MicroRNAs (miRNA) are a class of non-coding RNAs which play key roles inpost-transcriptional gene regulation. Previous studies indicate that miRNAs are dysregulated in patients with multiple sclerosis (MS). Th17 and regulatory T (Treg) cells are twosubsets of CD4+T-cells which have critical functions in the onset and progression of MS.The current study seeks to distinguish fluctuations in expression of CD4+T-cell derivedmiR-223 during the relapsing-remitting (RR) phase of MS (RR-MS), as well as the expressions of Th17 and Treg cell markers. This experimental study used real-time quantitative polymerasechain reaction (qRT-PCR) to evaluate CD4+ T cell derived miR-223 expression patternsin patients that experienced either of the RR-MS phases (n=40) compared to healthy controls (n=12), along with RNA markers for Th17 and Treg cells. We conducted flow cytometry analyses of forkhead box P3 (FOXP3) and RAR-related orphan receptor γt (RORγt) inCD4+T-cells. Putative and validated targets of miR-223 were investigated in the miRWalkand miRTarBase databases, respectively.RORγt, a master transcription factor of Th17, upregulated in the relapsing phase, whereas FOXP3 upregulated in the remitting phase. Additionally, potentialtargets of miR-223, STAT1, FORKHEAD BOX O (FOXO1) and FOXO3 were predictedby in silico studies. miR-223 significantly upregulated in CD4+T-cells during the relapsing phase ofRR-MS compared to the remitting phase (P=0.000) and healthy individuals (P=0.036).Expression of miR-223 may have a potential role in MS progression. Therefore, suppression of miR-223 can be proposed as an appropriate approach to control progression of the relapsing phase of MS. Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system with an unknown etiology ,2. Devel+T-cells, inhibits autoimmune responses by mediating immunological tolerance toself-antigens  are a new class of endogenous, noncoding RNAs which regulate expression of most genes in animals and plants ,20. Almo+T-cellderivedmiR-223 in RR-MS patients. The association of key transcription factors involved in development of Treg and Th17 cells with transcript levels of miR-223 was also considered. We used bioinformatics tools to reveal connotation of this miRNA in pathways of Th17 and Treg differentiation. Dysregulation of miR-223 in autoimmune diseases such as MS, rheumatoid arthritis (RA) and Crohn’s disease (CD) makes it a valuable diagnostic marker. miR-223 is upregulated in T lymphocytes of RA patients , hence tThe Institutional Review Board of Royan Institute approved the study protocol and informed consent form (Project Id. No. 91000618). All study participants provided written consent for participation. In this experimental study, 40 patients diagnosed with MS (age range: 19 to 46 years) were evaluated according to McDonald criteria  by a neu+ T-cells from the whole blood. In the first step, PBMCs were isolated by density gradient lymphoprep  according to the manufacturer’s instructions. In the second step, CD4+T-cells were isolated on PBMC by magneticactivated cell sorting (MACS) with a CD4+T-cell isolation Kit II human  based on the manufacturer’s protocol. This kit is an indirect magnetic labeling system for the isolation of untouched CD4+Thelper cells from human PBMC by elimination of cells that contain CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ, and glycophorin A with a purity of greater than 95%. Total RNA was extracted with the TRizol® reagent  from CD4+ T-cells according to the manufacturer’s protocol. The quantity and quality of extracted RNA were verified according to the ratio of absorbance at a 260/280 nm as measured by a NanoDrop spectrophotometer , and by electrophoresis on a 1% agarose gel. Total RNA samples were treated with RNA-free DNase  in order to eliminate trace amounts of contaminated DNA prior to real-time quantitative polymerase chain reaction (qRT-PCR) analysis. The obtained blood samples were immediately placed on ice and transferred to the laboratory for analysis. We used a two-step process to separate the CD4RNU48 as the reference gene . An ABI PRISM 7500 instrument  was used for the qRT-PCR analysis. All reactions were performed in triplicate using standard protocols. CDNA synthesis of key factors TGF-β, INTERLEUKIN 23R (IL23R) and IL17a was performed with a RevertAid First Strand cDNA synthesis Kit  according to the manufacturer’s protocol. The expression level of each gene was normalized vs. 18srRNA in the same sample. All measurements were performed for three independent replicates. Total RNA samples were divided into two parts, one batch for mRNA expression analysis of key factors in Treg and Th17 cell development and the second for the miRNA expression assay. A universal cDNA synthesis kit  was used for cDNA synthesis of miR-223, with nce gene  through The specificity of the miR-223 primers were assessed by electrophoresis of real time PCR products on a 12% poly acrylamide gel. For exact sequence matching of miR-223 to our product, the resultant electrophoresis bands were T/A cloned into a pTZ57R/T vector (Thermo Scientific) and sent for sequencing. + T-cells were evaluated forRORγt in Th-17 cells and FOXP3 expression inTreg cells. Briefly, the isolated CD4+ T-cells werefixed in 4% paraformaldehyde in phosphate buffersaline (PBS) for 20 minutes at room temperature,after which they were permeabilized in 0.2% Triton X-100 for 15 minutes. Then, samples were resuspended in PBS that contained bovine serum albumin  and subsequently stainedby mouse anti-human FOXP3-PE, rat anti-humanROR γ(t)-PE, and mouse anti-human CD4-AlexaFluor® 488 against the isotype controls .All experiments were run on a FACSCalibur flowcytometer  and analyzedby BD CellQuest Pro software (version 0.3).Green fluorescence was detected in a fluorescencedetector 1 (FL-1). Red fluorescence was detectedin FL-2.MACS-isolated CD4We used the Statistical Program for Social Sciences (SPSS) software (version 18) for all statistical tests. One-way ANOVA was utilized for comparison groups. A P<0.05 was considered statistically significant for all experiments. In order to identify validated and predicted targets of miR-223, we searched the miRTarBase  and miRW+ T-cells, the presence of miR-223 targets in the thymus and lymph nodes was validated by the Unigene database (http://www.ncbi.nlm.nih.gov/unigene/). We used CircuitsDB  phase and healthy controls (P=0.036). In contrast, miR-223 showed a nonsignificant downregulation in the remitting phase compared to the controls  and control(P=0.0003) groups. CD4+ T-cells that carriedFOXP3 as the main transcription factor for Tregcells significantly increased in the remittingphase compared to the relapsing (P=0.003) andcontrol (P=0.001) groups  groups . All samTGF-β and Il-23R, as main factors thatparticipate in Th17 and/or Treg pathogenicity. Wealso evaluated Il-17a, as a Th17 cytokine marker.There was a significantly elevated transcript levelof TGF-β in the relapsing (P=0.008) and remitting(P=0.029) groups compared to the control group.However, the difference between the relapsing andremitting groups was not significant (P=0.573).Expression of Il-23R increased in the relapsingphase versus the remitting phase (P=0.042), however this value was not significant between the relapsing and control (P=0.058) groups and betweenthe remitting and control groups (P=0.815). Il-17aRNA levels significantly elevated in the relapsing compared to the remitting (P=0.023) group,however differences between the relapsing phaseand control (P=0.329) groups, as well as betweenthe remitting phase and control group , FOXO3 and STAT1 mRNAs were considered as three mRNA targets of miR-223 that had vital functions for Th17 and Treg differentiation. Possible interactions between miR-223 and FOXO1, FOXO3 and STAT1 based on the RNAhybrid database are depicted in We performed molecular signaling pathway enrichment analysis to identify the potential role of miR-223 in MS pathogenicity by affecting Th17 and Treg differentiation. Based on miRWalk and miRTarBase databases, we identified 339 predicted and 24 validated target mRNAs . From alIn autoimmune diseases such as MS, the balancebetween Th17 and Treg cells is destroyed. However, miRNAs are critical post-transcription regulators that can modify differentiation pathways and potentially play a role in controlling the Th17/Tregbalance.+ T-cell derived miR-223 in the remittingand relapsing phases of RR-MS patients. Finding an association between the expressions ofmarkers for two of the most critical cells in autoimmune diseases (Th17 and Treg) and transcript levels of miR-223 in CD4+ T-cells wouldexpand our knowledge about the developmentof MS. We used bioinformatics methods toevaluate a possible mechanism where miR-223affected Th17/Treg differentiation. Based onthe predicted and validated targets of miR-223,we observed that STAT1, FOXO1 and FOXO3were considered miR-223 targets in MS. Up-regulation of miR-223 in CD4+ T-cells fromthe relapsing phase of MS agreed with previousstudies which showed increased expression ofmiR-223 in active brain lesions, PBMCs, andTreg cells of MS patients  which is a common mouse model for MS . BindingSTAT1 is a target of target of miR-223. As previously mentioned, the remitting group received INF-β as an immunomodulatory drug . IFN-β, Based on CircuitsDB, we have shown that there were two basic differences between humans and mice. MiR-223 has a different promoter sequence and STAT1 has different binding sites in the two species. FOXO1 and FOXO3. FOXO1 has been validated as a target of miR-223 (FOXO1 and FOXO3 by means of miR-223 could cause suppression of Treg cells and promote Th17 cells. Two other predicted targets of miR-223 are  miR-223 . FOXO pr miR-223 . In cont miR-223 . Resista miR-223 . We haveBased on flow cytometry results, FOXP3 protein significantly overexpressed in the remitting phase compared to the relapsing and control groups. The RORγt protein significantly overexpressed in the relapsing phase compared to the other phases. Il-23R and IL-17A are associated with Th17 cells. IL-23 is required for Th17 differentiation. Therefore increased IL-17A, IL23R mRNAs in the relapsing group has resulted in increased numbers of Th17 cells. Upregulation of TGF-β in both the relapsing and remitting patients is intriguing. This ambiguous pattern can be explained since TGF-β is required for both Th17 and Treg cell differentiation (Expression levels of ntiation ,54. +T-cell derived miR-223 in the relapsing phase of RR-MS along with elevated numbers of Th17 cells and decreased Treg cells. In silico molecular enrichment analysis has shown a potential role of miR-223 in Th17 and Treg cell differentiation via the chemokine signaling pathway. However, further in vitro and in vivo experiments are needed to confirm these observations. In this study, we observed upregulation of CD4


Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens, T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa, PppA and TagF are two distinct proteins that repress T6SS in TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein–protein interaction interfaces, we uncovered evidence that the TagF–Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens, but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa. Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control.The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens,  The delivery and activity of these T6SS toxins and effectors have a clear impact in interbacterial competition and/or pathogenesis during eukaryotic host infection (–The type VI secretion system T6SS)nfection , 2. The nfection –6 servesSSnfectiofection –, 8, withfection –. Upon cofection –, 11 are Pseudomonas aeruginosa H1-T6SS is post-translationally regulated, positively by PpkA and negatively by the cognate phosphatase PppA. Such control occurs via threonine phosphorylation at the Thr-362 residue on an FHA domain–containing protein, Fha1, in P. aeruginosa  (B)  and the C-terminal region (aa 231–471) encompassing the PppA domain (B). Overexpression of TagF-PppA, TagF, and tagged TagF-Strep in C58 completely abolished type VI secretion  (A). The T6SS antibacterial activity is also shut down, as shown by counting surviving Escherichia coli target cells. The number of survivors is indeed similar to that when the attacker is an A. tumefaciens T6SS mutant, ΔtssL (B). We also performed an in planta interbacterial competition assay with an A. tumefaciens prey strain lacking the three T6SS toxins  and cognate immunity proteins, Δ3Tis (3TIs was lower after co-infection with WT C58 harboring the vector pTrc200 (V). This was not seen when co-infecting with a T6SS inactive mutant ΔtssL-carrying pTrc200 (V) or C58 overexpressing the T6SS repressor TagF  (C). Interestingly, overexpression of PppA alone reduced but did not abolish Hcp secretion (A). The antibacterial activity of the PppA overexpression strain was also not completely abolished, whereas modest antibacterial activity was detected with our in planta interbacterial competition assay (C). In addition, the protein levels of all analyzed T6SS components encoded within the imp  and hcp (including ClpV and VgrG1) operons remained the same in all backgrounds tested, including overexpression of TagF-PppA, TagF, TagF-Strep, or PppA (A). Taken together, these data show that in A. tumefaciens, both TagF and PppA domains contribute the repressor function of TagF-PppA on T6SS effector secretion and antibacterial activity via a post-translational regulatory control.TPP regulation of T6SS has been demonstrated in only a few bacteria, including rain C58 . PpkA  . Atu4331eron (A) B (22). WA domain B. Overexnd Tde1) A. The T6t, ΔtssL B. We alss, Δ3Tis . PreviouF-Strep) C. Intereecretion A. The anon assay C. In add or PppA A. Taken tssL mutant also expressing His-tagged TssL (TssL-His). This TssL variant is functional and mediates Hcp, Tae, and Tde1 secretion   , which suggests that neither the full-length TagF-PppA nor any domain of TagF-PppA represses T6SS activity by controlling TssL phosphorylation.To explore the possible mechanisms of TagF-PppA-mediated T6SS repression at the post-translational level, we analyzed the impact on TssL phosphorylation. TagF-PppA, TagF, TagF-Strep, and PppA were overexpressed in the ΔA and B) . The TssFig. 3A) . In all B). Phosphorylated TssL protein was detected as a single protein band and found with the same abundance when comparing WT C58 and strains overexpressing TagF-PppA, TagF, TagF-Strep, and PppA. However, the pTssL protein band was not detected in ΔtssL and ΔppkA mutant strains (C). We concluded that TagF-PppA represses T6SS activity independently of TssL phosphorylation in A. tumefaciens.To determine whether the phosphorylation level detected from an overexpressed TssL-His is representative of an endogenous situation, we generated an antibody that specifically recognizes phosphorylated TssL (pTssL) B. Phosph strains C. We coni.e. both ppkA and tagF-pppA). We generated a ΔppkAΔtagF-pppA strain and separately overexpressed the TagF or PppA domain in this mutant background. Interestingly, unlike ΔppkA, resulting in decreased type VI secretion (ppkAΔtagF-pppA mutant retained type VI secretion activity comparable with that of the WT C58 (A). Overexpression of each of TagF or PppA domain in ΔppkAΔtagF-pppA abolished or greatly reduced the type VI secretion (B), suggesting that both TagF and PppA domains can inhibit type VI secretion in the absence of PpkA. As expected, the antibacterial activity was also abolished when the TagF or PppA domain was overexpressed in ΔppkAΔtagF-pppA, as shown by counting E. coli survivors and comparison with what is observed when the attacker is a T6SS mutant, ΔtssL (C). Interestingly, whereas the Hcp and effector (Tde1 and Tae) secretion levels in ΔppkAΔtagF-pppA were comparable with those of WT C58 (A), only partial antibacterial activity was observed (C). This phenotype is consistent with a previous observation in P. aeruginosa and S. marcescens, with ΔpppA showing reduced antibacterial activity despite elevated type VI secretion  experiments revealed that both A. tumefaciens full-length TagF-PppA and the isolated TagF domain specifically interact with Fha (A). We also detected an interaction between the P. aeruginosa H1-T6SS–encoded TagF and Fha1 by using both YTH (B) and bacterial two-hybrid (BTH) assays (C). The data demonstrate that TagF directly interacts with Fha, probably interfering with its function and thereby preventing type VI secretion in both A. tumefaciens and P. aeruginosa.Next, we investigated how TagF-PppA represses T6SS activity via a post-translational and TPP-independent pathway. We hypothesized that TagF-PppA may interact with the T6SS core component(s) to prevent T6SS activation. One plausible candidate is the forkhead-associated protein Fha because of its role in recruitment to a membrane-associated complex both independent of and dependent on TPP , 21, 22.with Fha A. We alsboth YTH B and bac) assays C. The daA. tumefaciens and P. aeruginosa, the two TagF proteins share only limited amino acid similarity (A). Yet, 14 amino acid residues are highly conserved among various TagF orthologs . We hypothesized that these residues may play important roles in TagF function. The structure of P. aeruginosa TagF is presented as a homodimer (Protein Data Bank entry 2QNU). The self-interaction of P. aeruginosa TagF was also confirmed by BTH and YTH . In contrast, the A. tumefaciens TagF probably does function as a monomer, as supported by several lines of evidence. First, the TagF protein is mostly a monomer when analyzed by gel filtration in vitro . Second, A. tumefaciens TagF lacks the residues required for dimer formation in P. aeruginosa , Leu-169 (L169), Leu-172 (L172), Ala-173 (A173), and Leu-195 (L195) in TagFPa)  (Protein Data Bank entry 2QNU). Finally, no self-interaction could be found for A. tumefaciens TagF using YTH . Other conserved residues are not involved in P. aeruginosa TagF homodimerization and were thus chosen for site-directed mutagenesis and characterization of the potential interface with other interacting proteins, namely Fha. A total of eight conserved residues (Gly-8 (G8), Lys-9 (K9), Asp-15 (D15), Phe-16 (F16), Ser-79 (S79), Asp-81 (D81), Arg-85 (R85), and Pro (P88) of P. aeruginosa TagF) form a specific loop, and the side chain of Asp-30 (D30) is outwardly exposed . Therefore, these are potential sites to interact with other proteins. In contrast, Phe-60 (F60), Gly-74 (G74), and Leu-139 (L139) are located inside the structure or the side chain wrapped in an internal structure to prevent interaction with other proteins. We generated four mutants with amino acid substitutions of alanine at GK , DF , DW , and SDR  (A and C). We performed YTH analysis with the various A. tumefaciens TagF variants to determine the roles of the substituted residues in the TagF–Fha interaction. The interaction was completely lost in TagFGK, TagFDW, and TagFSDR mutants . In contrast, TagFDF and TagFFD with mutations in two randomly selected nonconserved residues (Phe-141 and Asp-142 (F141D142) in A. tumefaciens TagF) still retained full capacity to interact with Fha as compared with the WT TagF . Western blot analysis revealed that the protein abundance remained the same or was even slightly higher for all analyzed TagF variants as compared with the WT TagF , which indicates that conserved G22K23, D44W46, and S93D95R99 residues but not D29F30 of A. tumefaciens TagF are critical for the TagF–Fha interaction.Although Fha is the target for TagF in both milarity A. Yet, 1FD-Strep, abolished Hcp, Tae, and Tde1 secretion. Remarkably, the secretion capacity remained high with overexpression of TagFGK-Strep, TagFDF-Strep, TagFDW-Strep, and TagFSDR-Strep in C58 (A). Upon Western blot analysis, the protein levels of all TagF variants were comparable with that of WT TagF-Strep, and overexpression did not affect the protein abundance of other T6SS components (A). These data suggest that the conserved residues GK, DW, and SDR of TagF are critical for TagF-mediated repression of type VI secretion via the TagF–Fha interaction. Intriguingly, TagFDF retained full binding capacity with Fha but lost the ability to repress type VI secretion, which suggests that D29F30 is not involved in binding Fha but is required for repressing T6SS activity. As expected, the type VI secretion activity of these TagF overexpression variants was consistent with their antibacterial activity (B and C). The survival of E. coli was reduced to a level similar to that with the strain harboring the empty vector control when TagFGK-Strep was overexpressed in ΔppkAΔtagF-pppA (B and C), which confirms that TagFGK-Strep lost the ability to repress T6SS-dependent antibacterial activity via a PpkA-independent pathway. To ensure that the overexpression phenotypes of the mutant alleles were also reflected when expressed at endogenous levels, we further generated chromosomal tagF-pppA alleles encoding the TagF amino acid substitution variants and further determined their T6SS suppression activity. All TagF amino acid substitution variants expressed from chromosomal alleles exhibited enhanced antibacterial activity (B), which is consistent with the derepressing T6SS activity demonstrated upon overexpression of these TagF variants (B and C). However, our secretion assay could not detect a significant difference in secretion levels of Hcp and two effectors (Tae and Tde1) of all analyzed tagF-pppA mutants as compared with WT C58 . Taken together, our data suggest that TagF negatively regulates T6SS via direct interaction with Fha in A. tumefaciens.To determine whether the TagF–Fha interaction is required for suppressing T6SS activity, we engineered the previously described mutations in the TagF-Strep variants and analyzed the impact of their overexpression on type VI secretion. As expected, overexpression of the WT controls, TagF-Strep or TagFp in C58 A. Upon Wmponents A. These activity , B and CagF-pppA , B and Cactivity B, which variants , B and CA. tumefaciens are also required in P. aeruginosa TagF for Fha1Pa interaction and T6SS activity. We generated two alanine substitution mutants in TagFPa, namely TagFPa-GK (Gly-8 and Lys-9) and TagFPa-SDR . Consistent with the results obtained with A. tumefaciens, TagFPa-GK and TagFPa-SDR mutants lost the interaction with Fha1Pa, although their expression level was comparable with that of WT TagFPa in yeast . To determine whether the TagFPa–Fha1Pa interaction is critical for TagF-dependent H1-T6SS repression, WT TagFPa, Strep-tagged WT TagFPa, and the TagFPa-GK and TagFPa-SDR variants were expressed from pRL662 in the P. aeruginosa ΔretS mutant, a constitutively H1-T6SS active strain (retS harboring the vector pRL662 (V), but H1-T6SS secretion was greatly reduced when TagFPa or TagFPa-Strep was overexpressed . Furthermore, overexpression of TagFPa-GK-Strep or TagFPa-SDR-Strep did not repress Hcp1 and Tse3 secretion. Interestingly, in contrast to A. tumefaciens, where overexpressed TagF mutant variants are at a level comparable with the overexpressed WT form, in P. aeruginosa, we observed significantly higher levels of WT TagFPa-Strep as compared with TagFPa-GK-Strep or TagFPa-SDR-Strep . Furthermore, protein levels of all analyzed T6SS components  were significantly reduced upon overexpression of WT TagF (TagFPa or TagFPa-Strep). Accordingly, protein levels of these T6SS components were restored to near WT levels in the presence of TagFPa-GK-Strep and TagFPa-SDR-Strep in P. aeruginosa .We then assessed whether these specific conserved amino acid residues of TagF required for binding to Fha and T6SS repression in e strain . As expePa-Strep was overexpressed in PAKΔretS, E. coli survival is similar to that for the T6SS-defective PAKΔretSΔtssB1 mutant. In contrast, the expression of TagFPa-GK-Strep and TagFPa-SDR-Strep conferred antibacterial activity similar to that of PAKΔretS (C). Because the reduced protein level/stability of both TagFPa-GK-Strep and TagFPa-SDR-Strep is associated with loss of ability to interact with Fha1Pa, we tested whether the presence or absence of Fha1Pa affected TagF abundance. Strikingly, TagFPa-Strep protein level was significantly reduced in PAKΔretSΔH1, which lacks endogenous Fha1Pa, as compared with PAKΔretS (D). TagFPa-Strep protein levels could be partially restored upon co-expression of TagFPa-Strep and Fha1Pa-HA in PAKΔretSΔH1 (D), which suggests that Fha1Pa may play a role in stabilizing TagFPa in P. aeruginosa. Taken together, our results indicate that the TagF domain represses T6SS activity via interaction with Fha but with distinct mechanisms when comparing A. tumefaciens and P. aeruginosa.Next, we performed an antibacterial activity assay and showed that when TagFPAKΔretS C. BecausPAKΔretS D. TagFPaΔretSΔH1 D, which A. tumefaciens TagF-PppA and P. aeruginosa TagF and provide compelling evidence that TagF specifically interacts with Fha to repress type VI secretion and antibacterial activity independently of TPP. The loss of interaction with Fha is associated with loss of repression of T6SS activity in both cases, yet we observed differences in the nature and impact of TagF and its variants in repressing T6SS. This may have strategic consequences for how bacteria from various species communicate and respond to each other during T6SS-dependent bacterial warfare.In the present study, we have characterized the A. tumefaciens and P. aeruginosa. A similar protein abundance of all TagFAt or TagFAt-Strep overexpressed  was observed in A. tumefaciens. In contrast, in P. aeruginosa, the protein level was significantly lower for TagFPa-GK-Strep or TagFPa-SDR-Strep than WT TagFPa-Strep . Furthermore, all analyzed T6SS proteins accumulated to a similar level in the presence or absence of endogenous or overexpressed TagFAt in A. tumefaciens . However, the protein levels of P. aeruginosa H1-T6SS components  were reduced with TagFPa or TagFPa-Strep overexpression but restored to nearly WT levels in the presence of mutant TagFPa-GK-Strep or TagFPa-SDR-Strep (B). Previous studies in P. aeruginosa suggested that TagFPa-mediated T6SS repression is mediated via a post-translational regulation mechanism  mutant  or activation of T6SS assembly (in A. tumefaciens).The role of Fha in activating T6SS in both TPP-dependent and -independent pathways may provide some clues to understand the mode of action by which TagF represses T6SS activity. Fha1 but not its phosphorylated form is required for ClpV1ruginosa , 21. Alsruginosa , 30, 36.) mutant . In A. tecretion . Togetheecretion , 38, thewn assay . Thus, F22K23, D44W46, and S93D95R99 residues but not D29F30 of A. tumefaciens TagF are critical for the TagF–Fha interaction, but all are required for repressing T6SS activity. This result led us to propose that D29F30 may repress T6SS function independently of binding to Fha. TagF may target Fha in repressing T6SS activity in two steps. The first step is to bind Fha via an interface involving G22K23, D44W46, and S93D95R99. Upon binding, TagF interferes with Fha recruitment to the membrane-associated complex for T6SS activation via D29F30 residues. Alternatively, it is possible that TagF can also target other T6SS components in addition to Fha. Future work to elucidate the molecular details underlying how TagF–Fha interaction influences T6SS activity or identifying additional TagF-interacting partners may provide answers to distinguish between two possible mechanisms.Of note, the conserved GA. tumefaciens and P. aeruginosa , Fha probably also functions as a core T6SS component via interaction with one or multiple T6SS components to activate T6SS assembly and secretion in the absence of PpkA and TagF-PppA. This proposed mechanism also explains the previous observation that type VI secretion is highly attenuated but not completely abolished in the absence of PpkA (ΔppkA)  , becauseP. aeruginosa, type VI secretion is significantly enhanced in ΔpppA or ΔtagF as compared with the parental strain, and PppA phosphatase negatively regulates type VI secretion in a TPP-dependent manner, whereas TagF represses type VI secretion independently of TPP , all Fha1 is available for activating T6SS assembly and secretion.In y of TPP , 21. Thu of PpkA , 21. Becormation , 21, nonIn conclusion, our proposed molecular model may provide answers to the longstanding question of how TagF mediates T6SS repression. We present compelling evidence suggesting that TagF specifically interacts with Fha and that such binding prevents Fha from being recruited to a T6SS membrane-associated complex. The TagF–Fha interaction has a different impact in different bacteria but ultimately prevents successful T6SS assembly. This study adds to our understanding of how bacteria deploy TPP-dependent activation and TPP-independent TagF-mediated repression mechanisms to control T6SS.Tables S1 and S2. The growth conditions are described in detail in supporting information S1.Strains, plasmids, and primer sequences used in this study are described in A. tumefaciens C58 via double crossover using the suicide plasmid pJQ200KS  containing the phosphorylated Thr-14 residue (The specific antibody for phosphorylated TssL (pTssL) was generated against the 15-mer peptide   and spotted onto LB agar plates. Where applicable, the mixture was spotted onto an LB agar plate containing 0.5 mm isopropyl β-d-thiogalactopyranoside (IPTG) to induce expression from the pTrc200 plasmid. 16 h after incubation at 25 °C, the spots were harvested, serially diluted, and plated on LB agar plate containing appropriate antibiotic to quantify surviving E. coli cells by counting cfu. An in planta bacterial competition assay was performed as described previously  and target (A600 of 0.5) strains were mixed in ½ Murashige and Skoog medium (pH 5.7) at a 10:1 ratio and infiltrated into leaves of 6–7-week-old Nicotiana benthamiana plants by use of a needleless syringe. After a 24-h incubation at room temperature, the infiltrated spot was punched out, ground in 0.9% NaCl, serially diluted, and plated in triplicates on LB agar containing appropriate antibiotic to select for the target cells. All assays were performed with at least two independent experiments, each with two biological replicates, or three independent experiments, each with one or two biological replicates.Type VI secretion assay was performed as described previously , 29, 30.(pH 5.5)  at 25 °Ceviously . An A. tP. aeruginosa type VI secretion assay was performed as described previously (P. aeruginosa strains harboring appropriate plasmids were grown in tryptone soy broth (TSB) overnight at 37 °C under agitation. Cells were harvested and subcultured to an A600 of 0.1, and then growth was continued in TSB to early stationary phase at 37 °C for 4–5 h to A600 of 5. Cells were separated from culture supernatants by centrifugation at 4000 × g at 4 °C. Cells were directly resuspended in 1 × SDS sample buffer. 10-Fold concentrated P. aeruginosa culture supernatant was prepared as follows. Proteins of the culture supernatant were precipitated using 6 m TCA (Sigma) at a final TCA concentration of 10%. Protein pellets were washed in 90% acetone, dried, and suspended in 1× SDS sample buffer; incubated at 96 °C for 20 min; and analyzed by SDS-PAGE. A P. aeruginosa antibacterial activity assay using E. coli as target cells was performed as described previously  in a 1:1 ratio on LB agar for 5 h at 37 °C. In addition, P. aeruginosa and E. coli strains grown alone on LB agar for 5 h at 37 °C served as negative growth controls. Subsequently, patches of bacteria were collected and resuspended in LB broth, and dilution series ranging from 100 to 10−7 were plated in triplicate on LB supplemented with 100 mg/ml 5-bromo-4-chloro-indolyl-β-d-galactopyranoside , allowing for colorimetric detection of lacZ-positive E. coli survivors. For quantitative analysis of the amount of E. coli survivors, the spots were harvested, serially diluted, and plated on LB agar plate containing X-gal and appropriate antibiotic to quantify surviving E. coli cells by counting. Data represent mean ± S.D. of all biological replicates.A eviously . In brieeviously . In brieSaccharomyces cerevisiae strain AH109. The transformants were selected by their growth on synthetic dextrose (SD) minimal medium lacking tryptophan (Trp) and leucine (Leu) (SD−WL medium). The positive interaction of expressed fusion proteins was then determined by their growth on SD lacking Trp, Leu, adenine (Ade), and histidine (His) (SD−WLHA medium) at 30 °C for at least 2 days.The Matchmaker yeast two-hybrid system was used as instructed  and as described previously . Each ofA600 of 0.4–0.6. Yeast cells were harvested, 100 μl of protein extraction buffer containing 0.1% Nonidet P-40, 250 mm NaCl, 50 mm Tris-HCl (pH 7.5), 5 mm EDTA (pH 8.0), 1 mm DTT, 2× protease inhibitor mixtures (Roche Applied Science), 4 mm phenylmethylsulfonyl fluoride, and 50 μl of acid-washed glass beads (Sigma-Aldrich) were added. The cells were broken by vortex at the highest speed for 30 s, and then tubes were placed on ice for 30 s. The same procedure was repeated six times to make sure cells had been completely broken. The supernatant above the glass beads was collected (the first cell extract). Then 50 μl of protein extraction buffer was added to wash the tube containing broken cells and glass beads by vortexing at the highest speed for 30 s. The supernatant above the glass beads was collected again (the second cell extract). The two extracts were mixed together, and the final protein extract was centrifuged at 13,000 rpm for 5 min at 4 °C; the resulting supernatant was collected, and protein concentration was measured. An equal volume of 2× SDS loading buffer was added to the final protein sample, incubated at 96 °C for 20 min, and analyzed by SDS-PAGE.In brief, to prepare the total protein from yeast , overnigP. aeruginosa PAK genomic DNA. DNA fragments encoding the proteins or protein domains of interest were cloned into plasmids pKT25 and pUT18C, which each encode for complementary fragments of the adenylate cyclase enzyme, as described previously  supplemented with 40 mg/ml X-gal in the presence of 100 mg/ml ampicillin, 50 mg/ml kanamycin, and 1 mm IPTG. Positive interactions were identified as blue colonies on LB agar plates containing X-gal after a 48-h incubation at 30 °C. The experiments were performed at least in duplicate, and a representative result is shown.The BTH assay was performed as described previously . In brieeviously , resultiFor quantitative analysis of BTH interactions, β-gal activity of co-transformants scraped from LB agar plates containing X-gal was measured as described previously, and activity was calculated in Miller units , 46.http://astatsa.com/OneWay_Anova_with_TukeyHSD/),p < 0.01 or p < 0.05).Data represent mean ± S.E. of all biological replicates. Statistics were calculated by one-way analysis of variance and Tukey's honestly significance difference test , 10 mm MgCl2, 1 mm DTT, and 1× protease inhibitor mixture (EDTA-free) with CIAP at 1 unit/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. The Phos-tag SDS-PAGE analysis was performed according to the user manual for Phos-tag acrylamide AAL-107  with minor modifications as described previously (m BisTris-HCl (pH 6.8), 35 μm Phos-tag acrylamide AAL-107, and 100 μm ZnCl2, with electrophoresis conducted at 40 mA/gel under a maximum voltage of 90 V 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 polyvinylidene difluoride membranes with a submarine blotting apparatus.Dephosphorylation analysis by calf intestinal alkaline phosphatase (CIAP) was performed according to the user manual  with minor modifications as described previously . Equal aeviously . ProteinE. coli BL21 (DE3) cells with the plasmid pET28a(+)-TagF 1–214. E. coli cells were grown in LB medium in the presence of kanamycin (20 μg/ml) at 37 °C until the cell density reached an A600 of 0.6–0.8. The cultures were induced with 1 mm IPTG for 16–20 h at 16 °C to induce production of His-tagged TagF aa 1–214 proteins. The cells were harvested by centrifugation followed by snap-freezing by liquid nitrogen and stored at −80 °C. Frozen bacterial pellets were resuspended with the lysis buffer , and then the cells were lysed by a microfluidizer. The cell lysate was clarified by centrifugation at 20,000 rpm for 40 min at 4 °C, and the supernatant was loaded onto an Ni-NTA column  pre-equilibrated with the lysis buffer. The column was washed with the washing buffer , and the bound protein was eluted by the elution buffer . The TagF aa 1–214 proteins were further analyzed by size-exclusion chromatography using a Superdex 75 16 × 60 column through a fast protein LC (FPLC) system . The column was equilibrated with 2 column volumes of buffer containing 50 mm Tris-HCl (pH 7.5) and 150 mm NaCl. To determine molecular weight, a parallel column was run with protein standards. The elution profiles were recorded as absorbance at 280 nm.N-terminal His-tagged TagF aa 1–214 proteins were expressed in 41, TssB, TssA, ClpV, Tae, VgrGs, and RpoA (P. aeruginosa Hcp1, TssB1, TagJ1, and Tse3 (Western blot analysis was performed as described previously  with theand RpoA , TssL (4and RpoA , ActC (4and RpoA , Tde1 (2and RpoA , P. aeruand Tse3 , polycloJ.-S. L. and E.-M. L. conceptualization; J.-S. L., P. P., and H.-H. W. data curation; J.-S. L., P. P., H.-H. W., M.-D. T., A. F., and E.-M. L. investigation; J.-S. L. and E.-M. L. writing-original draft; J.-S. L., P. P., H.-H. W., M.-D. T., A. F., and E.-M. L. writing-review and editing; M.-D. T., A. F., and E.-M. L. supervision; M.-D. T., A. F., and E.-M. L. funding acquisition.


Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion) into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration.Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs), is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and  As thmdx mice have been demonstrated to be increased to near wild-type levels by restoration of the dystrophin protein using exon-skipping therapies [mdx mice, miR-1 and miR-133a levels have been shown to be downregulated, whereas miR-206 levels are upregulated [mdx mice remain largely unknown. In the present study, we show that the release of EVs from C2C12 cells is regulated by ceramide metabolism in an intracellular Ca2+ concentration-dependent manner. The transport of miR-133a via EVs regulates the viability of C2C12 cells under conditions of cellular stress. In addition, the inhibition of ceramide synthesis by GW4869 (an inhibitor of exosome secretion), leading to the suppression of EV secretion, reduces muscle degeneration in mdx mice. Our findings demonstrate the potential of myomiRs as a novel noninvasive biomarker and the regulation of myomiR levels as a new therapeutic strategy for DMD.The increased levels of myomiRs in the sera of herapies . On the egulated , 27. Theegulated . These megulated , 30. Thuegulated –35. HowePrimary antibodies against mouse CD63 (3H1626), rabbit apolipoprotein A-I (apoA-I) (FL-267), and rabbit CD81 (H-121), and the anti-rat IgG- Horseradish peroxidase (HRP) secondary antibody were purchased from Santa Cruz Biotechnology . Mouse primary antibody against MHC class II (M5/114.15.2) was purchased from Novus Biologicals . Rabbit primary antibody against caveolin-3 was synthesized by Eurofins Genomics K.K. . Rabbit primary antibody against flotillin-1 was purchased from Bioss USA , and the anti-mouse IgG (H+L) HRP secondary antibody was purchased from Bio-Rad Laboratories, Inc. . Anti-rabbit IgG HRP-linked secondary antibody was purchased from Cell Signaling Technology, Inc. . Anti-LAMP2 (M3/84) and anti-GAPDH (EPR6256) antibodies were purchased from Abcam . Mouse monoclonal antibody against CD9 (IVA50) was purchased from Abnova . Mouse monoclonal antibody against calnexin (4F10) was purchased from MBL Laboratories CO., LTD . Mouse monoclonal antibody against HSP90 (H9010) was purchased from StressMarq Biosciences Inc. . Actinomycin D was purchased from Focus Biomolecules . Methyl-ß-cyclodextrin, 2-aminoethyl diphenylborinate (2-APB), A23187, C6-ceramide, and monensin sodium salt were purchased from Sigma-Aldrich . BAPTA-acetoxymethyl ester and EGTA-acetoxymethyl ester were purchased from DOJINDO Laboratories . C2-ceramide and simvastatin were purchased from Focus Biomolecules. Anhydrous caffeine was purchased from Nacalai Tesque . Cyclopiazonic acid, D-erythro-MAPP, ebselen, nocodazole, and U-0126 were purchased from Cayman Chemical Company . GW4869 was purchased from Calbiochem . Sphingosine-1-phosphate (S1P) was purchased from LKT Laboratories, Inc. .Five unrelated Japanese patients with DMD and four healthy controls were enrolled in this study with the approval of the Ethics Committee of the National Center of Neurology and Psychiatry , which followed regulation of Helsinki Declaration. All results are treated with standard medical confidentiality and confidential to the extent allowed by law. Written informed consent was obtained from all participants after explaining the details of the study, prior to the collection of peripheral blood.mdx mice with C57BL/6 background, transgenic mdx mice overexpressed dystrophin gene which lack coding region from exon 45 to exon 55 by CAG promoter with C57BL/6 background , and age-matched wild-type C57BL/6 male control mice were used in this study.All animals used in this study were housed in a specific pathogen free facility of the National Center of Neurology and Psychiatry and treated in accordance with the guidelines provided by the Ethics Committee for the Treatment of Laboratory Animals of the National Center of Neurology and Psychiatry , which has adopted the three fundamental principles of replacement, reduction, and refinement. Connsistent with the approvals stipulated by the protocol, all efforts were made to minimize suffering or discomfort to the animals. ®  was added to the sera, mixed well, and then incubated at 4°C for 60 min. The immunoprecipitated complexes were collected by centrifugation at 3,000 × g for 2 min at 4°C, and the supernatant was discarded. The pellet was washed with 1 mL of phosphate-buffered saline (PBS). The centrifugation and washing steps were repeated at least 3 times. The pellet was then resuspended in 100 μL of 5% sodium dodecyl sulphate (SDS) solution, heated at 95°C for 5 min, and centrifuged for 1 min at 12,000 × g at room temperature, and then the supernatant was collected as the immunoprecipitated samples.Two μg of antibodies were mixed with 0.1 mL of sera, and incubated at 4°C overnight. Protein G Sepharose™ Real-Time PCR System (ABI) according to the manufacturer’s protocol. Samples were subjected to real-time PCR in triplicate. The expression of each miRNA was represented as relative values normalized by the expression of miR-16 and cel-miR-39 used as an internal control and spike-in miRNA, respectively. Data analysis was performed by SDS 2.1 real-time PCR data analysis software (ABI). Expression data are shown as median values obtained from three samples together with the standard deviation.Total RNA was extracted from 0.1 μL of serum using the mirVana miRNA isolation kit  as 25 μL of RNA eluate according to the manufacturer’s protocol. RNA eluate (4.5 μL) was reverse transcribed using the TaqMan miRNA Reverse Transcription kit  and miRNA-specific stem-loop primers  as previously described (21). The expression levels of miRNA were quantified by real-time PCR using individual miRNA-specific primers  with StepOne™ , according to the manufacturer’s protocol. Briefly, 50 μL of Total Exosome Isolation solution or 63 μL of ExoQuick solution were added to 250 μL of serum. The mixture was vortexed for 15 seconds and then incubated at 4°C for 30 min. After centrifugation at 10,000 × g for 10 min at room temperature, the supernatant was discarded. The centrifugation and aspiration steps were repeated. The pellet containing the EVs was resuspended in 1× PBS. EVs were also isolated from C2C12 cells, which were conditioned in serum-free media for 24 hr at 90% confluence. Media were harvested, and then centrifuged at 2000 × g for 30 min to remove cells and debris. EVs were then isolated using Total Exosome Isolation reagent (Invitrogen). Briefly, the Total Exosome Isolation reagent (1/5 voulume) was added to cell-free culture media. The solution was vortexed for 15 seconds, and then incubated at 4°C overnight. After centrifugation at 10,000 × g for 1 hour at 4°C, the supernatant was discarded. Again, the centrifugation and aspiration steps were repeated. The pellet containing the EVs was resuspended in 1 × PBS and then filtered through a 0.22 μm filter, Millex-GV . The EVs were confirmed using antibodies against exosomal marker proteins as well as flow cytometry. Briefly, recovered exosomes were incubated with 4-μm diameter aldehyde/sulfate latex beads, 4% w/v,  for 15 min at room temperature. The mixture was then mixed in a final volume of 200 μL of PBS at room temperature under gentle agitation. Two hours later, the reaction was terminated by the addition of 100mM glycine for 30 min at room temperature. After centrifugation, the pellet was washed in PBS containing 0.5% of bovine serum albumin and then incubated with specific antibodies. Beads were analyzed by flow cytometry using a BD Biosciences FACSCalibur using FACSDiva version 6.0 software. Concentrations of the EVs were then determined indirectly by quantifying protein concentrations. The amount of released EVs was quantified by measuring the activity of acetylcholinesterase (AChE) (Sigma-Aldrich), as previously described [Serum was harvested from the peripheral blood of DMD patients in tubes by centrifugation at 3,000 × g for 15 min. Isolation of exosomes from serum was performed by Total Exosome Isolation reagent (Invitrogen) or ExoQuick-LPescribed . The totTen μg/μL of EVs extracted from mouse serum was serially diluted in PBS. One μL of the EV solution was dotted onto an Immobilon-P transfer membrane, which is a microporous polyvinylidene fluoride membrane . The membrane was dried at 60°C for 10 minutes, and then incubated with primary antibodies (2 ug) at 4°C overnight, and then with HRP-conjugated secondary antibodies at room temperature for 1 hr. Chemiluminescence was detected using ECL Prime Detection Reagent  and analyzed using LAS-3000 .mdx and wild-type (wt) mice were resolved by 12% SDS PAGE, and then transferred onto a Polyvinylidene Difluoride (PVDF) membrane . An anti-LAMP2 antibody  and Polysorbate 20 (TBST)) and an anti-rat IgG-HRP antibody  were used as first and secondary antibodies, respectively. Signal detection was performed using antibodies coupled to HRP and ECL Prime Detection Reagent , together with analysis by LAS-3000 (Fujifilm Corporation).For Western blotting analysis, 50 μL of sera from Serum CK levels were measured using the Fuji Dri-Chem system  according to the manufacturer’s protocol. Serum (10 μL) was deposited on a Fuji Dri-Chem slide and incubated at 37°C. The increase in absorbance by the generated dye was measured spectrophotometrically for 5 min at 540 nm and the activity was calculated according to the installed formula. Data were expressed as units per liter (U/L).2C12 murine myoblast cells were cultured in Dulbecco's Modified Eagle's medium  with 10% (v/v) fetal bovine serum  and 1% (v/v) penicillin/streptomycin  as the growth medium. The cells were grown at 37°C in controlled humidified air with 5% CO2. For myogenic differentiation of myoblasts into myotubes, the growth medium was changed to differentiation medium, namely, DMEM containing 2% heat-inactivated horse serum and 1% penicillin/streptomycin. Briefly, C2C12 cells were grown in 96-well plates  in growth medium until confluent and then changed to serum-free medium (DMEM containing 1% penicillin/streptomycin) and incubated with or without inducers or suppressors of EV secretion for the indicated times. The number of cells was determined by measuring the absorbance at 450 nm of the formazan product in the culture medium using a 680 microplate reader after incubation with 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt (WST-8) from Cell Counting Kit-8 (CCK-8) (DOJINDO Laboratories).Ccytomegalovirus promoter driving transcription of the dsRed-fluorescent protein-coding sequence interrupted by an intron. The sequences of the three fragments were as follows: pre-miR-1a: 5′-GTTTAAACCCAGGCCACATGCTTCTTTATATCCTCATAGATATCTCAGCACTATGGAATGTAAGGAAGTGTGTGGTTTTGGACTAGT-3′, pre-miR-133a: 5′-GTTTAAACAGAAGCCAAATGCTTTGCTGAAGCTGGTAAAATGGAACCAAATCAGCTGTTGGATGGATTTGGTCCCCTTCAACCAGCTGTAGCTGCGCATTGATCACGCCGCAACTAGT-3′, pre-miR-206: 5′-GTTTAAACGCTTGGGACACATACTTCTTTATATGCCCATATGAACCTGCTAAGCTATGGAATGTAAAGAAGTATGTATTTCAGGCACTAGT-3′. C2C12 cells cultured in 24-well dishes were transfected with these plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 0.2 μg of each plasmid was diluted in 25 μL of Opti-MEM®I Reduced Serum Medium (Invitrogen). The plasmid solutions were mixed with 4 μL of PLUS™ Reagent (Invitrogen), and then incubated at room temperature for 15 min. After incubation, the plasmid solutions were mixed with 25 μL of Lipofectamine 2000, diluted 25 times by Opti-MEM® I Reduced Serum Medum, and then incubated at room temperature for 15 min. The plasmid solutions were added to C2C12 cells in serum-free medium. After incubation at 37°C for 3 hrs, the medium was changed to growth medium, and then incubated at 37°C for the indicated times.Three DNA fragments, namely, precursors of miR-1a, miR-133a, and miR-206  were synthesized , and inserted into the intron site of the pEM-157 vector, which contains the 2C12 myoblast cells were cultured to confluence in growth medium in 6-well plates, and then the growth medium was changed to serum-free medium (DMEM containing 1% penicillin/streptomycin with 0.5 mM of H2O2). The cells were incubated with or without EVs (4 μg) for 24 hours. Then, caspase-3 activity was quantified using the Caspase-3/CPP32 Colorimetric Assay Kit . Briefly, cells were harvested, and the supernatant was removed by centrifugation. Pellets were suspended in 50 μL of chilled cell lysis buffer, and then incubated on ice for 10 min, and centrifuged for 1 min at 10,000 × g. Supernatants were transferred to fresh tubes, and protein concentrations were measured. Protein solutions were adjusted to a concentration of 100 μg/μL by dilution with PBS. Fifty μL of 2 × Reaction Buffer and 5 μL of 4 mM DEVD-pNA substrate were added to 50 μL of the protein solutions and then incubated at 37°C for 2 hrs. Caspase-3 activity was determined by measuring the absorbance at 405 nm using a 680 microplate reader.Cmdx mice. After 5 or 10 days, whole body blood was collected from the abdominal aorta. Twenty-four hours before sacrifice, mice were injected intraperitoneally with 1% evans blue dye  in PBS as described previously [GW4869 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1.5 mM, and then 2.5 μL of 5% methane sulfonic acid solution was added to 50 μL of the GW4869 solution, resulting in a final concentration of 1.43 mM of GW4869. The solution was then incubated at 37°C until the color of the solution became clear, and the GW4869 solution was further diluted to 100 μM in PBS. One-hundred μL of this final GW4869 solution was intraperitoneally injected into https://sg.idtdna.com/calc/analyzer.GC contents and ΔG were estimated using by a web tool, which is available free of charge at P-value test) followed by Bonferroni correction (Pc). A Pc-value or P-value of less than 0.05 were considered to indicate a statistically significant difference between two groups.Statistical analysis was performed by the Fisher’s exact probability test (tg) mdx mice, overexpressing a truncated dystrophin transcript with a deletion from exon 45 to 55, mdx mice, and wild-type (wt) mice were quantified by qRT-PCR. Consistent with a previous report [mdx mice were upregulated compared with those of wt mice, but the tg mice showed a decreased level compared with that of mdx mice  treatment (P < 0.01). In contrast, treatment with C2- or C6-ceramides significantly increased EV release from C2C12 myoblast cells . Moreover, ebselen, which acts as a glutathione peroxidase mimic, significantly reduced the amount of EVs released from C2C12 myoblast cells (P < 0.001). On the other hand, the ceramidase inhibitor d-erythro-MAPP (60 μM), significantly increased EV release (P < 0.001). Furthermore, S1P (40 uM) significantly increased the release of EVs from C2C12 myoblast cells (P < 0.001), and ameliorated the inhibitory effect of GW4869 (P < 0.001). These findings taken together suggested that EV release from C2C12 myoblast cells can be regulated by ceramide metabolism  , 38. To ytometry  and westytometry , were exst cells , and mittabolism .2C12 myoblast cells. To test this hypothesis, EVs released from C2C12 myoblast cells incubated with or without the calcium ionophores monensin and A23187 were quantified by AChE activity. These two calcium ionophores significantly ameliorated the inhibitory effect of GW4869 on EV release from C2C12 cells . Furthermore, a decrease in EV release by GW4869 was restored by caffeine, a ryanodine receptor activator . Furthermore, increases in EV release by S1P were significantly decreased in the presence of EGTA (P < 0.001). Loperamide, a calcium release-activated channel activator, and cyclopiazonic acid, a sarcoendoplasmic reticulum calcium transport ATPase inhibitor, significantly increased EV release from C2C12 myoblast cells (P < 0.001 for each). Taken together, these results indicate that EV release is regulated by intracellular calcium-dependent mechanisms in C2C12 myoblast cells , medium (2 μg), or high (7 μg) concentration of EVs extracted from the serum of mdx mice. The survival of myoblasts incubated for 24 hr with a high or medium concentration of EVs extracted from the serum was significantly increased compared with cells incubated without EVs . In addition, the number of myotube cells incubated with a high concentration of EVs for 48 and 72 hrs was significantly increased compared with cells incubated without EVs . Furthermore, the survival of myoblast cells incubated with a medium or high concentration of EVs extracted from C2C12 culture medium for 24 hrs was significantly increased compared with that without EVs . Significant increases in the survival of myotube cells were observed upon incubation with a high concentration of EVs extracted from C2C12 culture medium for 48 or 72 hrs . Furthermore, to determine whether EVs regulate the survival of C2C12 cells in response to apoptosis-associated stress, myoblasts were incubated with EVs from the serum of mice . In the presence of H2O2, the cell survival of myotubes incubated with EVs containing a large amount of miR-133a was significantly increased compared with cells incubated with EVs prepared from untransfected C2C12 cells (non-TF EVs) (P < 0.05). All of the C2C12 myotubes cultured with EVs (7 μg) from cells transfected with miR1, miR-133a, or miR-206 demonstrated significantly increased survival compared with cells cultured without EVs in serum-depleted medium for 48 and 72 hr (P < 0.001). Furthermore, we compared the survival of cells treated with two types of EVs, namely, EVs-133a and EVs-non-133a, which are EVs from C2C12 cells transfected with and without miR-133a, respectively. The cell survival of C2C12 myotubes cultured with EVs-133a from medium of C2C12 cells transfected with miR-133a, miR-1/miR-133a, miR-133a/miR-206, or miR-1/miR-133a/miR-206 were significantly increased compared with EVs-non-133a from cells transfected with miR-1, mir-206, miR-1/miR-206, or nontransfected cells in serum-depleted medium for 72 hours (P < 0.01), but not for 24 and 48 hours  or medium (2.0 μg) concentrations of EVs were significantly increased compared with that without EVs for 72 hr . However, no significant differences were observed in the cell survival of C2C12 myoblasts cultured with EVs-133a or EVs-non-133a for 48 or 72 hrs (data not shown). These results suggest that the survival of myotubes may be partially regulated by miR-133a via EVs.Next, to analyze the effect of myomiRs via EVs on cell survival, we performed gain-of-function experiments of miR-1, miR-133a, and miR-206, which were upregulated within EVs during the differentiation of C12 cells , using Cch miRNA , S8 Fig.48 hours . No signed cells . Further2C12 cells cultured with or without EVs containing a large amount of miR-133a. qRT-PCR demonstrated that the levels of miR-133a were significantly upregulated by the incubation of cells with the EVs  was intraperitoneally administered into mdx mice for 5 or 10 days . The tibialis anterior muscle of GW4869-injected mdx mice showed a significant decrease in the area of uptake of EBD, which accumulates in damaged cells, compared with that of control mdx mice  compared with those of mdx mice at 7 weeks of age. Moreover, Roberts et al. reported that restoration of the dystrophin protein by the skipping of exon 23 from the mature dystrophin transcript partially normalized serum myomiRs levels [The present study showed that myomiRs are noninvasive biomarkers for the diagnosis of DMD, and that the regulation of myomiR levels may possibly be established as a novel therapy for DMD. We found significant decreases in the levels of miR-1, miR-133a, and miR-206 in the serum of s levels . Furthers levels . These d2C12 cells was regulated by sphingolipids metabolism. In agreement with a previous report [mdx mice compared with controls. However, difference of serum myomiRs level in exosome and exosome-depleted supernatant fractions between our study and previously published data was observed [2C12 myotubes was increased by the addition of serum from DMD patients or the stimulation with fibroblast growth factors (FGF), transforming growth factor-β, or tumor necrosis factor-α (TNF-α) [2O2, and is inhibited by the antioxidant, glutathione-SH (GSH) [Next, we presented evidence that the secretion of EVs from Cs report , our stuobserved . The twoobserved . Moreoveobserved . The miRobserved . The GC  (TNF-α) . Further (TNF-α) , 50, andSH (GSH) , 52. Thu2C12 cell stress, via the suppression of the expression of apoptosis-associated genes, despite the EVs including various bioactive molecules, such as mRNA, protein, and DNA. It was reported that the overexpression of these myomiRs had little effect on the proliferation or apoptosis of cardiac progenitor cells (CPCs) under basal conditions, whereas miR-133a increased cell survival under oxidative stress in part through targeting of the potent proapoptotic factors, Bim and Bmf [2C12 cells cultured with EVs in growth medium did not exhibit a significant difference in survival compared with untreated control cells (data not shown). These findings indicate that miR-133a within EVs in the absence of cellular stresses may not affect muscle survival.In this study, miR-133a in EVs exerted protective effects against C and Bmf . In addi and Bmf . In our Bax expression levels have been shown to be significantly upregulated in the muscle of mdx mice [mdx mice [TAGLN2 gene in cardiomyocytes [DAPK2 gene [2C12 cells under conditions of cell stress. These findings indicate that modification of apoptosis by miR-133a might be a novel therapeutic strategy for DMD.In addition, some previous reports indicated that caspase-9 is a possible direct target of miR-133a, and the inhibition of miR-133a increases caspase-9 and caspase-3 activity as well as the number of apoptotic cells in cardiomyocytes , 55–57. mdx mice . An imbamdx mice . Moreovemdx mice . Moreovemyocytes . It was PK2 gene . In our 2+ levels. In the present study, it was shown that the secretion of EVs from C2C12 cells may be modulated by intracellular Ca2+ concentrations. A chronic increase in Ca2+ concentrations in skeletal muscles fibers of DMD patients and mdx mice has been observed [2+ concentrations, which is induced by IP3RII targeting by miR-133a.Expression of the inositol 1,4,5-triphoshate receptor II (IP3RII) calcium channel was constitutively suppressed by miR-133a . In cardobserved –65. Thesmdx mice with GW4869 resulted in a significant decrease in myomiR levels in the serum compared with untreated mdx mice. In addition, myomiR levels in both EV-containing and EV-depleted supernatant fractions from the sera of GW4869-treated mdx mice were significantly lower than in untreated mdx mice. It was reported that the local injection of myomiRs or exosomes enhanced muscle regeneration and prevented fibrosis via the expression of myogenic factors in skeletal muscle of injured rat and mouse models [Treatment of e models , 66. Exoe models , 53. TakSmpd3 in C2C12 cells. Moreover, the transport of miR-133a via EVs into C2C12 cells increases cell survival under conditions of cellular stress. The inhibition of ceramide synthesis resulted in the suppression of EV secretion, and improved muscle dystrophy in mdx mice. Taken together, these findings suggest that EV-mediated myomiR delivery may have potential as a cell-free therapeutic strategy for DMD, by enabling intracellular communication.In summary, our study demonstrated that the release of myomiRs into the blood circulation was associated with the degeneration of skeletal muscle. The secretion of EVs may be regulated by ceramide metabolism by S1 Figmdx, and tg mice . miR-21, miR-29, and sno202 are used as ubiquitous-expressed miRNAs. miR-302 is specifically expressed in embryonic stem cell. Data are represented as means + S.E. *: P < 0.05, **: P < 0.01, ***: P < 0.001 for mdx vs wt or mdx vs tg.(A) Levels of miR-1, miR-133a, and miR-206 in the sera of wt, (TIFF)Click here for additional data file.S2 Figmdx mice using antibodies of the exosome markers, CD63, CD9, caveolin-3, and MHC class II. HSP90, apoA-I, and calnexin was used as a positive or negative control for cell lysate from SHSY-5Y cells (cell lysate), exosome and exosome-depleted supernatant (sup). (B)Western blot of Lam2, exosome marker, using 50 μL of serum from 5-weeks old mdx or wt mice using antibody against the exosome marker Lamp2. Transferrin was used as a positive control. (C) EV content in the sera of wt, mdx, and tg mice. EVs were extracted from the sera of wt, mdx, and tg mice  by using Total Exosome Isolation kit. Ten μg/μL of EVs were serially diluted in PBS. One μL of EV solutions  were subjected to dot blot analysis using anti-MHC class II and anti-CD63 antibodies.(A) Western blots of exosomes isolated from 200 μL of serum from 5-week old (TIFF)Click here for additional data file.S3 Figmdx, and tg mice . miR-16, miR-21, and miR-212 were used as ubiquitous-expressed miRNAs. miR-122a and miR-323 are specifically expressed in liver and brain, respectively. Data are represented as means + S.E. *: P < 0.05, **: P < 0.01, ***: P < 0.001 for mdx vs wt or mdx vs tg.(A) miR-16 level in the EVs separated by immunoprecipitation with anti-caveolin-3 (Cav3), anti-CD63, anti-CD81, anti-flotillin-1 (Flot1), or anti-MHC class II (MHC II) antibodies from the sera of DMD patients and controls . (B) miR-1, miR-133a, or miR-206 levels in the EVs separated by immunoprecipitation with anti-caveolin-3, anti-CD63, anti-CD81, anti-flotillin-1, or anti-MHC class II antibodies from the sera of wt, (TIFF)Click here for additional data file.S4 Fig2C12 myoblast cells using by Total Exosome Isolation Reagent with anti-CD63, anti-caveolin-3, anti-flotillin-1, anti-GAPDH, anti-HSP90, and anti-calnexin antibodies.(A) EVs covalently bound by aldehyde/sulfate latex beads were mixed with antibodies against caveolin-3, CD9, and CD81, followed by the anti-rat IgG Alexa 488 secondary antibody, and then subjected to flow cytometry analysis. (left) Gating was performed to define area containing EVs, and percentages are indicated. (right) Representative flow cytometric histograms showing caveolin-3, CD9, and CD81-labeled exosome-bead complexes, with percentages indicated for each subpopulation. Staining without secondary antibody was used as a negative control. (B) Western blot analysis of cell lysate and exosomes from C(TIFF)Click here for additional data file.S5 Fig2C12 cells (90% confluent) were incubated for 24 hr with GW4869 and monensin (A), GW4869 and A23187 (B), or GW4869 and caffeine (C), D-erythro-MAPP and BAPTA (D), D-erythro-MAPP and EGTA (E), or D-erythro-MAPP and 2-APB (F) in serum-depleted medium. (G-I) C2C12 cells (90% confluent) were incubated for 24 hr with S1P and EGTA (G), loperamide (H), and cyclopiazonic acid (I) in serum-depleted medium. (J) Schematic figure of the effects of calcium on exosome release. CRAC: calcium release-activated channels; Rab: Rab GTPase activating protein; SERCA: sarco/endoplasmic reticulum Ca2+-ATPase; RyR: ryanodine receptor; InsP3R: Inositol trisphosphate receptor. The amounts of released exosomes were quantified by measuring AChE activities. Data represent means + S.E. of absorbance at 405 nm. *: P < 0.05, **: P < 0.01, ***: P < 0.001.(A-F) C(TIFF)Click here for additional data file.S6 Fig2C12 myoblasts (left) and myotubes (right) were differentiated for 3 days and then incubated for the indicted times in serum-depleted medium with low (0.7 μg), medium (2 μg), or high (6 μg) concentrations of EVs that were extracted from C2C12 culture medium. (B-D) C2C12 myoblasts were differentiated for 2 days in 2% serum-containing DMEM, followed by incubation with/without EVs (2 μg) extracted from mouse serum, in serum-free medium with or without 1.0 mM (+) or 2.0 mM (++) of methyl-ß-cyclodextrin (MßCD) for 48 hr (B), or 20 mg/mL of nocodazole (Nocod.) or 2 mM of Simvastatin (Simv.) for 48 hr (C), or 500 mM of U0126 for 24 hr (D). Data are represented as mean + S.D. for absorbance at 450 nm by CCK-8. *: P < 0.05, **: P < 0.01, ***: P < 0.001.(A) C(TIFF)Click here for additional data file.S7 Fig2C12 cells were cultured with growth medium until 90% confluency and then changed to differentiation medium for 1 to 6 days. miRNAs were isolated from EVs extracted from the media of C2C12 cells on the indicated days and miR-1, miR-133a, and miR-206 levels were measured by RT-quantitative PCR.C(TIFF)Click here for additional data file.S8 Fig2C12 cells transfected with miR-1, miR-133a, or miR-206, and their four possible combinations  were measured by RT-quantitative PCR. Levels are shown relative to that of the non-transfected cells, which was set to 1.miR-1, miR-133a, and miR-206 levels within EVs extracted from the medium of C(TIFF)Click here for additional data file.S9 Fig2C12 cells transfected with miR-1, miR-133a, or miR-206, or non-transfected (non-TF EVs) for 24 hrs in serum-depleted medium (A) or in the presence of H2O2 (10 mM) (B). Data represent mean + S.E. *: Pc < 0.05.Myoblasts (A) and myotubes (B), differentiated for 4 days, were incubated with or without low (0.7 μg), medium (2 μg), or high (6 μg) concentrations of EVs extracted from the medium of C(TIFF)Click here for additional data file.S10 Fig2C12 cells transfected with miR-1, miR-133a, miR-206, or their four possible combinations  for the indicated times. Data are represented as mean + S.D. for absorbance at 450 nm by CCK-8. *: P < 0.05, ***: P < 0.001.Myotubes were incubated in serum-depleted medium, with 0.8 μg (A), 2 μg (B) of EVs extracted from the medium of C(TIFF)Click here for additional data file.S11 Figmdx (mdx), GW4869-treated mdx for 5 days (GW4869-5d), GW4869-treated mdx for 10 days (GW4869-10d), and wt mice were measured by RT-quantitative PCR. Data represent mean + S.E. *: P < 0.05, **: P < 0.01, ***: P < 0.001.miR-1, miR-133a, and miR-206 levels in EVs (A), or EV-depleted supernatants (B) from sera of untreated control (TIFF)Click here for additional data file.S12 Figmdx mice treated with GW4869 for 10 days or untreated control mdx mice were subjected to western blotting to analyze utrophin expression. Gapdh  and tublin were used as loading controls.TA muscle lysates of (TIFF)Click here for additional data file.S13 Fig(TIFF)Click here for additional data file.S1 TableGC contents and ΔG for mature sequences of myomiRs were estimated.(XLSX)Click here for additional data file.


Escherichia coli Tat system to recognize and translocate de novo–designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b–induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model  The transport of proteins across membranes is one of the great challenges faced by the cell. In prokaryotes, two major pathways are used to achieve protein translocation across the cytoplasmic (inner) membrane. The general secretion (Sec)The majority of Tat substrates are cofactor-containing proteins that require assembly in the cytoplasm –6 includ–Escherichia coli the Tat apparatus comprises TatA/B family proteins and the TatC protein. Single point mutations in either TatA or TatC were identified that allow translocation of unfolded substrates, and point mutations in both TatB and TatC enabled export of a broader range of unfolded substrates, suggesting that the TatABC subunits cooperatively assess the folding state of proteins independently of protein translocation . Histidine residues within the maquette cavity ligate hemes, producing artificial proteins resembling b-type cytochromes and myoglobin  imparts changes in the extent of folding of the maquette variants. The archetypal trimethylamine-N-oxide reductase (TorA) Tat signal peptide was fused to the N terminus of the three proteins and the E. coli Tat machinery was challenged to differentiate between the folding variants. We show that the Tat apparatus is able to sense the conformational flexibility of the different maquette substrates, and that increasingly well-folded maquettes are exported with enhanced efficiency.Here, three different maquette variants were utilized, each with a different heme capacity . Using nE. coli Tat system could recognize and export a de novo–designed di-heme protein, and then to use variants of this protein with experimentally confirmed differences in conformational flexibility to test if the Tat apparatus selectively processes the more folded proteins.The aim of this study was to test whether the b cofactors. The requirement for bis-histidine ligation of heme b in the artificial constructs enabled the generation of the one and no heme-binding variants through H53A, and H53A/H88A  point mutations, respectively  because the native tetrapyrrole biosynthesis pathway cannot keep pace with the induced synthesis of maquette . Apoproteins were reconstituted in vitro with an excess of heme and unbound pigment was removed by anion exchange chromatography. The Soret absorption bands for the BT6, BT6M1, and BT6M0 maquettes were normalized for protein concentration (absorbance at 280 nm) and had maxima at 413, 412, and 396 nm, respectively (A), with the amplitudes corresponding to binding two, one, and no hemes, respectively. The weak, blue-shifted absorption of the BT6M0 maquette suggested a low level of adventitiously bound heme. We compared heme absorption in the maquette-bound, and solvated (buffer or DMSO) states, which had respective maxima at 413, 404, and 384 nm . The blue-shifted absorption maxima for solvated heme are consistent with the weak, blue-shifted absorption for BT6M0 (A) arising from residual, weakly bound heme; ligation into the internal cavity of BT6 and BT6M1 causes a red-shift absorption of the heme.Maquettes are typically overproduced in ectively A, with tor BT6M0 A arisingFig. S2, black lines). Likewise, irrespective of heme binding heating to 80 °C resulted in spectra typical of that of a denatured protein .Temperature-dependent CD spectroscopy was performed to assess the effect of heme ligation on the thermal stability of each maquette scaffold. At 15 °C the CD spectra of all three maquette variants, with or without bound heme, were typical of α-helical structures . For BT6, heme incorporation displaced the midpoint of the melting curve from 22 to 56 °C, the 34 °C difference indicating increased stabilization following heme binding. The same procedure with BT6M1 increased stabilization by only 16 °C (25 to 41 °C), and had very little effect on the BT6M0 scaffold (32 to 35 °C)  . Thus, b1H) NMR spectroscopy was used to assess conformational changes in tertiary structure upon heme binding to maquettes. A 1H NMR spectrum characteristic of a protein with limited tertiary structure was observed for all maquette scaffolds in the absence of heme . Addition of heme to BT6 showed greater dispersion of resonances in the amide proton region, with a notable increase in the number of peaks at around 10 ppm, suggesting that the heme is binding to the scaffold and stabilizing the protein tertiary structure . BT6M1 showed a small increase in resonance dispersion following heme addition , whereas BT6M0 showed no significant change in amide proton dispersion . The increase in the chemical shift dispersion observed in the methyl proton region (at around 1.0–0.0 ppm) for BT6, BT6M1, and BT6M0 mirrored the behavior observed for the amide proton resonances . Together, the increase in 1H NMR dispersion in both backbone amide and methyl regions confirm the ligation of heme into the BT6 and BT6M1 variants and indicate an increase in protein folding upon ligation. These results are consistent with a previous NMR study that showed poor dispersion for a related apo-maquette, and progressive structuring of the maquette when 1 then 2 eq of heme were added  to the E. coli periplasm via the Tat system  were synthesized and cloned into the pEXT22 vector (Table S1). In the TorA–BT6 variants residues His-95 and His-130 correspond to His-53 and His-88 in the maquette scaffolds presented in The t system . ConstruE. coli BL21(DE3) along with empty pET-21a(+). The pEXT22 vector was used as it allows isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible expression of the synthetic gene under the control of a tightly regulated tac promoter. The low copy number of pEXT22  (E. coli Tat apparatus with substrate.The resulting plasmids were co-transformed into omosome) , 33 limi6 tag allows immunodetection of the recombinant protein; the predicted molecular mass of the unprocessed precursor protein is ∼21 kDa, whereas the Tat-processed mature protein is ∼17 kDa. Anti-GroEL was used to probe the degree of cytoplasmic contamination in the periplasmic preparations (A); for all samples only a faint signal was detectable in the periplasm compared with that detected in cell-free extracts, indicating the level of contamination was very low. The Tat export assays were performed in cells that also maintained the pET-21a(+) vector, allowing immunoblot signals to be normalized by immunodetection of β-lactamase (B), allowing direct comparison of the degree of Tat transport between samples.Following growth and protein production, periplasmic fractions were prepared from harvested cells as described under “Experimental procedures” and analyzed alongside cell-free extracts prepared from identically grown cells. The C-terminal Hisarations A; for alactamase B, allowiC) preparations with no signal detected at 21 kDa, indicating complete processing and export of TorA–BT6. To confirm the periplasmic localization was Tat-dependent, a R12K/R13K (KK) variant of the TorA signal peptide was generated and fused to BT6 (C). Together these data indicate that the BT6 scaffold is able to acquire heme from the native biosynthetic pathway and fold in vivo, and that this folding event can be detected by the Tat system, with the TorA signal peptide sufficient for Tat-mediated recognition and transport of heme-loaded BT6 to the E. coli periplasm.For TorA–BT6, a 17-kDa signal was present for both periplasmic and cell-free extract C preparad to BT6 ; it has d to BT6 . When thd to BT6 C. TogethC). Conversely, blots of cell-free extracts of the same strains reveal a significant signal for the unprocessed maquettes for BT6M0, and to a lesser extent BT6M1, as observed for the TorA–KK–BT6 control, showing that the preprotein accumulates in the cytoplasm when it is less well folded .To probe the proofreading ability of the Tat machinery to recognize and efficiently export only folded proteins, similar constructs were generated in which the TorA signal peptide was fused to the BT6M1 and BT6M0 maquettes, which, in the presence of heme, are either partially folded (BT6M1) or unfolded (BT6M0) relative to BT6  and 3. CFig. 4C shows that the total signal (preprotein plus exported protein where relevant) for all three maquettes is similar. However, to further confirm that the differential periplasmic targeting of the three BT6 variants by the Tat system was due to differences in their heme loading and associated folding and not in expression, synthesis, or stability, we performed two additional control experiments. First we produced each maquette under the same conditions as for the Tat export assays but without the TorA signal peptide, and immunoblotting confirmed approximately equal levels of each , ruling out that BT6M1 and BT6M0 are produced at lower levels or are more rapidly degraded in the cytoplasm than BT6.The cellular levels of maquette variants should be very similar as all the proteins are identical apart from 1 or 2 point mutations and were expressed from the same plasmid in the same cell line under the same conditions. Indeed, the data presented in Fig. 4Table S1) in-frame with an N-terminal PelB (Erwinia carotovora pectate lyase B) Sec leader peptide (22 amino acids)  vector (o acids) , 36 (Tabo acids) . Stop coE. coli W3110-TatExpress cell line (inset panels). SDS-PAGE of the concentrated eluates revealed the same pattern as the Tat exports presented in Fig. S7). This also ruled out any leakage of unprocessed protein across the cytoplasmic membrane as the larger precursor species were not purified . The absorbance spectra of Tat-exported BT6, BT6M1, and BT6M0 samples normalized at 280 nm are shown in in vitro heme-reconstituted BT6. There is a close match between the spectra of the Tat-exported heme-loaded BT6 purified from the periplasm and the reconstituted BT6 sample (solid red versus dashed red lines). The high affinity of BT6 for heme . The number of bound hemes therefore provides a simple, well-defined method to alter the stability of a protein substrate. The consequent effects of heme content on the thermostability and folding of each maquette were measured using temperature-dependent CD spectroscopy .The export data shown would only be obtained if the BT6 maquettes were able to ligate heme,  E. coli , the spein vitro A.E. coli Tat translocase will never have encountered the artificial protein substrates described here, our data show that it is able to recognize and distinguish between them, even to the extent of processing the intermediate state of BT6M1 differently from the folded BT6 and the least structured BT6M0. Thus, the Tat proofreading process must involve a generic form of discrimination; the correlation between the structural flexibility of the Tat substrate and its suitability for export suggests that there are initial encounters between Tat components and the substrate at the membrane surface. Such interactions might sense flexible motions of the substrate that are transmitted to other components of the Tat machinery, preventing transport across the membrane.Although the native b from a native biosynthetic pathway, can be exported from the cell in its correctly folded state. Development of this concept may contribute a significant advance in biotechnology, where the principle could be applied to other organisms and to important biomolecules and protein-cofactor complexes, particularly those that may cause toxicity to the host.This proof-of-concept study shows that an artificial protein, engineered to bind heme et al.  were checked for convergence before proceeding to production MD runs of 50 ns. Following production runs, trajectories were corrected for periodicity and centered on the protein. Snapshots of the trajectories at 50 ns were exported to .pdb files. Electrostatics and solvent-accessible surface calculations were performed using APBS  pre-processing, production runs, and post-processing were performed with Gromacs version 4.6 . MD simuing APBS . VisualiTables S1 and S2, respectively. Synthetic genes encoding maquette protein variants codon optimized for expression in E. coli were purchased from DNA2.0 (now ATUM) or Integrated DNA Technologies. Point mutations were generated using the QuikChange II Site-directed Mutagenesis Kit (Agilent). The TorA Tat signal peptide was amplified from E. coli genomic DNA and joined in-frame to maquette constructs by overlap extension-PCR using Q5 High-Fidelity DNA Polymerase (New England Biolabs). All plasmids were sequence verified by automated DNA sequencing (GATC Biotech). Competent E. coli JM109 (Promega) was used for cloning and was grown in Luria-Bertani (LB) broth/agar supplemented with the appropriate antibiotic(s) (Table S1).Plasmids and primers used in this study are provided in E. coli BL21(DE3) containing the desired plasmid was grown with shaking (230 rpm) at 37 °C in LB broth with 100 μg ml−1 ampicillin to an absorbance at 600 nm (A600) of ∼0.6. At this point IPTG was added to a final concentration of 1 mm to induce expression and the cultures were incubated for a further 16 h at 37 °C. Cells were harvested by centrifugation  and resuspended in buffer A . Cells were lysed by sonication on ice and the lysate was clarified by centrifugation . The supernatant was filtered through a 0.45-μm filter and applied to a Chelating Sepharose Fast Flow column  pre-equilibrated with 10 mg ml−1 of nickel sulfate. The column was washed with 5 column volumes of buffer A with the flow-through and wash was collected and pooled. The pooled sample was buffer exchanged into buffer B  and further purified by ion exchange chromatography on a Fast Flow Q-Sepharose column  with a linear gradient of 0–1 m NaCl in buffer B. Where required maquettes were further purified by size exclusion chromatography on a Superdex 200 Increase column  in buffer C . Where necessary protein was concentrated using Vivaspin centrifugal concentrators (Sartorius).The high copy number pJexpress414 plasmid was used for recombinant protein production under control of an IPTG-inducible T7 promoter. −1) were prepared in 100% DMSO. Protein concentrations were calculated by absorbance at 280 nm following the method described by Gill and von Hippell  DMSO) and incubated for 45 min at 25 °C, before being transferred to ice and buffer exchanged into buffer B using Vivaspin centrifugal concentrators. Unbound cofactor was removed by ion exchange chromatography on a DEAE-Sepharose (Sigma) column.Hemin (Sigma) stocks  and UV-visible absorption was measured in a 1-cm path length UV cuvette in a Cary 60 UV-visible spectrophotometer (Agilent) at room temperature.Protein samples were buffer exchanged into buffer E (5 mMRW) of protein samples was measured in a 1-mm path length quartz cuvette on a Jasco J-810 spectropolarimeter with a Jasco PFD-425S Peltier to enable temperature control. Spectra were obtained from 15 to 80 °C at 5 °C intervals. Spectra were recorded continuously at a scan speed of 100 nm min−1, with 1-nm resolution and a 4-s response with 4 accumulations. Ellipticity ([θ]) at 222 nm was measured every 1 °C from 15 to 80 °C at 1 °C min−1 with a 4-s response. Melting temperatures (mT) and refolding temperatures (fT) are the temperature at which 50% of the protein is unfolded or folded, respectively, as determined by fitting melting data to a sigmoidal Boltzmann distribution according to the following equation, where T is the temperature, mT is the melting (or folding) temperature, and T0 is the initial temperature of the experiment.Mean residue ellipticity , and 1 mm trimethylsilyl propanate (reference standard). 1H NMR spectra were recorded using a Bruker Avance 800 MHz spectrometer fitted with a 5-mm QXI room temperature probe, equipped with z axis gradients. One-dimensional experiments were acquired as accumulations of 4096 transients over a spectral width of 24.038 kHz, corresponding to a proton spectral width of 30.0 ppm. All data were processed using an EM window function and 5-Hz line broadening, without linear prediction in TopSpin (Bruker). Spectra were referenced to trimethylsilyl propanate at 0 ppm prior to overlay and analysis.Spectra were recorded at 298 K on 0.2–0.5 min vivo transport assays, maquette genes were cloned into the KpnI and XbaI sites of the low copy number pEXT22 plasmid for expression from a tightly controlled tac promoter (Table S1) were co-transformed into E. coli BL21(DE3) cells along with empty pET-21a(+). Cultures (50 ml) were grown at 37 °C with shaking (230 rpm) in LB medium with 30 μg ml−1 of kanamycin and 100 μg ml−1 of ampicillin in 250-ml Erlenmeyer flasks. At an A600 of ∼0.6 expression was induced with 0.5 mm IPTG for 2 h. Cells were harvested by centrifugation  and washed with buffer F . To prepare cell-free lysates, cells were re-suspended to A600 ∼2 in 2 ml of chilled Buffer C and lysed by sonication on ice. The supernatant, following clarification by centrifugation , was collected as the cell-free extract. Periplasmic fractions were obtained using a procedure based on the EDTA/lysozyme/osmotic shock method described by Randall and Hardy  microcentrifuge and the supernatant was collected as the periplasm.To avoid overloading the Tat system for promoter . The pEXnd Hardy . Briefly6 , anti-β-lactamase (Abcam), or anti-GroEL (Sigma) primary antibodies followed by an appropriate horseradish peroxidase-conjugated secondary antibody (Sigma). Chemiluminescence was detected using the WESTAR ETA C 2.0 chemiluminescent substrate (Cyanagen) on an Amersham Biosciences Imager 600 .Immunoblotting was performed essentially as described previously . BrieflyE. coli W3110 TatExpress cell line (Table S1). 500-ml cultures were grown in 2-liter Erlenmeyer flasks at 30 °C with 220 rpm agitation. At an A600 of ∼0.6, protein production was induced with 0.5 mm IPTG and cultures were incubated for 24 h, after which cells were harvested by centrifugation . To obtain periplasmic fractions, cells were resuspended in 10 ml of chilled buffer F. 10 ml of chilled milliQH2O was added followed by 800 μl of 1 mg ml−1 of lysozyme and samples were incubated on ice for 10 min. 800 μl of 1 m MgSO4 was added and the solution was centrifuged  with the supernatant collected as the periplasm.The ell line  was tran−1 of nickel sulfate. The column was washed with 20 ml of buffer A and 20 ml of buffer G . Protein was eluted with 10 ml of buffer H  and the elution fractions were collected.Periplasmic fractions were applied to a Chelating Sepharose Fast Flow column  pre-equilibrated with 10 mg mlG. A. S., K. J. G., N. B. P. A., D. M. J. M., A. S. J., A. J. R., D. B. A., A. A. B., F. S., P. T., P. D., P. L. D., C. R., A. H., and C. N. H. data curation; G. A. S., K. J. G., N. B. P. A., D. M. J. M., A. S. J., A. J. R, D. B. A., A. A. B., F. S., P. T., P. D., P. L. D., C. R., A. H., and C. N. H. formal analysis; G. A. S., K. J. G., N. B. P. A., D. M. J. M., A. S. J., A. J. R, D. B. A., A. A. B., F. S., P. T., P. D., P. L. D., C. R., A. H., and C. N. H. methodology; G. A. S., K. J. G., A. H., and C. N. H. writing-original draft; G. A. S., N. B. P. A., A. H., and C. N. H. writing-review and editing; K. J. G., A. S. J., F. S., P. T., P. D., P. L. D., C. R., A. H., and C. N. H. investigation; N. B. P. A., F. S., P. T., and P. D. software; K. J. G., A. S. J., F. S., P. T., P. D., P. L. D., C. R., A. H., and C. N. H. conceptualization; C. N. H. project administration.