S-phase checkpoint The aim of this study was to determine some of the requirements for SMC1/3 phosphorylation as well as its consequences on the spatiotemporal responses of these cohesin subunits following DNA damage. We show by siRNA depletion of RAD21 that only the fully assembled complex is phosphorylated and furthermore, that in humans ATM-dependent cohesin phosphorylation is mediated by H2AX, 53BP1 and MDC1, suggesting that only loaded cohesins close to DSBs are phosphorylated. We used fluorescence recovery after photo-bleaching (FRAP) to investigate the mobility of phosphorylated SMC1 in G2 phase cells. Our data suggest that ATM-dependent phosphorylation of cohesin is required for efficient mobilization of the complex after DNA damage.The following antibodies were used in this study: rabbit polyclonal against SMC1, SMC1pS966, SMC3, SMC3pS1083, MDC1 and H2AX , anti-actin and anti-mouse/rabbit-HRP , mouse monoclonal anti-Rad21 and anti-γH2AX , anti-CENP-F (ab5) and anti-53BP1 , anti-mouse AlexaFluor 488  and all other secondary antibodies for immunofluorescence .l-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and cultured at 37 °C and 5% CO2. HeLa cells stably expressing EGFP-SMC1 wt or EGFP-SMC1S966A were generated by transfecting SMC1wt-pEGFP-N1 HeLa cells  were grown in Eagles minimum essential medium with 10% foetal calf serum, 2 mM Cell synchronization at the G1/S transition was achieved by a double thymidine block as previously described siRNA transfections were carried out using Oligofectamine (Invitrogen) according to the manufacturer's protocol. The siRNAs were either purchased from Dharmacon or from MWG Biotech (for details see Supplementary Table S1). All siRNAs were used at a final concentration of 100 nM. Cells were assayed 20–24 h after Rad21 siRNA transfection and 48 h after H2AX, 53BP1 and MDC1 siRNA transfection.Plasmid transfections were carried out using polyethylenimine . For each well of a 6-well plate, 2.5 μg DNA and 10 μg PEI were mixed in 0.5 ml medium without FBS and antibiotics, incubated for 20 min at room temperature and slowly added to the cells. The medium was changed 4 h after transfection and cells were cultured with 700 μg/ml G-418  to establish stable cell lines. Clones were isolated and checked for their EGFP fluorescence. Clones with a stable nuclear expression of the EGFP-SMC1 were taken for subsequent FRAP experiments. Cells stably transfected with SMC1-EGFP have previously been shown to still form an intact cohesin complex containing the EGFP-SMC1 protein A single point mutation (T2896G) in the cDNA sequence was generated in SMC1 wt to mutate Ser966 to Ala. Site-directed mutagenesis was carried out with the QuikChange® II XL Site-Directed Mutagenesis Kit  according to the manufacturer's instructions. The following primer was used 5′AGG ACT CAG TGA GTG GTG CAC AGA GAA TTT CCA GT-3′.137Cs γ-ray irradiator  at a dose rate of ~ 1.7 Gy/min. Partial cell irradiation using partially shielded ultrasoft X-rays Conventional X-ray exposures were performed at room temperature with a 250 kV  X-ray set (Pantak) with a compound filter of copper and aluminium  at a dose rate of ~ 1 Gy/min. Gamma-ray exposures were performed using a GSR D1 Immunofluorescence was performed as described elsewhere 2 independent medium without phenol red  supplemented with 10% FCS and antibiotics for another 30 min before imaging was started. Cells were kept at 37 °C on a heated stage insert  during imaging. Single cells were captured automatically with open pinhole using a Zeiss LSM710 microscope, Plan-Apochromat 63×/1.40 Oil DIC M27 objective. Nine iterations of photo-bleaching at 100% transmission of 488 nM laser were used for all FRAP experiments.SMC1-EGFP cells were grown in T25 flasks and synchronized by double thymidine block All cells that moved during the imaging process were excluded from analysis. To analyse FRAP data, fluorescence recovery was measured in user-defined cellular regions. The background-subtracted mean intensities were normalized to the initial fluorescence intensity distribution, thereby correcting for fluorescence decay due to post-bleach acquisition .The diagrams shown in Immuno-precipitations were carried out as described previously Cell pellets were lysed in ice-cold lysis buffer  containing protease inhibitors and 50 nM okadaic acid . Proteins were separated on NuPage Novex 3–8% Tris–Acetate or 12% Bis–Tris gels  and then transferred onto a PVDF membrane according to the manufacturer's protocol . Membranes were blocked in phosphate-buffered saline (PBS) containing 5% BSA. Primary and secondary antibodies were diluted in Tris-buffered saline/0.1% Tween20 (TBST) containing 2.5% BSA over night at 4 °C or for 1 h at room temperature. The proteins were visualised using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and imaging was carried out using either a Kodak Image Station 4000MM system or Hyperfilm ECL films . Quantification was performed using Kodak Molecular Imaging V4.0.1 software. The level of RNAi knockdown was determined using the level of actin as a standard.g and the cells were resuspended in PBS containing propidium iodide  at a final concentration of 10 μg/ml.Cells were harvested and fixed with ice-cold methanol at 4 °C for a minimum of 30 min. For cell cycle analysis the fixative was removed by centrifugation at 250 The samples were run on a Becton-Dickinson FACScan. Analyses were carried out using Flowjo .We used a partially shielded ultrasoft X-ray system A to deteTo address whether phosphorylated SMC1 and SMC3 are part of the cohesin complex, we immuno-precipitated SMC3pS1083 from un- or irradiated HeLa cell extracts C. SMC3pSTo test whether the cohesin complex had to be assembled to enable efficient SMC phosphorylation, siRNA was used to deplete the Rad21 subunit of cohesin D. SurpriWe wanted to know whether RAD21 depletion results in changes of the cell cycle distribution and therefore this could explain the strongly reduced levels of SMC1 and SMC3 phosphorylation.A cell cycle analysis was performed where HeLa cells were transfected with Rad21 or control siRNA, fixed 24 h post transfection and stained for PI to analyse cell cycle distribution. Rad21 depleted cells have a marginally different cell cycle distribution with more cells in G1 and less in S and G2/M. This marginal difference is statistically significant as shown by a t-test. The observed difference in relative numbers means 5% more G1 cells (55.94% vs. 50.75%), 3% less S cells and 2% less G2/M cells in Rad21 depleted cells . We do not think that this observed difference explains the strongly reduced levels of SMC1 and SMC3 phosphorylation.To determine the spatial distribution of SMC phosphorylation in relation to the DNA damage marker γH2AX in different cell cycle phases, co-immunofluorescence microscopy was performed with centromer protein F (CENP-F) which is absent in G1, increasingly abundant during S and peaks in G2/M To test whether generation of SMC1pS966 and SMC3pS1083 is H2AX-dependent in human cells and at which step in the signalling cascade the cohesin phosphorylation takes place, we used siRNAs to deplete H2AX, MDC and 53BP1 (for details see Supplementary Table S1).HeLa cells were transfected with either a single siRNA or any combination of the three. Cells were irradiated 48 h following transfection and then left for 1 h to allow for DNA damage signalling and repair. The siRNA-treated extracts A were usHowever, SMC1pS966 and SMC3pS1081 were still present following any individual siRNA treatment and any combination of two of the siRNAs reduced the SMC3pS1083 signal to about 50% whereas the SMC1pS966 did not seem to be affected B and C. FRAP experiments were performed to elucidate whether the phosphorylation of Ser966 has an influence on the mobility of SMC1. Stable cell lines were established expressing either wt EGFP-SMC1 or mutant EGFP-SMC1 where S966 was changed into an A. Relative expression levels of EGFP-SMC protein in these cells were assessed  and the expression ratio of EGFP-SMC1wt to endogenous SMC1 (1.40) is similar to the ratio of EGFP-SMC1S966A to endogenous SMC1 (1.37).These cells were synchronized by a double thymidine block, irradiated or sham-treated 7 h after release from the second thymidine arrest and incubated for 1 h before imaging was started.To determine the cell cycle stage during FRAP imaging, synchronized cells were fixed at indicated time points and stained with propidium iodide B. TherefDuring FRAP imaging, one part of the nucleus in each cell was photo-bleached and the signal recovery was followed C. Our exThe bleaching introduced with the FRAP experiments was 75% +/− 2.2% for the un-irradiated and 79% +/− 3.4% for the irradiated cells. The calculated immobile fractions are 64% +/− 6.4% for the wt SMC1 and 51.2% +/− 8.8% for the mutant SMC1 .We analyzed the redistribution kinetics of irradiated and un-irradiated G2 cells by plotting recovery curves over time D and E. It has been shown that SMC1 and also SMC3 are phosphorylated by ATM Using siRNA-mediated knockdown of Rad21, which is required to form a functional cohesin complex, we show that the phosphorylation of SMC1 and SMC3 requires fully assembled cohesin. It was shown previously that SMC1 phosphorylated on S957 can be immuno-precipitated with SMC3 indicating that it is part of the cohesin complex H2A phosphorylation is required for cohesin accumulation at DNA double-strand breaks in yeast We used FRAP and measured the dynamic process of binding of cohesin after IR and its exchange over time where we focused on the influence of S966 on the mobility of SMC1. Cohesin consists of two populations, one fast pool with a residence time of 8.4 ± 3.3 min and a slow population with a residence time of more than 6 h in G2-phase cells How SMC1 and 3 modifications influence cohesin binding mechanistically is currently not clear. We speculate that one functional consequence of SMC phosphorylation is to regulate the ATPase activity of the cohesin complex because the phosphorylation sites Ser966 on SMC1 and S1083 on SMC3 are close to the ATP hydrolysis sites in each SMC protein. Posttranslational modifications close to the ATP hydrolysis site might regulate this distinct step of the ATPase cycle. This is consistent with the observation that the ATPase function of the SMC1/3 heterodimer is essential for cohesin binding to chromatin and sister chromatid cohesion We speculate that ATP hydrolysis in the SA mutant is impaired and therefore movement of SMC1 after IR is slower than of wt SMC1. While SMC1S966A turned out to be slower in the beginning of the FRAP measurements the overall fraction of mobile protein has not changed (immobile fractions of wt and mutant SMC1 are similar). We speculate that the ATP hydrolysis is impaired and the mobility of the cohesin complex is altered because in the complexes with SA SMC1 and wt SMC3 only the SMC3 phosphorylation can influence the ATP hydrolysis.One possible scenario could be the accumulation of cohesin at DNA DSBs due to the mobilized phospho-cohesin complex. Alternatively the observed enhanced mobility may suggest a release of phosphorylated cohesin subunits. This would result in more phospho-SMC1 floating freely in the nucleus which in turn would speed up fluorescence recovery. This may occur at sites of completed DSB repair, given that half of all DSBs are repaired within 1 h after irradiation.This may only be one function of the phosphorylation sites of the cohesin complex and further investigation is needed to obtain a more complete picture of their functional relevance.


Innate immunity to viruses involves receptors such as RIG-I, which senses viral RNA and triggers an IFN-β signaling pathway involving the outer mitochondrial membrane protein MAVS. However, the functional status of MAVS phosphorylation remains elusive.Here we demonstrate for the first time that MAVS undergoes extensive tyrosine phosphorylation upon viral infection, indicating that MAVS phosphorylation might play an important role in MAVS function. A tyrosine-scanning mutational analysis revealed that MAVS tyrosine-9 (Y9) is a phosphorylation site that is required for IFN-β signaling. Indeed, MAVS Y9F mutation severely impaired TRAF3/TRAF6 recruitment and displayed decreased tyrosine phosphorylation in response to VSV infection compared to wild type MAVS. Functionally, MAVS Y9 phosphorylation contributed to MAVS antiviral function without interfering with its apoptosis property.These experiments identify a novel residue of MAVS that is crucially involved in the recruitment of TRAF3/TRAF6 and in downstream propagation of MAVS signaling. The innate immune system, which is characterized by the production of type-1 interferon (IFN)-α/β cytokines and by the activation of natural killer (NK) cells, plays important roles in invading pathogens detection and elimination The MAVS molecule has emerged as the key regulatory in RIG-I signaling, signal transduction downstream of MAVS culminates in the activation of the IRF3/7 (interferon regulatory factors 3 and 7) and NF-κB transcription factors HEK293T, MCF-7 and HepG2  cells were grown in Dulbecco's modified Eagle's medium  supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Vectors and Epitope Tagging of Flag-tagged MAVS, Myc-tagged MAVS, Myc-tagged TRAF3, Myc-tagged TRAF6 and Myc-tagged STING were expressed by cloning the genes into the pcDNA3-based vector (Invitrogen). Flag-MAVS mutation plasmids were made based on the pcDNA3-Flag-MAVS plasmid using Quick-Change Mutagenesis kit (Invitrogen). Transient transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.MAVS−/− mice on a 129/Sv/C57BL/6 background were a kind gift from Dr. Zhijian. Chen Cell lysates were prepared in lysis buffer  containing 1% Nonidet P-40. Soluble proteins were subjected to immunoprecipitation with anti-Flag , anti-Myc , or anti-mouse IgG antibody (Sigma). An aliquot of the total lysates  was included as control. Immunoblotting analysis was performed with anti-Myc, HRP-conjugated anti-Flag (Sigma), anti-β-Tubulin (Sigma) and anti-MAVS . The antigen-antibody complexes were visualized by chemiluminescence .HEK293T cells were transfected with 0.2 µg of the Luciferase reporter pNF-κB-LUC or IFN-β-LUC plus 0.02 µg of the internal control reporter pRL-TK, with or without MAVS expression vector. Transfected cells were collected and Luciferase activity was assessed. All experiments were repeated at least three times in duplicate.Cells were washed briefly in PBS, fixed in 4% paraformaldehyde (PFA) in PBS for 10 min, and the nuclei were stained for 10 min with DAPI. After a final washing in PBS, samples were preserved in glycerol and images were captured using a digital camera under a confocal microscope (Zeiss LSM510).3VO4, protease inhibitor mixture (Roche), 1 mM DTT, 1 mM PMSF] and passed through a 25G needle 15 times. Nuclear fractions were collected by centrifugation at 1,000×g for 5 min at 4°C. Mitochondrial fractions were collected by further centrifugation of the supernatants at 10,000×g for 5 min at 4°C. The supernatants were further fractionated by centrifugation at 100,000×g for 30 min at 4°C to collect membrane and cytosolic fractions.Cells were harvested in low-salt buffer [10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM NaF, 1.5 mM NaFirst-strand cDNA was generated from total RNA using random priming and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen). Real-time PCR was performed using QuantiTect SYBR Green PCR Master Mix  in triplicate experiments and analyzed on an ABI Prism 7700 analyzer . All real-time values were normalized to 18S ribosomal RNA. IFN-β using the following primers:5 -CACGACAGCTCTTTCCATGA-3;IFN-β S, 5 -AGCCAGTGCTCGATGAATCT-3.IFN-β AS, 6 cells/plate and incubated overnight. Viral infection was performed when 60% cell confluence was reached. Culture media was replaced by serum-free DMEM, and VSV was added into the media at multiplicity of infection (MOI) as indicated, respectively. After 1 h incubation, extracellular virus was removed by washing cells twice with serum-containing media. Cells and supernatant were harvested at indicated times post-infection.Cells were plated in 10 cm plates at a density of 1×10For Trypan blue exclusion test of cell viability, 10 ml of cell suspension was mixed with 10 ml of 0.4% trypan blue solution (Sigma), and both unstained (viable) and stained (dead) cells were counted on the hemacytometer. The percentage of dead cells was calculated by dividing the number of stained cells by the total number of cells.For propidium iodide staining, cells were harvested by trypsinization, fixed with ice-cold 70% ethanol and resuspended in a solution containing 50 µg/mL propidium iodide, 0.1% Triton X-100, 50 µg/mL RNase A, and 5 mM EDTA at room temperature for 1 h. Cells were then diluted 1∶1 in 1% BSA PBS for cytometric analysis. DNA content was assessed by staining 70% ethanol-fixed cells with propidium iodide and monitoring by FACScan . Annexin V staining was performed as indicated with the Annexin V-Biotin Apoptosis Detection Kit (BD Bioscience). Briefly, cells were washed with cold PBS, resuspended in 1× Binding Buffer and then Annexin V Dye was added, after 15 min incubation, cells were washed and propidium iodide was added, cells were analyzed by flow cytometry. The results were expressed as the mean of three independent experiments.Analyses were done using the statistical software SAS/STAT. Data analyses over time were undertaken by repeated measures analysis using SAS/STAT. P<0.001 was considered the threshold value for statistical significant significance.Recent studies showed that ubiquitination at MAVS K500 mediates recruitment of IKKε to MAVS, leading to MAVS phosphorylation and negative regulation of the NF-κB activation, raising the possibility that phosphorylation may be involved in this process In order to identify the relevant tyrosine residue(s) of MAVS required for its phosphorylation activity, all the tyrosine amino acids in human MAVS were substituted with phenylalanine residue individually. Plasmids encoding individually the 10 variants were generated by site-directed polymerase chain reaction (PCR) mutagenesis using a WT MAVS (accession number NM_020746) vector as a template. We first studied the impact of those mutations on the expression pattern of MAVS protein. To avoid a nonspecific cell-type effect, WT and YF mutational MAVS were expressed in two different human cell lines, the human embryonic kidney cell line HEK293T and human breast cancer cell line MCF-7. Western-blotting revealed that none of the YF mutations altered MAVS expression profile . InteresMAVS is a mitochondrial outer membrane protein, which is required for its self-association and IFN-β signaling. Thus we first analyzed the subcellular localization of MAVS Y9F mutant. We employed confocal microscopy to visualize Hela cells transfected with an expression vector encoding Flag-tagged MAVS or Flag-tagged MAVS Y9F mutant. Fluorescent immunostaining with a Flag-specific antibody showed a punctuate pattern of MAVS localization that overlapped with the staining pattern of Mito Tracker, a fluorescent marker taken up specifically by mitochondria. We also found by confocal microscopy that MAVS Y9F mutant was a mitochondria protein that overlapped with Mito Tracker staining just as WT MAVS .To further determine if MAVS Y9F mutant is localized to the mitochondrial membrane, we carried out subcellular fractionation experiments. HEK293T cells were homogenized in an isotonic buffer that preserved mitochondria and other organelles, and the cell lysates were then subjected to differential centrifugation to separate cytosol and nucleus from mitochondria, MAVS Y9F mutant was found only in the mitochondria pellet, together with other mitochondrial proteins including HSP60 just as WT MAVS . Y9F MAVNext, we investigated the mechanism by which Y9F resulted in altering MAVS-mediated immune response. We hypothesized that such a mutation might alter MAVS conformation, thus modifying its interaction with downstream signaling molecules. Among them, TRAF3, TRAF6 and STING (stimulator of interferon genes) are critical MAVS signaling effectors Since MAVS Y9 is a phosphorylation acceptor tyrosine that mediates TRAF3 and TRAF6 recruitment to MAVS, the ability of MAVS Y9F mutant to undergo phosphorylation was then examined. Phosphorylation was examined using a tyrosine-specific phosphorylation antibody with ectopically expressed wild-type MAVS or MAVS Y9F in HEK293T cells. A sharp reduction of tyrosine phosphorylation was observed in Y9F mutant compared to wild type MAVS upon VSV infection, indicating that tyrosine-9 of MAVS was the phosphorylation site . Both thTo confirm the relevance of the loss-of-function effect of Y9F MAVS in virus-infected cells, we examined the responsiveness of WT or Y9F MAVS to VSV viruses in MAVS−/− MEFs introduced by either WT MAVS or Y9F mutant. While WT MAVS rescue highly enhanced antiviral response triggered by viral infections, Y9F transfection dramatically impaired these responses. Remarkably, Y9F mutation resulted in complete loss-off induction on IFN-β mRNA and protein levels .We next sought to determine whether MAVS Y9F mutant regulated the replication of VSV virus replication, as RIG-I-mediated IFN-β signaling is critical in restricting replication of this RNA virus. We therefore used VSV to infect MAVS−/− MEFs transfected with an expression vector encoding Flag-tagged MAVS or Flag-tagged MAVS Y9F mutant. MAVS Y9F mutant increased the production of VSV by about 6 fold , suggestPrevious reports have shown that the MAVS-induced caspase-dependent apoptosis and MAVS-induced apoptosis are independent of type-1 interferon production or NF-κB activity Ubiquitination and phosphorylation have emerged as key posttranslational modifications that control induction and shutdown of the interferon response A recent report demonstrated that the transmembrane domain is essential for MAVS to both self-associate and signal et al. recently demonstrated that K63-linked ubiquitination of C-terminal residues of IRF7 occurred as a prelude to IRF7 phosphorylation and activation et al argued that IKKε directly phosphorylated MAVS and that this serine or threonine phosphorylation modification of MAVS played a negative control in IFN-β signaling In addition to K63-ubiquitination at MAVS K500, MAVS can link K48-polyubiquitin chains following virus infection and is the target for proteasomal degradation by the E3 ligase RNF125, suggesting that MAVS may undergo multiple ubiquitination events. Ning et al demonstrated that MAVS-mediated apoptosis was not a consequence of IFN-I induction by MAVS It is known that MAVS is a potent inducer of IFN-I responses and that IFN-I can activate host apoptotic responses, therefore it was important to determine whether the cell death response observed in the current study was a consequence of IFN-I secretion. Lei In summary, here we have demonstrated that phosphorylation modification is involved in the control of MAVS activity in innate immune response, which thereby provides positive regulation of the innate antiviral response against infection by RNA viruses. Phosphorylation at tyrosine-9 is essential events to regulate the activity of MAVS. Uncovering the mechanism of MAVS phosphorylation state controlled by protein kinases and phosphatases will help us to further understand the function of MAVS at the molecular level.


Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13.Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by 148–E166) and S22 (A243–K274) were recognized by sera from 90% and 100% of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T261–K274), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity.Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (Ain silico-predicted residues involved in an important antigenic region of Pen c 13. The G270A mutant of Pen c 13 has the potential to serve as an additional tool for the diagnosis/prognosis of mold allergy, and the K274A mutant, as a hypoallergenic form of the epitope, may provide a framework for the design and development of a safe and efficient therapeutic strategy for treating human allergic diseases.Experimental analyses confirmed  Penicillium sp. is one of the most frequently encountered species Penicillium citrinum among patients with mold allergy Fungi are ubiquitous in our environment, and many have been identified as causative factors in IgE-mediated allergy. Among the fungi associated with allergic disorders, Understanding of IgE-binding epitopes on allergens should help elucidate allergen-antibody interactions and may contribute to the development of more effective immunotherapeutic strategies. It has been demonstrated that immunotherapy induces strong allergen-specific IgG responses and that protective IgG antibodies are important for the success of allergen-specific immunotherapy Among the strategies for identifying allergen epitopes are fragmentation of allergens, either enzymatically Pharmacological agents have been the mainstay of allergy symptom management, but allergen-specific immunotherapy is the only long-lasting, allergen-specific approach for treating type I allergy and preventing its progression to severe disease manifestations In the present study, we mapped the IgE-binding epitopes on recombinant Pen c 13 (rPen c 13) using enzymatic digestion and chemical cleavage methods, followed by dot-blotting and mass spectrometry. A concentration-response inhibition assay was performed to provide evidence of IgE binding. We also integrated results from a B-cell epitope-predicting server and molecular modeling to minimize the experimental validation process. Subsequent site-directed mutagenesis of the residues involved in the major epitope, S22, led to the identification of amino acids that are important for allergen-IgE interactions. Identification of immunodominant regions of Pen c 13 may facilitate the development of specific peptide-based diagnostics and possibly lead to therapies for mold allergy.Penicillium notatum recognized by human IgE antibodies≥class 2 (0.70 kU/l). Among these patients a total of 10 individuals who had a positive skin prick test  and stored in aliquots at −80°C until use. The allergic response was confirmed by clinical history and diagnosis, and characterized by measuring specific IgE reactivity using the ImmunoCAP® system . Mold-allergic patients were initially included on the basis of an ImmunoCAP score for the crude extract of ick test  responseP. citrinum DNA library by polymerase chain reaction (PCR), and the amplified fragment was ligated into the vector pQE 30  to generate an expression plasmid for N-terminally His6-tagged Pen c 13 (pQE-30/Pen c 13), which was then used to transform E. coli M15 cells (Qiagen). Cultures of transformed cells were grown to an OD600 value of 0.6, induced with 1 mM isopropyl-β-D-thio-galactoside , and harvested after 2 h. The His-tagged rPen c 13, isolated as inclusion bodies, was bound to a Ni2+-chelate affinity column, washed with binding buffer , and then eluted with elution buffer . Finally urea was removed by sequential dialysis with reducing urea concentrations from 8 M to 4 M, 2 M, 1 M, and 0 M against phosphate-buffered saline (PBS). The dialyzed rPen c 13 was concentrated using the concentrators with 10 kDa cut-off membrane . Protein concentrations were determined using a bicinchonic acid protein assay reagent kit  using BSA as the standard. The purity of rPen c 13 was verified by Coomassie Brilliant Blue staining after SDS-PAGE and mass spectrometric analysis.rPen c 13 was purified as described previously p-nitrophenyl phosphate substrate. Plates were allowed to stand in the dark for 30 min before reading at 405 nm. The cutoff was determined as two times the mean of the 30 controls. To adjust for inter-plate variability, normalized ELISA values were calculated by dividing the optical density (OD) of the test samples by the mean OD of the reference sera on each plate.The detection of specific IgE antibodies reactive with Pen c 13 was examined by enzyme-linked immunosorbent assay (ELISA) using sera from 80 patients and 30 healthy controls. Microtiter plates  were coated with rPen c 13 by incubating with a 0.5-µg/ml solution of rPen c 13 in PBS. Each serum sample was serially diluted in PBS/0.1% Tween-20 and added to plates, which were then incubated for overnight at 4°C. Bound IgE was detected by incubating sequentially with alkaline phosphatase-labeled anti-human IgE antibodies  and a Pen c 13 peptides were obtained through enzymatic and chemical cleavage. rPen c 13 was digested overnight at 37°C with lysyl endopeptidase (EC 3.4.21.50), trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), or Glu-C endopeptidase (EC 3.4.21.19) at an enzyme/substrate ratio of 1∶50. Lysyl endopeptidase, trypsin, and chymotrypsin digestions were carried out in 0.1 M pyridine/acetate/collidine (pH 8.2), and Glu-C endopeptidase digestions were performed in 0.1 mM calcium chloride and 0.1 M pyridine/acetate (pH 6.5). The resulting peptides were fractionated by reversed-phase high-performance liquid chromatography (HPLC) on a Beckman ODS (C18) column  using a linear gradient of 0–44% acetonitrile in 0.06% trifluoroacetic acid and a flow rate of 1 ml/min. Cyanogen bromide (CNBr) cleavage was performed at 37°C for 24 h in 70% formic acid with a 100-fold molar excess of CNBr over methionine residues. After cleavage, the fragments were separated by HPLC on a YMC-Pack C4 column  using the same elution system as for the Beckman column. Peptide elution was monitored at 220 nm, and all fractions were collected and analyzed for peptide mass and purity using a Procise ABI 494 protein sequencer  and a QSTAR XL mass spectrometer (Applied Biosystems).Approximately 30 pmol each of lysyl endopeptidase-, trypsin-, chymotrypsin-, Glu-C endopeptidase-, and CNBr-cleaved peptides from rPen c 13 was spotted onto a polyvinylidene fluoride (PVDF) membrane. A total of 24 peptides covering most of the Pen c 13 sequence were screened for their ability to bind human IgE in individual serum samples from allergic subjects. Bound antibodies were detected using alkaline phosphatase-conjugated goat anti-human IgE (Biosource) and the CDP-Star detection system , according to the manufacturer's instructions.To evaluate the major epitopes S16 and S22, using three individual patient sera , with a high, medium and low ranges of the ELISA OD values were previously incubated overnight at 4°C with different concentrations of S16, S22, or both. For inhibition control, a pool of sera from the 10 patients was previously incubated overnight at 4°C with different concentrations of rPen c 13 as a positive control or albumin as the negative one. Microtiter plates  were coated with rPen c 13 by incubating for overnight at 4°C with 100 µl of a 5-µg/ml solution of rPen c 13 in PBS, then incubated for overnight at 4°C with aliquots of separate sera (diluted 1∶10) that had previously been incubated with different concentrations of peptides or proteins as mentioned above. The amount of specific IgE bound to the wells was determined as described above.To assist in the identification of potential residues that are critical in IgE binding, we utilized an epitope conservancy analysis tool. This tool was implemented as a component of the Immune Epitope Database and Analysis Resources (IEDB) Lecanicillium psalliotae, Ver112 (Protein Data Bank code 3F7M), which is the mostly closely related to Pen c 13  in the structure database Pen c 13 was modeled based on the structure of the alkaline serine protease from 243–K274), (A243–A260) and (T261–K274), is amplified using pQE-30/Pen c 13 as a template and the PCR with oligonucleotide primers  designed specifically for amplification of the piece of cDNA of interest. Incorporating restriction sites into the primers facilitates subcloning into the multiple cloning site of the pGEX-2T vector. The PCR products were gel-purified and then ligated into the pGEX-2T expression vector  at BamHI and EcoRI sites. The fidelity of the cloned sequence was confirmed by DNA sequencing. For production of recombinant proteins, all plasmids were transformed into Escherichia coli strain BL-21 (DE3) . Cultures of transformed cells were grown to an OD600 value of 0.6, induced with 1 mM IPTG, and harvested after 4 h. Cells were resuspended in binding buffer  and sonicated on ice for 5 min, after which the suspension was centrifuged and the supernatant was loaded onto a Glutathione Sepharose 4 Fast Flow column  pre-equilibrated in binding buffer. After adsorption, the non-bound material was removed by washing with binding buffer, and bound protein was eluted using elution buffer . Eluted fractions were collected and analyzed by SDS-PAGE.The region of the Pen c 13 targeted for (A261–K274) was cloned into the pGEX-2T vector as described above and used as a template for the preparation of all Pen c 13 (T261–K274) mutants. These mutants were generated using the QuikChange Site-Directed Mutagenesis kit . All mutated genes were confirmed by direct DNA sequencing. The primers used for mutagenesis were as follows:The gene fragment for wild-type (WT) Pen c 13 (TGCTGGCACTACAAGCAAG-3′5′-ATCTCTAGGGCTCCCS269A: GCCACTACAAGCAAGTAA-3′5′-TCTAGGGCTCCCTCTG270A: GCTACAAGCAAGTAAGAA-3′5′-AGGGCTCCCTCTGGCT271A: GCAAGCAAGTAAGAATTC-3′5′-GCTCCCTCTGGCACTT272A: GCCAAGTAAGAATTCATC-3′5′-CCCTCTGGCACTACAS273A: GCGTAAGAATTCATCGTG-3′5′-TCTGGCACTACAAGCK274A: 261–K274) mutants were expressed and purified as described above.The nucleotide sequences were obtained from GenBank (accession no. AF084546); the mutated sites are underlined. The nucleotide sequences of all clones were confirmed by DNA sequencing. All GST fusion rPen c 13 (T243–K274), (A243–A260) and (T261–K274), and mutants of rPen c 13 (T261–K274) were separated by SDS-PAGE along with GST only (control). For specific IgE immunodetection, resolved GST fusion peptides were transferred to a PVDF membrane, which was then blocked with skim milk, and incubated with serum (diluted 1∶10) from mold-allergic patients. Bound human IgE was detected using horseradish peroxidase-conjugated goat anti-human IgE antibodies (BioSource). Bands were visualized using enhanced chemiluminescence reagents (Millipore) and autoradiography. Densitometric analysis was performed using the LabWorks software . The relative density of the bands normalized to loading control.GST fusions of WT rPen c 13  . The intTo analyze the relevance of IgE-binding sequences identified by dot-blotting, we performed IgE ELISA inhibition experiments. Three serum samples that recognized the epitopes S16 and S22 of Pen c 13 in dot-blots were used for ELISA inhibition analysis. Sera from patients preincubated with different concentrations of the peptide  were evaluated for IgE binding to rPen c 13. The results showed that IgE reactivity with Pen c 13 was inhibited in a concentration-dependent manner by epitope S16 and/or S22 . For inhLecanicillium psalliotae, as a suitable template (PDB ID: 3F7M). The identity and similarity between the target and template were 48% and 67%, respectively. As shown in According to the results of IEDB prediction, shown in 243–K274) and narrow down the candidate epitope residues predicted by the in silico-based method, we expressed and isolated the GST fusion peptides, rPen c 13 (A243–A260) and rPen c 13 (T261–K274). Immunoblotting unambiguously showed that IgE reactivity is located in the rPen c 13 (T261–K274)  GST fusion, and assayed their ability to bind IgE. The result of the immunoblot strip in patient 10 was shown in To validate the IgE-binding region of Pen c 13 (A61–K274) , consistIdentification of IgE-binding epitopes might provide a better understanding of the functional role that allergens play in the disease and could have implications for immunodiagnosis and immunotherapy. A number of approaches are currently used to predict linear epitopes, including sequence-based prediction software and 3D structure-based prediction Lecanicillium psalliotae Ver112, the IgE-binding determinant 261TSSISRAPSGTTSK274 assumes a loop-like structure on the surface of the protein. This finding is consistent with the known importance of loop-like structures in IgE binding and the reported allergenicity of other allergens, such as transaldolase, a fungal allergen whose IgE-reacting fragment is located in a loop-like structure of the C-terminal region In the present study, we mapped IgE-binding epitopes using enzymatically/chemically cleaved fragments to obtain more detailed information on the binding of IgE to the Pen c 13 molecule. Cleaved peptides covering most of the sequence of Pen c 13 were analyzed by dot-blotting using sera from mold-allergic patients. Twelve distinct IgE-binding epitopes distributed throughout the entire molecule were found, although additional epitopes may also exist. Only a few epitopes may have been missed because epitopes are located on cleavage sites and small gaps in the sequence coverage. In molecular modeling studies, these regions were predicted to lie on the protein surface and contained mostly solvent-exposed residues. The results suggest that IgE predominantly recognizes linear epitopes, the S22 peptide (residues 243–274), accounts for a high percentage of Pen c 13-specific IgE binding. In a 3D model of Pen c 13 based on the structure of 269SGTTSK274) and generated six mutants with single alanine substitutions to test the role of the predicted residues. The K274A mutation largely abolished IgE-binding ability, reflecting charge and/or size differences between lysine and alanine, implying that K274 is a major contributor to this interaction. Dramatic effects of single amino acid substitutions on the IgE-binding properties of allergens have been previously reported Using computational prediction, we further narrowed the responsible sequence to a six-residues region . An SPT was performed on 10 patients who had serum IgE reactive with Pen c 13 in a specific IgE ELISA and a history of mold allergy. The skin testing was performed using 50 µl of purified Pen c 13 from Penicillium citrinum at concentrations of 10 mg/ml and 100 mg/ml in physiological saline (0.9% NaCl) or physiological saline solution alone as a negative control. Skin reactions  were recorded 15 min after prick. Wheals with a diameter of at least 1.5 mm greater than that produced by the negative control were regarded as positive responses.(DOC)Click here for additional data file.


Autophagy is a cellular response to intracellular pathogens including mycobacteria and is induced by the direct inhibitors of mammalian target of Rapamycin (mTOR), a major negative regulator of autophagy. Autophagy induction by mTOR inhibition (mTOR dependent autophagy), through chemical means or starvation, leads to mycobacterial killing in infected cells. However, previous work by our group has shown that mycobacterial infection of macrophages naturally induces both autophagy and mammalian target of Rapamycin (mTOR) activity (mTOR independent autophagy). In the current work, we further explore the relationship between mTOR activity and mycobacterial killing in macrophages.Mycobacterium smegmatis killing. The growth of M. smegmatis was also inhibited by high concentrations of Rapamycin in LC3B and ATG5 deficient bone marrow derived macrophages, suggesting that non-autophagic mechanisms might contribute to killing at high doses. Since mycobacterial killing could be observed only at fairly high concentrations of the mTOR inhibitors, exceeding doses necessary to inhibit mTOR, we hypothesized that high doses of Rapamycin, the most commonly utilized mTOR inhibitor for inducing autophagic killing, may exert a direct bactericidal effect on the mycobacteria. Although a short-term treatment of mycobacteria with Rapamycin did not substantially affect mycobacterial growth, a long-term exposure to Rapamycin could impact mycobacterial growth in vitro in select species.While low concentrations of the mTOR inhibitors, Rapamycin, Torin 1, and Torin 2, can effectively reduce or block mTOR activity in response to lipopolysaccharides (LPS) or mycobacteria, higher concentrations (10 uM) are required to observe This data, coupled with previous work from our laboratory, further indicates that autophagy induction by mTOR inhibition is an artificial means to increase mycobacterial killing and masks more relevant endogenous autophagic biochemistry that needs to be understood. M. tuberculosis, a protein essential for the processing of LC3B, results in increased bacterial burdens and enhanced inflammatory responses in comparison to ATG5 expressing mice [The autophagy pathway was first identified as a stress response that allowed cells to survive when nutrients were scarce . Under sing mice . Thus, iPrevious work by our laboratory has documented that mycobacterial infection naturally induces autophagy in RAW264.7 cells . mTOR inM. smegmatis and BCG induced similar levels of P-S6 as well. Rapamycin (1 uM-10 uM) inhibits P-S6 induction in response to Mycobacterium bovis Bacille Calmette-Guérin (BCG) while 25 uM Rapamycin was used to confirm the ability of M. smegmatis to induce mTOR activity. In the current work, we aim to expand upon previous data and further define the connection between mTOR inhibition and mycobacterial killing. A panel of mTOR inhibitors that target mTOR kinase directly (Torin 1 and Torin 2) or indirectly (Rapamycin) was chosen to confirm the capacity of these agents to both inhibit mTOR activity and to induce autophagy [Escherichia coli-derived LPS. All compounds were effective at both 1 uM and 10 uM concentrations to reduce the induction of phosphorylated ribosomal S6 (P-S6), a bona fide mTOR target . As a result, we identify 10 uM as the minimum dose required for robust mycobacterial killing. Lastly, while not used as extensively as Rapamycin, Torin 1, and Torin 2, preliminary work with KU0063794, an additional mTOR inhibitor [M. smegmatis, yet the same doses were effective at reducing mTOR activity.Since as little as 1 uM of each inhibitor was sufficient to demonstrate mTOR inhibition and DQ-BSA hydrolysis in RAW264.7 cells, we evaluated whether equally low concentrations of mTOR inhibitors could produce observable levels of mycobacterial killing. RAW264.7 cells were infected with hibitors . While 1nhibitor , has thuM. smegmatis killing, we wanted to determine if the mycobacterial killing induced by higher doses of mTOR inhibition was autophagy specific or due to an unrecognized secondary effect. Similar killing assays were performed in LC3B and ATG5 [M. smegmatis. As shown in Figure M. smegmatis killing in these assays. Similar assays were independently applied to LC3B and ATG5 deficient macrophages. It has been established that ATG5 is critical for efficient autophagic responses to mycobacteria, and that LC3B coated vesicles co-localized with mycobacteria to deliver toxic payloads [M. smegmatis or the response to Rapamycin, western blots from infected cells with or without Rapamycin treatment were performed. As expected, LC3B deficient macrophages could induce P-S6 in response to M. smegmatis, and P-S6 induction was blocked by Rapamycin  were essential to produce consistent and robust and ATG5 ,21 deficpayloads ,22,23. TM. smegmatis and BCG were cultured in DMEM containing 10% HI-FBS under mammalian cell conditions with either DMSO (control) or Rapamycin. After 3 hours, the cultures were harvested and the mycobacteria were plated for CFU determination. As shown in Figure M. smegmatis nor BCG viability was impacted by Rapamycin in these assays when cultured with up to 25 uM Rapamycin. We conclude that Rapamycin does not appear to directly impact the mycobacteria tested during a standard autophagy assay condition in the absence of the host cells.The observation that higher of doses of mTOR inhibitors were required to elicit robust mycobacteria killing in RAW264.7 cells led us to suspect that Rapamycin might directly impact mycobacteria in autophagy assays. To test this hypothesis, a modified autophagy assay was performed whereby RAW264.7 cells were eliminated from the assay. Mycobacterium avium subspecies paratuberculosis (MAP) [M. smegmatis does not appear to be altered by Rapamycin, we wondered if longer exposures of Rapamycin could alter the growth properties of mycobacteria. Several species of mycobacteria were exposed to Rapamycin for longer durations in their typical growth media (7H9-OADC) and the OD600 was monitored for changes in culture growth. M. smegmatis, which is a non-pathogenic, fast-growing mycobacterium, was not impacted by Rapamycin through 10 hours of exposure  as indicated by Student’s T test, the magnitude of the differences was modest. There was however no differences observed at shorter time-points  . While te Figure A. In cone Figure B. While s Figure C. Thus, The autophagy pathway has emerged as a versatile cellular mechanism that allows mammalian cells to defend themselves from an array of intracellular microbes ,9. AutopM. smegmatis in RAW264.7 macrophages  [M. kanasii, and laboratory and clinical isolates of M. tuberculosis, did have altered growth properties when exposed to Rapamycin for extended periods of time  , it seeme Figure . While nThroughout this study we utilized the phosphorylation of ribosomal S6 protein (P-S6) as the indicator of mTOR activity in our assays. While this is a well-known and highly bona fide mTOR target that has been widely utilized as a measure of mTOR activity ,25, we cThe use of mTOR inhibitors to induce mTOR dependent autophagic pathogen killing has become the gold-standard assay within the autophagy field. This is somewhat counterintuitive given that nutrient sensing and pathogen sensing utilize unique, and presumably non-overlapping, biochemical mechanisms to affect stimulus specific responses. Taken in sum, our current work strongly suggests that the use of mTOR dependent autophagy to study mycobacterial killing (and possibly other pathogen killing) is artificial and casts shadows on the endogenous host-pathogen biochemistry that naturally occurs during infection. This is consistent with our previous studies indicating that mycobacteria induce mTOR independent autophagy during infection. Future efforts on our part will continue to study mycobacterial autophagy in the absence of artificial influences/inducers to identify specific biochemical events that can be exploited to bolster host defenses. This could be accomplished by a number of methodologies including: proteomics approaches that identify specific post-translational modifications induced shortly after mycobacterial infection; the identification of mycobacterial transposon mutants that are susceptible to macrophage autophagy; the continued use of newly created mouse model systems that are more or less susceptible to mycobacterial infection; and the identification of pharmacological agents that induce autophagy and mycobacterial killing without inhibiting the immunologically sensitive mTOR pathway. Lastly, while mTOR inhibition does carry with it substantial global effects on cellular metabolism, it can not be overlooked that finely tuned mTOR inhibition, especially if restricted to macrophages , could provide a valuable means to favor host defense against mycobacterial infection.M. smegmatis. In contrast, high doses of Rapamycin, the most common mTOR inhibitor used in autophagy research, induces substantial M. smegmatis killing in wildtype macrophages and macrophages from autophagy deficient mice. As it does not appear that Rapamycin has a direct effect on mycobacteria in the short time frames of standard autophagy assays, it suggests that high dose inhibition of mTOR may be acting through an unappreciated cellular mechanism to elicit killing activity. When combined with our previous studies demonstrating that mycobacterial infection naturally induces both autophagy and mTOR signaling, this data reinforces the idea that mTOR inhibition through drugs or starvation is an artificial means of studying mycobacterial killing. We contend that the use of mTOR inhibition to study the molecular mechanisms of host-pathogen interactions is masking the relevant biochemistry that needs to be understood and exploited to favor host defense. However, additional studies further examining the connection between mycobacteria, the mTOR pathway, and host defense need to be performed, as fine-tuning mTOR activity to favor host defense without additional effects would be advantageous and could be developed as a valuable therapeutic.While low doses of several mTOR inhibitors are sufficient to reduce mTOR signaling as measured by a reduction in phosphorylated ribosomal S6, the same doses of these compounds are incapable of eliciting robust killing of Wildtype and LC3B knockout mice ,21 were Rapamycin (Sigma), Torin 1, and Torin 2, (Tocris) were dissolved in DMSO to a concentration of 10 mM, aliquoted, and stored at −20°C. Inhibitors were diluted fresh in culture media immediately before use. Lipopolysacharride (LPS) was purchased from Sigma, dissolved in DMEM, aliquoted, and stored at −20°C.Mycobacterium bovis Bacille Calmette-Guérin (BCG) and M. smegmatis have been described previously [eviously . This steviously . Unless Murine RAW264.7 macrophages have been described previously . Cells wThe following antibodies were used in this study: anti-Actin plus anti-mouse HRP were purchased from GenScript and Anti-phospho-S6, anti-S6, and anti-rabbit-HRP were purchased from Cell Signaling. Blotting conditions and chemiluminescence have been described .RAW264.7 macrophages were loaded with DQ-BSA as described previously . After aA549 cells were cultured on glass coverslips, fixed with 4% formalin, stained with either rabbit anti-LC3B  or isotype control, and visualized with Alexa Fluor-594 conjugated anti-rabbit secondary antibodies as described . CounterAnalysis was performed with GraphPad Prism using an analysis of variance (ANOVA) with a Tukey post-test or Students T test. P values of p ≤ 0.05 were considered to be significant.The authors declare that they have no competing interests.AZ and KS designed assays, performed experiments, and analyzed data. SL aided in assay design and data analysis. AZ, KS, and SL contributed to the writing and editing of the manuscript. All authors read and approved the final manuscript.