MS experiments identified three lysine residues as acetylation targets in the host cells and suggested that Lys-31 and Lys-90 are acetylated by PCAF and GCN5, respectively. RNAi-mediated silencing of GCN5 and PCAF did not change acetylation levels of NP. However, interestingly, viral polymerase activities were increased by the PCAF silencing and were decreased by the GCN5 silencing, suggesting that acetylation of the Lys-31 and Lys-90 residues has opposing effects on viral replication. Our findings suggest that epigenetic control of NP via acetylation by host acetyltransferases contributes to regulation of polymerase activity in the influenza A virus.Histone acetylation plays crucial roles in transcriptional regulation and chromatin organization. Viral RNA of the influenza virus interacts with its nucleoprotein (NP), whose function corresponds to that of eukaryotic histones. NP regulates viral replication and has been shown to undergo acetylation by the cAMP-response element (CRE)–binding protein (CBP) from the host. However, whether NP is the target of other host acetyltransferases is unknown. Here, we show that influenza virus NP undergoes acetylation by the two host acetyltransferases GCN5 and P300/CBP-associated factor (PCAF) and that this modification affects viral polymerase activities. Western blot analysis with anti–acetyl-lysine antibody on cultured A549 human lung adenocarcinoma epithelial cells infected with different influenza virus strains indicated acetylation of the viral NP. A series of biochemical analyses disclosed that the host lysine acetyltransferases GCN5 and PCAF acetylate NP  NP receives multiple posttranslational modifications, which play crucial roles in regulating NP functions. Phosphorylation of NP inhibits its oligomerization and, consequently, ribonucleoprotein (RNP) activity and viral growth , 12. NP In this study, we succeeded in finding two acetyltransferases in host cells, GCN5 and PCAF, other than CBP, which acetylated NP at target lysine residues and consequently affected viral transcriptional activities. Mass spectrometry identified different candidate lysine residues that may have undergone acetylation by the two enzymes. Interestingly, viral transcriptional activities were increased by the RNAi against PCAF but were decreased by that against GCN5, suggesting that the different lysine residues targeted for acetylation caused these opposing results. Our findings provide insights to understand the epigenetic molecular mechanisms that regulate viral growth through posttranslational modifications.A). By infecting cells with the H3N2 strain, two positive sets of bands of different sizes were strongly detected at around 50 kDa and 25 kDa, as well as proteins of host cells at around 37 kDa and 45 kDa (F). The viral titer of this strain was lower than that of the H1N1 strain. Therefore, it was suggested that the signal intensity of the acetylated NP of the H3N2 was not strong enough to eliminate the extra signals of host proteins. The two bands exhibiting strong signals at 50 kDa and 25 kDa were concluded to be viral proteins because they were both detected 8 h after infection (A and F). To determine which viral proteins were acetylated, a Western blot analysis using antibodies for several viral proteins was performed. Strong signals were detected at around 50 kDa by anti-NP antibody in both strains of influenza virus (B and G). In addition, bands were observed above the 25 kDa marker bands by anti-NS1 antibody (C and H). Both expression and acetylation of NP and NS1 started 8 h after infection (A–C and F–H). Acetylation of NS1 was limited to the H3N2 strain as reported previously (C and H). We reported previously that PB2 interacted with acetyl-CoA . Western blot analysis with anti-actin antibody showed that there was no difference in the amount of loaded samples among the lanes (E and J). We used the other virus of the H3N2 strain (A/Hiroshima/52/2005) and obtained the same results of Western blot analysis with those of A/Uruguay/716/2007 (data not shown). These results suggested that NP of both strains was acetylated in cultured A549 cells.To identify acetylated proteins during viral growth in host cells, we performed a Western blot analysis using anti–acetyl-lysine antibody. Cultured A549 human lung adenocarcinoma epithelial cells were infected with two different strains of influenza A virus (A/Puerto Rico/8/34 (H1N1) or A/Uruguay/716/2007 (H3N2)) and prepared for SDS-PAGE at 0, 4, 8, 12, 24, 36, and 48 h after infection. In cells infected by the H1N1 strain, a strong signal was detected as single bands of around 50 kDa in mass using the anti–acetyl-lysine antibody without any extra bands A. By infd 45 kDa F. The vinfection , A and Fza virus , B and Gantibody , C and Heviously , C and Hetyl-CoA , but acehe lanes , E and JK). Western blot analysis with anti–acetyl-lysine antibody showed that NP was precisely acetylated in host cells (K). Overexpressed NP in cells was also acetylated . Cotransfection of other viral proteins and vRNA with NP did not have any effect on its acetylation. These results showed that NP was acetylated in host cells without any interference from other viral proteins or from vRNA.Although Western blot analysis with anti–acetyl-lysine antibody suggested that there was acetylation of NP in host cells, this technique was not enough to specify which proteins were acetylated. To determine that this acetylated protein was indeed NP, a combination of immunoprecipitation using anti-NP antibody and Western blot analysis with anti–acetyl-lysine antibody was performed. Substitution of anti-NP antibody with random IgG or distilled water as negative controls failed to immunoprecipitate NP, showing that NP precipitation was specific K. Westerst cells K. Overexin vitro acetylation assay. The nuclear extract and recombinant proteins of three different acetyltransferases were incubated with the recombinant protein of NP (recombinant NP). The nuclear extract, which mainly contains P300/CBP with intrinsic histone acetyltransferase (HAT), and the recombinant protein of P300/CBP both did not acetylate NP . Histone H1 as a positive control was heavily acetylated, showing that the enzymatic activities of the nuclear extract and P300/CBP were conserved . On the other hand, the recombinant proteins of PCAF and GCN5 acetylated the NP . PCAF and GCN5 are grouped in the superfamily of GCN5-related N-acetyltransferases (GNAT) and their secondary and tertiary structures are highly similar to each other . Next, to investigate whether the NP that constructs part of the RNP was acetylated by PCAF and GCN5, these HATs were incubated with RNP purified from virions of the H1N1 strain (A/Puerto Rico/8/34). Acetylation of the NP within the RNP was clearly observed .To identify the enzymes in the host cells that acetylate NP, we performed an ch other , 23. TheA) . Embelin and garcinol were effective inhibitors at concentrations of 50 μm .PCAF and GCN5 contain Val-Lys-Gly (VKG) and Val-Arg-Gly (VRG) sites, respectively, which are required for acetyl-CoA interaction , 24. ResFig. 3A) . TherefoTM accession no. AKU37741). This technique identified three lysine residues that were candidates of acetylation targets within NP in infected cells (m/z of 402.22 (z = 3) that matched NP residues 27–38 contained acetylated Lys-31 (A). However, this peptide was detected as an isotopic peak, suggesting that another modification might be present on this lysine residue (m/z of 828.42 (z = 2), matching NP residues 78–91, harbored acetylated Lys-90 (B). Finally, in SGAAGAAVKGVGTMVMELVR with the precursor m/z of 655.01 (z = 3), matching NP residues 176–195, Lys-184 was acetylated (C). Ambiguous acetylation because of low scores by MASCOT search of MS was observed at Lys-103 ; GenBanked cells : Lys-31,d Lys-31 A. Howeve residue . The nexd Lys-90 B. Finalletylated C. Ambigu Lys-103 .in vitro. Among the three lysine residues detected as candidates of acetylation targets in host cells (A and C and m/z of each peptide and the corresponding amino acid sequences were 602.32 (ASVGKMIGGIGR with Lys-31 acetylated by PCAF) (A), 547.61 (YLEEHPSAGKDPKK containing Lys-90 acetylated by GCN5) (C), and 974.01 and 649.50  (B and D). In vitro incubation of the recombinant proteins of NP and two kinds of HATs showed that other lysine residues, such as Lys-77, Lys-87, Lys-91, Lys-103, Lys-236, and Lys-273, together with three residues detected in the samples prepared from infected cells  A, 547.61by GCN5) C, and 97ctively) , B and Ded cells . Acetylam/z of the peptide containing Lys-90 purified from infected cells  was different from that of the recombinant protein . In the peptides purified from the infected cells, the methylation on Lys-91 was detected together with acetylation on Lys-90 , and the molecular mass of this peptide was larger than that of the peptide from the recombinant protein in the mass of methyl group. Variable modifications, such as ubiquitination and methylation, on lysine residues in NP were also detected in infected cells (data not shown). Acetylation on Lys-77 and Lys-103 in the recombinant proteins was ambiguous because of low MASCOT scores  (A–C) (A). All three lysine residues—Lys-31, Lys-90, and Lys-184—were localized in the RNA-binding groove (A–C), suggesting that acetylation of these three lysine residues is involved in regulation of the interaction with vRNA, mRNA, and/or cRNA. Although Lys-184 was localized in an RNA-binding groove (B) (green in B and C) was exposed to the surface of the groove. This lysine residue was a target of ubiquitination, as reported previously  , A–C 304IRY (A–CY) (A–C) A. All thg groove , A–C, sug groove B 18), o, o4IRY) green in , B and Ceviously , 18. UbiA). There were no changes in the amount and acetylation levels of NP (A and B). We performed a minigenome assay to measure alterations to the polymerase activity after transfection of the siRNAs. Transfection of random siRNA increased the relative polymerase activity significantly (p < 0.01). However, interestingly, compared with the cells injected with random siRNA, viral transcription increased with the addition of PCAF-specific siRNA (p < 0.01), but decreased by that of GCN5 (p < 0.05) . These results suggested that GCN5 and PCAF regulated viral transcriptional activity in host cells.In general, acetylation of histones causes alterations to the chromatin structure and stimulates transcriptional activity , 32. To Fig. 7In vitro experiments indicated that the host acetyltransferases, GCN5 and PCAF, were responsible for acetylating the NP of the influenza A virus. We succeeded in determining the candidate lysine residues for acetylation targets by MS. Treatment with siRNAs specifically targeted against PCAF and GCN5 did not alter the acetylation levels of NP in host cells. However, inhibition of PCAF and GCN5 expression by RNAi increased and decreased viral transcriptional activity, respectively, suggesting that the activities of these acetyltransferases are important for controlling viral growth. Generally, transcriptional activities in eukaryotic cells are regulated by acetylation of histone-constituting nucleosomes. In this study, we showed that the transcription of the influenza virus was regulated by acetylation of NPs, which interact with the vRNA of the influenza virus as well as with the histones that bind to genomic DNA. Here, we suggested that this epigenetic control also influences the transcriptional activity in the influenza virus.In this work, a series of biochemical studies showed that the NP of the influenza A virus was acetylated in infected cells. C). We suggested that this was because of different lysine residues being targeted for acetylation. Although mass spectrometric analyses using infected cells identified three lysine residues as candidates of acetylation targets , suggesting that the acetylation of Lys-90 by GCN5 has a biological function different from that of Lys-31 by PCAF, which is involved in the defense responses of the host. Similar opposing effects of acetylation by PCAF and GCN5 were reported in tumor cells  , which hml-glutamine, at 37 °C in the presence of 5% CO2.A549 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Wako) supplemented with 10% fetal bovine serum (Life Technologies), 50 units/ml penicillin and 50 μg/ml streptomycin (Life Technologies), and 4 m5 cells/well), and then infected with a multiplicity of infection (m.o.i.) of three influenza A virus strains (A/Puerto Rico/8/34 (H1N1), A/Uruguay/716/2007 (H3N2), and A/Hiroshima/52/2005 (H3N2)) at 37 °C under 5% CO2. After incubation for 0, 4, 8, 12, 24, 36, or 48 h, the cells were lysed in a buffer containing 125 mm Tris-HCl, pH 6.8, 5% SDS, 25% glycerol, 0.1% bromophenol blue, and 10% β-mercaptoethanol and boiled for 5 min. Samples of infected cells were separated by SDS-PAGE and transferred onto pieces of PVDF membranes . To detect acetyl-lysine NP, NS1, PB2, or β-actin proteins, we used the mouse monoclonal anti–acetyl-lysine IgG , the mouse monoclonal anti-influenza A NP IgG , the rabbit anti-PB2 anti-serum , the goat polyclonal anti-influenza A NS1 IgG , and the rabbit monoclonal anti-β-actin IgG  as the primary antibodies. The horseradish peroxidase–conjugated goat anti-mouse IgG , donkey anti-goat IgG , and goat anti-rabbit IgG  were used as the secondary antibodies. The luminescent signals were detected using Western Lightning ECL Pro (PerkinElmer Life Sciences).A549 cells were seeded in a 24-well plate (2 × 106 cells/well) were infected with m.o.i. of five A/Puerto Rico/8/34 at 37 °C under 5% CO2. After postincubation for 24 h, the cells were lysed in a buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, and the cOmplete ULTRA Tablets, Mini, EDTA-free, Protease Inhibitor Cocktail (Roche). The cell lysates were centrifuged at 15,000 rpm at 4 °C for 10 min and the supernatant was collected. Protein G magnetic beads  were incubated with monoclonal anti-NP antibody (1-μl/sample) at 4 °C for 30 min. The cell lysate was incubated with the antibody–protein G bead complex at 4 °C overnight. The beads were washed three times with lysis buffer, added to 30 μl SDS sample buffer, boiled at 95 °C for 5 min then separated by SDS-PAGE. Western blotting was performed using either the mouse monoclonal anti–acetyl-lysine IgG  or the mouse monoclonal anti-influenza A NP IgG (SouthernBiotech), using the standard Western blotting procedure.A549 cells seeded in a 6-well plate (1 × 10Escherichia coli and purified as previously published (NP_003875) (NP_066564) were partially amplified from cDNA of HaCaT cells. Nucleotide sequences of primers for cloning of NP were as follows: forward primer, 5′-GCTAGC ATG GCG TCC CAA GGC ACC AAA CGG-3′ (containing restriction site for NheI) and the reverse primer, 5′-CTCGAG TTA ATT GTC GTA CTC TGC ATT-3′ (containing restriction site for XhoI). Nucleotide sequences of primers for cloning of PCAF were as follows: the forward primer, 5′-GCTAGC GTA ATT GAA TTT CAC GTG GTT GGC-3′ (containing restriction site for NheI) and the reverse primer, 5′-GGATCC TCA CCG TGG ATT TAG CTC ACA TCC-3′ (containing restriction site for BamHI). Nucleotide sequences of primers for cloning of GCN5 were as follows: forward primer, 5′-GCTAGC ATC GAG TTC CAT GTC ATC GGC-3′ (containing restriction site for NheI) and the reverse primer, 5′-GGATCC TTA CTC CGT GTA GGG GAT GCG GGG-3′ (containing restriction site for BamHI). The underlined sequences are the restriction enzymes' recognition sites. Each PCR product was ligated into the pET28a(+) vector (Novagen). The recombinant proteins of full-length human GCN5 and the catalytic domain of human PCAF  were also purchased.The recombinant proteins of full-length influenza NP and HAT domains of human PCAF and GCN5 were synthesized in ublished . The ful_003875)  and GCN514C]acetyl-CoA (Perkin Elmer) at 30 °C for 2 h in buffer containing 50 mm Tris-HCl, pH 8.0, 10% glycerol, 1 mm DTT, and 10 mm sodium butyrate. To detect [14C] radioactivity, reactions were separated in 10% SDS-PAGE gels, after which an imaging plate was exposed to the gel for several days. Signals were detected using a fluoroimage analyzer . Anacardic acid (Sigma), garcinol (Enzo Life Sciences), and embelin (Sigma) prepared in DMSO were incubated with NP and a HAT for 30 min at 30 °C. The RNP was purified from virions of A/Puerto Rico/8/34  as described previously   was incubated with PCAF or GCN5. The amount of recombinant NP, the concentration of reaction solution, and the temperature and duration of incubation were the same as the acetylation assays, except for substitution of [eviously . Next, i4IRY) (4IRY) was obtained from the Protein Data Bank.Acetylated lysine residues were mapped in the tertiary structure of the dimer of the NP (PDB ID: 4IRY)  using thA), A549 cells (5 × 105 per dish) seeded in a 6-well plate were transfected with 10 nmSilencerTM Select Negative Control No. 2 siRNA  (Ambion/Thermo Fisher Scientific) KAT2A: GCN5 , or KAT2B: PCAF  using LipofectamineTM RNAiMAX reagent (Thermo Fisher Scientific) and incubated for 48 h. The siRNA-treated cells were incubated at 37 °C with A/Puerto Rico/8/34 at m.o.i. 5 under 5% CO2 for 24 h. The cells were lysed in a buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, and a cOmplete ULTRA Tablet, Mini, EDTA-free Protease Inhibitor Cocktail (Roche). The cell lysates were centrifuged at 15,000 rpm at 4 °C for 10 min, and the supernatant was collected. Western blotting analysis was performed as described above. The intensity of the bands was measured using the software for image processing and Java and ImageJ.For Western blotting analyses A, A549 cRLuc , which express Renilla luciferase and are regulated by thymidine kinase (TK) promoter as an internal control. The NP ligated into the pCA plasmid was derived from the WSN strain (H1N1), and all lysine residues for acetylation targets were conserved. The luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) at 24 h post transfection. Polymerase activity was calculated by normalizing the Firefly luciferase activity to the Renilla luciferase activity. Polymerase activity of WT was set to 100%.Procedures of culturing and siRNA treatment of cells were same as those of Western blotting analyses mentioned above. Minigenome assay based on the dual-luciferase system was performed as described previously –55. Brie–D. H. resources; D. H., M. S., S. Y., R. Y., N. O., S. T., A. S., Y. A., A. M., T. Komatsu, R. N., M. N., T. N., and T. Kuzuhara data curation; D. H., M. S., S. Y., R. Y., N. O., S. T., A. S., Y. A., A. M., T. Komatsu, R. N., M. N., T. N., Y. K., and T. Kuzuhara formal analysis; D. H. funding acquisition; D. H., M. S., S. Y., R. Y., N. O., S. T., A. S., Y. A., A. M., T. Komatsu, R. N., M. N., and T. Kuzuhara investigation; D. H. writing-original draft; D. H., T. N., Y. K., and T. Kuzuhara writing-review and editing; S. Y. methodology; M. N., Y. K., and T. Kuzuhara supervision; M. N. validation; T. Kuzuhara conceptualization; T. Kuzuhara project administration.


Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural Wiskott-Aldrich syndrome protein (N-WASp) is a scaffold protein which can directly bind to actin monomers and Arp2/3 and is a crucial player in the formation of an invadopodium precursor. Expression modulation has pointed to an important role for N-WASp in invadopodium formation but the role of its C-terminal VCA domain in this process remains unknown. In this study, we generated alpaca nanobodies against the N-WASp VCA domain and investigated if these nanobodies affect invadopodium formation. By using this approach, we were able to study functions of a selected functional/structural N-WASp protein domain in living cells, without requiring overexpression, dominant negative mutants or siRNAs which target the gene, and hence the entire protein. When expressed as intrabodies, the VCA nanobodies significantly reduced invadopodium formation in both MDA-MB-231 breast cancer and HNSCC61 head and neck squamous cancer cells. Furthermore, expression of distinct VCA Nbs (VCA Nb7 and VCA Nb14) in PC-3 prostate cancer cells resulted in reduced overall matrix degradation without affecting MMP9 secretion/activation or MT1-MMP localisation at invadopodial membranes. From these results, we conclude that we have generated nanobodies targeting N-WASp which reduce invadopodium formation and functioning, most likely via regulation of N-WASp—Arp2/3 complex interaction, indicating that this region of N-WASp plays an important role in these processes. In this process cancer cells leave a primary tumour to disseminate through the entire body . In doinin vivo . A matur[in vivo . Inside [in vivo . They co[in vivo –9.Activation of the Arp2/3 –N-WASp pathway, found to be important in invadopodium formation, has been observed in a broad spectrum of cancers . Arp2/3 in vitro that N-WASps VCA domain is needed during the formation of the invadopodium precursor [The neural variant of WASp (N-WASp) is a ubiquitously expressed member of the Wiskott-Aldrich syndrome protein (WASP) family , 13, 14.recursor , 22, 23.recursor .We generated alpaca nanobodies against the VCA domain of N-WASp (VCA Nb) to investigate the role of this region in formation of cancer cell invadopodia. Nanobodies are single-domain antibody fragments or, more specifically, the variable part of the heavy chain of Camelid heavy chain antibodies . A nanob420KKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED505-NH2, used for raising nanobodies (see further), was chemically synthesized by Caslo (Denmark).Rabbit polyclonal anti-N-WASp (H100) was obtained from Sigma  and Santa Cruz Biotechnology . Mouse monoclonal anti-actin clone C4 (0869100) was obtained from MP Biomedicals . Rabbit monoclonal anti-ARPC2 antibody (ab133315) and anti-MMP14 (ab51074) were obtained from Abcam . Rabbit monoclonal anti-MMP9 was obtained from Epitomics . Alexa Fluor-labeled secondary goat anti-rabbit or anti-mouse IgG antibodies were obtained from Molecular Probes . Mouse monoclonal anti-V5 antibody, Alexa Fluor labeled phalloidin and Mitotracker Orange were purchased from Invitrogen . The synthetic VCA—hN-WASp peptide biotin-Ahx-Nbs were obtained in collaboration with the Vlaams Instituut voor Biotechnologie (VIB) Nanobody Service Facility (NSF). All animal work was performed by the VIB NSF and has PHS approved animal welfare assurance from OLAW (F16-00131(A5593-01)). Briefly, two alpacas were immunized by a subcutaneous injection of human VCA-N-WASp peptide on days 0, 7, 14, 21, 28 and 35. On day 39, anticoagulated blood was collected for lymphocyte preparation. A nanobody library was constructed by extracting total RNA from peripheral blood lymphocytes and screened for the presence of antigen-specific nanobodies. The obtained Nbs were subcloned into the phagemid vector pMECS. In order to isolate N-WASp nanobodies, several rounds of phage panning were performed. These experiments suggested that the phage population was enriched for antigen-specific phages from round 3 panning onwards. In total, 380 colonies from 3rd and 4th rounds were randomly selected and analyzed by ELISA for the presence of antigen-specific nanobodies in their periplasmic extracts. The positives ones were sequenced. Based on those sequence data, the positive colonies represented 5 different groups of nanobodies in each of the two alpacas.6 and pEGFP-N1 vectors , as described before [6 vector by using the following primers: forward 5’-TCGATTCTACGCGTACCGGTGCCCAGGTGCAGCTGCAGGAG-3’ and reverse 5’-GGTGATGATGACCGGCTAGCTGGAGACGGTGACCTG-3’. Cloning of the Nbs in the pEGFP-N1 vector was done using the following primers: forward 5’-CGAGCTCAAGCTTCGGCCACCATGCAGGTGCAGCTGCAGGAG-3’ and reverse 5’-GGCGACCGGTGGATCCTTGCTGGAGACGGTGACCTG-3’.Nbs were cloned into mitochondrial outer membrane (MOM) V5 pcDNA3.1 Hisd before –28. The 5’-TGGAGAAGGATCCGCGGCCGCGCCACCATGGCCCAGGTGCAGCTGCAGGAGTCTGGG-3’ and reverse 5’-CTACCCGGTAGAATTCTTACTTGTACAGCTCGTCCATGCC-3’.To obtain inducible Nb expressing cells, a third cloning of the EGFP-tagged VCA Nbs into the pLVX-TP vector (Clontech) was done using following primers: Forward BL21 cells were transformed with VCA Nbs in pMECS vector or N-WASp in pTrcHis TOPO. They were grown in TB (with 100 μg/ml ampicillin) at 37°C. After induction with 1 mM IPTG, incubation was performed at 28°C (overnight for nanobody production or 4 h for N-WASp production).Nanobodies were extracted from the periplasm, where they are located due to an N-terminal PelB signal, by means of an osmotic burst of the outer membrane using TES buffer . Next an IMAC and/or gel filtration was performed for a higher purification, as described before .To extract recombinant N-WASp, pelleted cells were dissolved in lysis buffer  supplemented with 200 μg/ml lysozyme and incubated during 30 min at room temperature. After sonication and pelleting, IMAC purification was performed.2 incubator, HNSCC61 cells were grown at 5% CO2. All cells (except PC-3) were grown in DMEM. We obtained the HNSCC61 cells from Prof. Dr. Weaver [MDA-MB-231 (ATCC HTB-26), PC-3 (ATCC CRL-1435) and HEK293T (ATCC CRL-11268) cells were maintained at 37°C in a humidified 10% CO. Weaver   was used to perform transient expression according to the manufacturer’s protocol. For virus production HEK293T cells were transfected using the calcium phosphate method as described before . InducibCells were lysed with ice-cold lysis buffer  . ProteinIn the pull down assays to analyse binding between N-WASp and the 10 VCA Nbs, recombinant HA-tagged nanobodies were immobilized onto HA-agarose beads  (1.5 h at 4°C). Next, 650 μg crude cell lysate was added and followed by incubation (1.5 h at 4°C).In the pull down to analyse binding between N-WASp and VCA Nbs using EGFP-tagged VCA Nb expressing cancer cells, we performed overnight incubation of cell lysate with anti-GFP antibody (at 4°C), followed by an incubation with Protein G Sepharose beads  .In the binding assay, recombinant HA-tagged VCA Nbs, in a concentration range, were incubated with the synthetic biotin-tagged VCA domain (1.5 h at 4°C). Next, 550 μg crude MDA-MB-231 cell lysate was added, as source for Arp2/3 and actin, and an incubation was followed (1.5 h at 4°C). Last, an immobilization on STREPTactin beads  was performed (1h at 4°C).After washing steps, elution of the beads was obtained by boiling for 5 minutes in Laemmli SDS sample buffer . This was followed by SDS-PAGE and Western blot analysis. The H100 N-WASp antibody from Santa Cruz Biotechnology was used to detect N-WASp on blot.ITC was performed as described before –29. BindCells were seeded on a 100 μg/ml gelatin  or 50 μg/ml rat tail type I collagen  coated coverslip. Cells were fixed using 3% paraformaldehyde, permeabilized using 0.1% Triton for 5 min and neutralized in 0.75% glycin for 20 min. Next, a blocking step was performed using 1% bovine serum albumin (Sigma-Aldrich), followed by an incubation with primary antibodies (1 h at 37°C) and Alexa Fluor-conjugated secondary antibodies (30 min at room temperature). DAPI  and Alexa Fluor labeled phalloidin (Invitrogen) were used to stain nuclei and actin filaments, respectively. Cells were mounted using VectaShield . For imaging, a Zeiss Axiovert 200 M Apotome epifluorescence microscope equipped with a cooled CCD Axiocam camera  and Axiovision 4.5 software (Zeiss) was used at room temperature.In matrix degradation assays, coverslips were coated with Cy3-labeled gelatin according to the manufacturer’s protocol . 24h after induction with doxycycline, cells were seeded and allowed to degrade the matrix for another 24h. Degradation is found on the places where black holes were present in the matrix underneath the cells. Two parameters were used to analyze the degradation. The parameter ‘degraded area per cell area’ was obtained by dividing the total degraded area by the total cell area per picture. The second parameter ‘degradation area per cell’ is determined by the total degradation area divided by the amount of cells per picture.2, pH 7.5). Band intensities were analyzed with ImageJ.24 h after induction with 500 ng/ml doxycycline, cells were seeded in a 1 mg/ml collagen matrix which was allowed to polymerize for 1 h at 37°C. After approximately 20 h incubation in serum-free medium, equal amounts of conditioned medium proteins were analyzed on 10% SDS-PAGE without (for secretion levels) or with 0.1% gelatin (for activity). In the SDS-PAGE containing 0.1% gelatin, proteins were enabled to renature by removing SDS with 2% Triton X-100 washing buffer. To analyse the activity, digestion was allowed during an overnight incubation in MMP buffer  was performed with ImageJ . Statistical analysis was performed with GraphPad Prism  using Kruskal-Wallis test and Dunns post tests with p = 0.05. Bar plots were made using mean and SEM.in vitro, recombinant HA-tagged nanobodies were used in a pull down assay. MDA-MB-231 breast cancer cell lysate was used as a source of endogenous N-WASp. Seven nanobodies out of 10 bound with the endogenous N-WASp , lymphocyte collection and library construction. Based on their amino acid sequence, 10 different groups could be discerned. One nanobody was chosen from each group for biochemical characterisation. To detect antigen-nanobody binding s N-WASp .in vitro  delocalisation assay. In this experiment, the nanobodies were equipped with a MOM- and V5-tag at their N-terminus. The MOM-tag originates from yeast TOM70  and rediin vitro  are alsoThe mitochondrial patterns of the MOM-tagged VCA Nb13 and VCA Nb14 show a dissimilar pattern compared to the control and the other VCA Nb conditions (VCA Nb2 and VCA Nb7) . These pd values were determined at 0.699 ± 0.154 μM for VCA Nb2, 0.820 ± 0.201 μM for VCA Nb7, 0.685 ± 0.133 μM for VCA Nb13 and 1.597 ± 0.249 μM for VCA Nb14  was performed at physiological pH by using recombinant VCA Nbs . As thesVCA Nb14 . These rVCA Nb14 .N-WASps VCA domain can directly interact with actin monomers via its V region and with Arp2/3 via its CA regions. To analyse if the VCA Nbs are able to interfere with the binding between N-WASp and either actin or Arp2/3, a pull down assay was performed by adding a concentration range of VCA Nb2, 7, 13 or 14 together with biotinylated VCA peptide and STREPTactin beads. As an Arp2/3 and actin source, we used MDA-MB-231 cell lysate . Via WesBefore a fully mature invadopodium can be formed, an invadopodium precursor has to be created. N-WASp plays an important role in the latter –10. The To analyse if the VCA Nbs could influence invadopodium formation in different cancer cells, lentiviral, stable doxycycline-inducible MDA-MB-231 breast and HNSCC61 head and neck squamous cancer cell lines were made in which EGFP-tagged VCA Nbs are expressed as intrabodies. As a control, an EGFP-only expressing stable cell line was made. After nanobody induction overnight, the cells were fixed with paraformaldehyde and stained with phalloidin. When VCA Nb2, 7, 13 and 14 were expressed a significant reduction in invadopodia numbers was observed in both MDA-MB-231 and HNSCC61 cancer cells .A conspicuous and important link to metastasis is the proteolytic activity of a mature invadopodium . Once thMatrix remodelling is caused by proteolytic activity of invadopodia , 8. The In this study, nanobodies were generated against the VCA domain of N-WASp, an actin and Arp2/3 interaction partner. Out of all binders, four VCA nanobodies  were selected and expressed in 3 different cancer cell lines. Pull down experiments and immunofluorescence revealed their ability to bind endogenous N-WASp. Their submicromolar affinity for the VCA peptide was determined with ITC. Previous studies found actin binding affinities to the VCA domain of WASP family proteins ranging from 50 to 250 nM, also using ITC , 44. TheN-WASp participates in the formation of an invadopodium precursor which activates the actin polymerisation process. It was previously demonstrated that N-WASp is a key player in invadopodium genesis . The canAfter invadopodium maturation, matrix remodelling will follow. Our results revealed that VCA Nb7 and 14 were able to significantly reduce the overall degradation . Presumaet al. concluded that N-WASp plays a role in matrix degradation by optimising the organisation and positioning of MT1-MMP, the master switch of other MMPs. At the cytoplasmic tail of MT1-MMP, N-WASp creates an actin network to specifically stabilize MT1-MMP to increase the effectiveness of degradation [The properties of N-WASp in this pathway may also be related to the transportation of vesicles , 42, 53.radation . Moreoveet al. succeeded in eradicating endogenous proteins via the proteasome degradation pathway using nanobodies and monobodies [et al. very recently demonstrated that CRISPR/Cas9 genome editing can trigger hundreds of unintended mutations and deletions in the genome [Recently, Fulcher nobodies . If thisnobodies . In thise genome . It is tIn conclusion, nanobodies against the VCA domain of N-WASp affect invadopodium formation and overall matrix degradation, most likely via regulation of N-WASp—Arp2/3 complex interaction, indicating that this region of N-WASp critically contributes to these processes.S1 FigThe EGFP-tagged VCA Nbs were pulled down using a GFP Ab and Protein G Sepharose beads. N-WASp is detected by N-WASp antibody and the VCA-Nbs by GFP Ab.(TIF)Click here for additional data file.S2 FigRepresentative epifluorescence images showing the mitochondrial patterns indicating that the MOM-tag directs the nanobody to the mitochondrial outer membrane (compare with the Mitotracker channel). MOM-tagged EGFP nanobody was used as a negative control (upper panel). Nuclei were visualized with DAPI (blue), nanobodies with anti-V5 antibody (green) and the mitochondria with Mitotracker Orange (red). .(TIF)Click here for additional data file.S3 FigRepresentative epifluorescence images showing the mitochondrial patterns. Nuclei were visualized with DAPI (blue) and the mitochondria with Mitotracker Orange (red). .(TIF)Click here for additional data file.S4 FigITC profiles of recombinant HA-tagged VCA Nbs with synthetic VCA peptide of human N-WASp. VCA peptide was titrated once with VCA Nb7 and once with VCA Nb14. VCA Nb2 and VCA Nb13 were titrated with VCA peptide. The upper panel shows the raw data of heat release in function of time, while the lower panel shows the fitted binding curve of total heat release per injection as a function of the molar ratio.(TIF)Click here for additional data file.S5 Fig(A) Quantification of MMP9 levels in medium was determined using ImageJ after SDS-PAGE and Western blotting. As control the uninduced cell line was used. (B) Activity was obtained after digestion in 0.1% gelatin gel. Quantification was performed using ImageJ and a Kruskal-Wallis and Dunns post tests were performed. The bars represent mean and SEM (n = 3). (ns = not significant).(TIF)Click here for additional data file.S6 FigMT1-MMP containing invadopodia were counted when MT1-MMP dots were overlapping with F-actin dots in HNSCC61 cells, in which VCA Nbs expression could be induced. The number of MT1-MMP containing invadopodia was divided by the total amount of invadopodia for each cell and Kruskal-Wallis and Dunns post tests were used. The bar plot represents mean and SEM (n = 3). (ns = not significant).(TIF)Click here for additional data file.


Clostridium botulinum group III-producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of the first five strains of Clostridium botulinum type D/C isolated in Brazil and used for vaccination purposes.Animal botulism is mainly associated with  Clostridium botulinum strains that produce type C and D toxins, or a chimeric fusion of C and D termed C/D or D/C toxins , producing 105 to 190 contigs  medium, using the DNeasy blood and tissue kit , according to the manufacturer’s instructions for Gram-positive bacteria. Libraries were prepared using the Nextera XT kit (Illumina). Whole-genome sequencing was performed using an Illumina MiSeq platform (Illumina), according to the manufacturer’s instructions. Five MiSeq runs were carried out, three with paired-end 150-nucleotide (nt) reads on MiSeq version 2 nano, one with version 2 micro, and the last with version 2 standard chemistry. The raw reads were trimmed  and assembled in CLC Genomics Workbench 8.0.2 by  contigs . The medd 47 kbp . The seqC. botulinum group III genomes, as defined previously (The average size of the genomes in this study is 2.94 Mb, with 2.57 Mb being the smallest genome size, an average G+C content of 28.1% (isolate 1277, Clostridium botulinum group III strains were deposited in DDBJ/ENA/GenBank under the accession numbers listed in The annotated draft whole-genome sequences of these


One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 , which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells.The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γIMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity achieved by tropism retargeting to selected cancer receptors. However, the retargeted oncolytic HSVs strictly depend on cancer receptors for infection. Here, we devised a strategy for in vitro cultivation of retargeted HSVs in non-cancer cells. The strategy envisions a double-retargeting approach: one retargeting is via gD to the cancer receptor, and the second retargeting is via gH to an artificial receptor expressed in Vero cells. The double-retargeted HSV uses alternatively the two receptors to infect cancer cells or producer cells. A universal non-cancer cell line for growth of clinical-grade retargeted HSVs represents a step forward in the translational phase. GM-CSF or T-VEC . ONCO-VEXGM-CSF represents the prototypic oncolytic HSV and is characterized by a combination of attenuation and cancer specificity, and hence by a high safety profile. The attenuation was achieved through the deletion of the virulence genes γ134.5. The further deletion of the α47 gene, designed to enable a better presentation of antigenic peptides by major histocompatibility class II peptides, placed the US11 gene under the control of an immediate-early promoter (134.5 genes and an intact α47 gene (GM-CSF to replicate in some cancer genotypes (GM-CSF encodes granulocyte-macrophage colony-stimulating factor (GM-CSF), which boosts activation of monocytes, antigen-presenting cells, and macrophages. ONCO-VEXGM-CSF and all of the Δγ134.5 attenuated oncolytic HSVs gain their cancer specificity, and hence their safety profile, from the fact that most cancer cells are defective in mounting innate immune responses against the viruses with these deletions.There is an intense interest in viruses as therapeutic agents against cancer; these are termed oncolytic viruses, and there is an even greater interest in oncolytic viruses that can be administered in combination with immunotherapeutic compounds or that encode immunotherapeutic molecules –4. The l–5–or T-VEC , went thor T-VEC  and was or T-VEC . It has α47 gene . The attAn alternative strategy to achieve cancer specificity consists of retargeting the virus tropism to cancer-specific receptors, coupled with detargeting from the natural receptors –19. Thes–21–The HER2-retargeted virus stocks employed in the preclinical studies consisted of viruses grown in the HER2-positive SK-OV-3 ovarian tumor cell line. Inasmuch as cancer cells may not be approvable for the growth of clinical-grade oncolytic HSVs (o-HSVs) and the retargeted HSVs require the HER2 oncogene to infect cells, we reasoned that even a non-cancer cell line, transgenically expressing HER2, might not be considered approvable by the regulatory agencies.ad hoc cell line. Briefly, the double retargeting takes advantage of the fact that not only gD but also gH are suitable tools for retargeting  are described in reference 1P4B), and (iii) the scFv bound the GCN4 peptide with very high affinity (20 pM). The GCN4 peptide has the sequence GSKNYHLENEVARLKKLVGS. The core amino acids (YHLENEVARLKK) (We engineered the 20-aa-long peptide named GCN4 in the gH of R-LM113 . This peEVARLKK) . The corEVARLKK) . These mTo engineer R-213, the oligonucleotide encoding the GCN4 peptide and the GS linkers were engineered in the gH of the bacterial artificial chromosome (BAC) LM113, between aa 23 and 24 . The recTo generate the Vero-GCN4R cell line, the scFv derived from a monoclonal antibody that binds the GCN4 peptide with 20 pM affinity  was engiwt-Vero cells were transfected with the scFv GCN4-nectin plasmid, selected for resistance to 400 μg/ml hygromycin, enriched by use of microbeads, and then cloned by limiting dilution. The cell surface expression of the artificial GCN4R was detected in an indirect immunofluorescence assay (IFA) with the HA tag . To evalTo verify whether R-213 exhibits the expected dual tropism for GCN4R and HER2, we assayed its ability to infect the Vero-GCN4R cells, its wt-Vero counterpart, the HER2-positive SK-OV-3 cancer cells, and the J-HER2 cells. J-cells express no receptor for HSV and cannot be infected by wt HSV . J-nectiA key feature for the employment of Vero-GCN4R cells for virus production is the extent of R-213 replication. Vero-GCN4R cells were infected with R-213 and also with R-LM5 for comparison. R-LM5 carries a wt gD  and doesNext, we analyzed effects on cell-to-cell spread capability in Vero-GCN4R cells, as measured in a plaque assay. Replicate aliquots of each virus were plated on wt-Vero, Vero-GCN4R, and SK-OV-3 cells. R-213, but not R-LM113 or R-LM5, exhibited a much higher plating efficiency in Vero-GCN4R cells than in SK-OV-3 cells ; the avein vitro. Here, we compared the ability of R-213 to that of R-LM113 to kill SK-OV-3 cells, as measured in an alamarBlue reduction assay. Vero-GCN4R cells were included for comparison. SK-OV-3 or Vero-GCN4R cells were infected with R-213, R-LM113, R-LM5, and HSV-1 (strain F) at the indicated multiplicity of infection (MOI). alamarBlue was added at the indicated days after infection. The killing ability was determined as the percent reduction in the viability of the infected cell samples, relative to viability of uninfected cells. A key property for any candidate oncolytic HSV is its ability to kill cancer cells In earlier work, we and others showed that it is possible to readdress HSV tropism by means of deletions in gD which remove critical residues for interaction with HVEM or nectin-1 and the replacement of deleted sequences with a ligand to a cancer receptor of choice –19. Rece–We successfully retargeted HSV tropism by insertion of a 20-aa-long peptide, made up of a 12-aa-long epitope plus bracketing residues and GS linkers. The scFv that forms the N terminus of the artificial GCN4R was derived from a monoclonal antibody that binds the epitope at very high affinity (20 pM) . The lenHSV can infect cells that express either GCN4R or HER2 as the sole receptor. The novel finding to emerge is that R-213 makes alternative use of the two receptors as portals of entry. Taking into account that the interaction with HER2 is via gD and the interaction with GCN4 receptor is via gH, the determinants of the viral tropism are alternatively gD or gH, depending on the specific receptor that the virus uses to infect each specific cell. This property opens the way to engineer oncolytic HSVs that are simultaneously retargeted to two different receptors of choice.in vitro growth of clinical-grade retargeted HSV in non-cancer cells.A comparison of the extent of growth and cell-cell spread caused by R-213 versus R-LM113 in SK-OV-3 cells showed that R-213 was not hampered relative to R-LM113, indicating that the addition of the short GCN4 peptide in gH was not deleterious to gH functions. A comparison of R-213 growth and spread in Vero-GCN4R cells relative to that SK-OV-3 cells highlighted a somewhat lower titer in Vero-GCN4R cells, as expected, given that SK-OV-3 are cancer cells. Altogether, the Vero-GCN4R cell line exhibits promising properties for the The GCN4 receptor is an artificial receptor that we generated by fusion of an scFv to the GCN4 peptide with domains II and III, TM, and the C tail of nectin-1. Nectin-1 does not play any role in GCN4R-mediated entry, for two reasons. First, it is well established that the only nectin-1 domain that mediates virus entry and interacts with gD is domain I –34; henc–The strategy employed to design the Vero-GCN4R cell line differs from previous methods used to generate producer cell lines for retargeted adenovirus and measles virus , 36. TheThe Vero-GCN4R cell line and gH retargeting via the GCN4 peptide can be applied to oncolytic HSVs retargeted not only to HER2 but also virtually to any cancer receptor of choice. Thus, the Vero-GCN4R cell line may be a universal cell line for cultivation of retargeted oncolytic HSVs.The capacity of a retargeted HSV to use alternative receptors of choice via gD or via gH has made it possible to grow the retargeted oncolytic HSV in non-cancer cells. A few properties of these cells are worth noting.The availability of a universal, non-cancer cell line for growth of clinical-grade retargeted oncolytic HSVs represents a step forward in the translational phase of these viruses.1P4B) (in vitro and cloned in BamHI and XhoI restriction sites of a pcDNA3.1/Hygro(+) vector by using GeneArt (Thermo Fisher Scientific).To generate the chimeric receptor specific for the GCN4 peptide, a single-chain antibody specific for the peptide (Protein Data Bank accession number 1P4B)  was fuseVero cells (ATCC CCL-81) were transfected with scFv GCN4-nectin by means of Lipofectamine 2000 (Thermo Fisher Scientific) and selected for resistance to hygromycin B (400 μg/ml for 7 days). The selected cells were enriched with microbeads (Miltenyi Biotech) following incubation with an anti-HA antibody (Sigma-Aldrich Corporation) diluted 1:100 and with secondary anti-mouse antibodies (Jackson Laboratories). For the IFA, cells were reacted with an anti-HA antibody (Sigma-Aldrich Corporation) diluted 1:100 and secondary anti-mouse antibodies (Jackson Laboratories). Single-cell clones were obtained by limiting dilution. Clones were checked for stability for up to 40 passages in cell culture by flow cytometry (BD Accuri).The following recombinant viruses were previously described , and the1P4B)  . In R-211P4B) .galK recombineering, in two steps . In the second step, the galK insert was replaced with the GCN4 peptide. The resulting recombinant BAC DNA was then transfected in Vero-GCN4R cells, for virus reconstitution. In detail, to carry out the first step, the galK cassette, with homology arms to gH, was amplified by means of primers gH6_galK_f, ATGCGGTCCATGCCCAGGCCATCCAAAAACCATGGGTCTGTCTGCTCAGTCCTGTTGACAATTAATCATCGGCA, and gH5_galK_r, TCGTGGGGGTTATTATTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGGTCAGCACTGTCCTGCTCCTT, using plasmid pgalK as the template. The PCR-amplified galK cassette was then electroporated into SW102 bacteria, which carry the BAC LM113, to generate BAC 212. To exclude galK false-positive colonies, the recombinant clones were plated on MacConkey agar base plates supplemented with 1% galactose and 12 μg/ml chloramphenicol and checked by colony PCR with primer galK_129_f, ACAATCTCTGTTTGCCAACGCATTTGG, and galK_417_r, CATTGCCGCTGATCACCATGTCCACGC. To carry out the second step, a cassette encoding the GCN4 peptide (GenBank accession number AF416613.1), bracketed by the downstream and upstream Gly-Ser linkers and by homology arms to gH, was generated through annealing and extension of the partially overlapping oligonucleotides GCN4gH_23_42_fB, TCGTGGGGGTTATTATTTTGGGCGTTGCGTGGGGTCAGGTCCACGACTGGGGATCCAAGAACTACCACCTGGAGAACGAGGTGGCCAGACTGAAGAAGCTGGTGGGCAGC, and GCN4gH_23_24_rB, ATGCGGTCCATGCCCAGGCCATCCAAAAACCATGGGTCTGTCTGCTCAGTGCTGCCCACCAGCTTCTTCAGTCTGGCCACCTCGTTCTCCAGGTGGTAGTTCTTGGATCC. The oligonucleotides contained a silent BamHI restriction site, for screening purposes. The amplimer encoding the GCN4 cassette, with homology arms to gH, was electroporated into SW102 bacteria carrying BAC 212. The recombinant BAC was named BAC 213. Positive bacterial clones were checked by means of BamHI restriction analysis on colony PCR fragments amplified with primers gH_ext_r, GTTTCTTCCTTTTCCCCACCCCACCCC, and gH_2176_2200_f, CAGGTAGGTCTTCGGGATGTAAAGC. BAC 213 DNA (500 ng) was then transfected into Vero-GCN4R cells by use of Lipofectamine 2000 (Life Technologies) in order to reconstitute the R-213 recombinant virus. Virus growth was monitored by fluorescence of the EGFP marker, cloned within the BAC sequences. The recombinant gH of R-213 was checked by sequencing. Viral stocks were generated and titrated in Vero-GCN4R and SK-OV-3 cells.The starting material for the engineering of R-213 was BAC LM113, . The engwo steps , 39. In The indicated cells were infected with R-213 or R-LM113, at 1 PFU/cell, and monitored 24 h later with a Nikon Eclipse TS100 fluorescence microscope. Infection was carried out in the presence of monoclonal antibody (MAb) to HER2 (trastuzumab) at a concentration of 28 μg/ml, where indicated.Vero-GCN4R and SK-OV-3 cells were infected with R-LM5, R-LM113, or R-213 at 0.1 PFU/cell. Unabsorbed virus was inactivated by rinsing the cells with a pH 3 solution . Replicate cultures were frozen at 24 and 48 h after infection. Progeny virus (intracellular plus extracellular) was titrated in SK-OV-3 cells. Results are expressed as the mean findings of three independent experiments ± the standard deviation (SD).In virus release experiments, replicate cultures of Vero-GCN4R or SK-OV-3 cells infected with R-LM5, R-LM113, or R-213 at 0.1 PFU/ml were harvested 48 h after infection as cell lysates plus medium. Alternatively, medium and cell-associated fractions were harvested separately. Progeny virus was titrated in SK-OV-3 cells. Results are expressed as the mean findings of three independent experiments ± SD.Replicate aliquots of R-LM5, R-LM113, or R-213 were plated on wt-Vero, Vero-GCN4R, and SK-OV-3 cells, and the number of plaques was counted 3 days later. Results represent the mean findings of three independent infections ± SD. For plaque size determination, pictures of 5 individual plaques from each of the above samples were taken 3 days after infection. Plaque areas were measured with Nis Elements-Imaging software (Nikon). Each result represents mean areas ± SD.3 cell/well) and infected with R-LM5, R-LM113, or R-213 (6 or 3 PFU/cell) or mock infected. alamarBlue  was added to the culture medium at the indicated times after infection and cultures were incubated for 4 h at 37°C. Plates were read at 560 and 600 nm with GloMax Discover system (Promega Corporation). For each time point, cell viability was expressed as the percentage of alamarBlue reduction in infected versus uninfected cells, after subtraction of the background value . Each point represents the average results of at least triplicate samples ± SD.SK-OV-3 and Vero-GCN4R cells were seeded in 96-well plates (8 × 10


Either RAGE-blocking antibody or Sp1-knockdown can partially block the AGEs-induced effects. Moreover, AGEs increased the phosphorylation of ERK, and reducing the phosphorylation level of ERK by MEK1/2 inhibitor decreased the expression of Sp1. These results indicate that accumulation of glucose-derived AGEs may act as one of potential risk factors for GC progression and promote the invasion and metastasis of gastric cancer partially through the activation of RAGE/ERK/Sp1/MMP2 pathway.Advanced glycation end products (AGEs) have been reported to take part in many cancer processes. Whether AGEs contribute to gastric cancer (GC) course and the underlying mechanism are still unclear. Here, glucose-derived AGEs are detected to be accumulated in tumor tissues and blood of patients with GC. As the receptor for AGEs, RAGE is highly expressed in cancer tissues, and closely associated with the depth of cancer invasion, lymph node metastasis and TNM stage. Both  Both morbidity and mortality of gastric cancer (GC) are very high among all the cancers worldwide. Many pathological factors are correlated with the development and advance of GC, such as Helicobacter pylori infection, smoking, and inflammation . The ideAs products of a non-enzymatic reaction, where lysine residues are glycation, advance glycation end products (AGEs) are implicated in progressions of age-related diseases, diabetic complications, as well as cancer course -4. CrossAs the member of Sp family, Specificity Protein 1 (Sp1) is overexpressed in a variety of cancers, such as gastric cancer, pancreatic cancer, and prostate cancer, and is closely associated with poor prognoses of cancers. By regulating the expression of effector molecules including SREBP-1c, VEGF, MMP2, Sp1 plays pivotal roles in cancer cell metabolism, proliferation, and metastasis . HoweverHere, we design a series of experiments to investigate the role of glucose-derived AGEs on the invasion and migration of gastric cancer. AGEs were found to be accumulated in blood of GC patients and gastric cancer tissues. AGEs-RAGE signaling cascade induced the cancer cell invasion and migration partially through increasing expressions of RAGE, Sp1, and MMP2. Our results uncover glucose-derived AGEs as a new risk factor for gastric cancer, which may help us improve the progression and prognosis of gastric cancer.p=0.025) and TNM stage (p=0.004), but not to other factors  is the second most common cause of cancer-related death worldwide , 15. Howin vivo [As now well known, AGEs abnormally accumulate in diabetic patients with six subtypes, including glucose-derived AGEs (AGE-1), glyceraldehyde-derived AGEs AGE-2), glycolaldehyde-derived AGEs (AGE-3), methylglyoxal-derived AGEs AGE-4), glyoxal-derived AGEs (AGE-5), and 3-deoxyglucosone-derived AGEs (AGE-6), which play important roles in diabetic complications , 16. Lik, glyoxal, glycolaThe receptor for advanced glycation end products, popularly known as RAGE, were also indicated to be overexpressed in many cancers including hepatocellular, breast cribriform, colorectal, gastric carcinomas and so on . MoreoveSp1 was detected to be over-expressed in many cancers, such as gastric, breast, pancreatic, glioma, and thyroid cancers, and is closely associated with the stage, invasive potential and metastasis . HoweverThe primary antibodies for RAGE, MMP2 were obtained from Abcam and antibodies against Sp1, AKT, P-AKT, ERK, p-ERK, JNK, p-JNK, p38, p-p38, Alexa Fluor®488 and Alexa Fluor®594 were purchased from Cell signaling technology. DAPI was purchased from Thermal Fisher Scientific (USA). MEK1/2 inhibitor, U0126, was purchased from Selleckchem .160 sets of gastirc tumors and paired non-tumorous tissues (more than 5 cm away from primary tumor margin) were obtained from patients who underwent curative surgery in Ruijin Hospital affiliated to Shanghai Jiaotong University School of Medicine from 2009 to 2014. The patients didn’t receive any preoperative radiotherapy or chemotherapy before surgery. The pathological stage of tumor was evaluated strictly according to the criteria of UICC TNM classification. Two pathologists independently determined the histological type of tumor in a double–blind way. The blood samples of patients were collected with tubes containing EDTA before surgery. Serum was obtained and preserved at -80°C after blood was centrifuged at 3000 rpm, 4°C, for 5 minutes. The level of serum glucose-derived AGEs was detected with glucose-derived AGEs specific antibody  by Western blot. All of the related studies were carried out according to the Code of ethics of the World Medical Association (Declaration of Helsinki) and were approved by Medical Ethics Committee of Shanghai Ruijin Hospital.RNA extraction of tissue samples and SGC7901 cells and qRT-PCR analysis were carried out as previously described . The forThe protein lysates of cells or tissue specimens and Western blotting were performed as previously described .Immunohistochemistry was performed as previously described .The gastric cancer cell line, SGC7901, was kindly provided by Shanghai Digestive Surgery Institute. SGC7901 was cultured in RPMI-1640 with 10% FBS under 37°C, 5% CO2 and saturation humidity. When treated with 100 μg/ml glucose-derived AGEs for 2 days, SGC7901 cells were culture in RPMI-1640 with 2.5% FBS. Glucose-derived AGEs were prepared and purified as previously described . And 5 μ2000 cells were seeded at every well in 96-well plate for 24 hours and were designed with six copies each group. After incubated with serum-poor medium (SPM) supplemented with 2.5% FBS in the presence of 100 μg/ml AGEs or non-modified BSA for different times, cell viability was measured with CCK8 . After incubation with CCK8 at 37°C for two hours, OD was assessed at 490 nm using spectrophotometer .In order to stably knock-down RAGE expression of SGC7901 cells, lentiviral expression vectors containing RAGE-specific shRNAs and green fluorescent protein (GFP) were constructed by GeneChem . Briefly, SGC7901 cells were seeded on 6-well plates. 24 hours later, cells were transfected with lentiviral expression vectors according to manufacturer’s instructions . For selection of stable clones, the infected cells were treated with 5 μg/ml puromycin for 7 weeks. The interference efficiency was detected by Western bolt analysis. The base sequences of RAGE shRNA were as follows: S1 forward: 5′-CCGGGTGCCAGGCAATGAACAGGAACTCGAGTTCCTGTTCATTGCCTGGCACTTTTTG-3′, S1 reverse: 5′-AATTCAAAAAGTGCCAGGCAATGAACAGGAACTCGAGTTCCTGTTCATTGCCTGGCAC-3′; S2 forward: 5′-CCGGCACCTGACACATCTTGCAAAACTCGAGTTTTGCAAGATGTGTCAGGTGTTTTTG-3′, S2 reverse: 5′-AATTCAAAAACACCTGACACATCTTGCAAAACTCGAGTTTTGCAAGATGTGTCAGGTG-3′.In order to assess the ability of invasion and migration, transwell migration and invasion assay were carried out as previously described .2000 cells were plated on every well of Millicell® EZ slides  and cultured in complete medium for 24 hours. After treated with or without 100 μg/ml AGEs for 2 days, cells were rinsed with 1×PBS for twice, fixed using 4% paraformaldehyde for 10 minutes at room temperature (30 min for human GC tissues) and then increased permeability of cells using 0.1% Triton X-100 for 5 minutes at room temperature. Incubation of cells or GC tissues with primary antibody overnight at 4°C and subsequently with fluorescent secondary antibody for 1 hour at room temperature were performed in turns. Finally, the slides were washed with 1×PBS for 3 times, subsequently mounted with Prolong Gold with DAPI  and imaged on fluorescence microscope .Sp1 siRNA were obtained from Obio Technology Co., Ltd . The siRNA transfection was carried out using lipofectamine 2000 according to manufacturer’s protocol. The control siRNA sequence was 5′-CCAACAGAUUAUCACAAAUDTDT-3′. The Sp1 siRNA sequence was 5′-AUUUGUGAUAAUCUGUUGGTDTD-3′.To assess the matrix metalloproteinase (MMP) activities, the medium used for cell invasion was collected and centrifuged at 1000 rpm, 4°C for 5 minutes. Equivalent volume supernatant was mixed with non-denatured gel sample loading buffer and subjected to electrophoresis on 10% SDS-PAGE gels containing 1mg/ml gelatin . The gels were washed with the Novex zymogram renaturing buffer  for 30 min at room temperature and then incubated in Novex zymogram developing buffer  at 37°C overnight. The Coomassie brilliant blue staining, photographed and quantified by densitometry with Image J were performed in turns.The peritoneal metastasis models of gastric cancer were established in four-week-old male BALB/C nude mice  as previously described . Three dP<0.05 and P<0.01 were respectively considered as statistically significant.All of experiments were done in triplicate. Data were expressed as mean±SD and analyzed by SAS 8.0 statistical software. Correlations between the level of RAGE expression in GC tissues and clinicopathological parameters were analyzed by chi-square or Fisher’s exact tests. Student’s T test was used to analyze the statistical differences between two groups.


T cell-mediated alloresponses were correlated with genetic markers such as HLA-C mRNA expression and the number of amino acid (aa) MMs in the α1/α2 domains (peptide-binding region). Because of the high rate of HLA-DPB1 incompatibilities in HLA-A-, B-, C-, DRB1-, and DQB1-matched unrelated HSCT patient/donor pairs, the impact of HLA-DPB1 mismatching, a potential bystander of CD4+ T cell activation, was also considered. Heterogeneous alloresponses were measured in 63 HLA-C-mismatched pairs with a positive assay in 52% of the combinations (2.3–18.6% activated CTLs), representing 24 different HLA-A~B~DRB1~DQB1 haplotypes. There was no correlation between measured alloresponses and mRNA expression of the mismatched HLA-C alleles. The HLA-C*03:03/03:04 MM did not induce any positive alloresponse in five MLRs. We also identified HLA-C*02:02 and HLA-C*06:02 as mismatched alleles with lower immunogenicity, and HLA-C*14:02 as a more immunogenic MM. A difference of at least 10 aa residues known to impact peptide/T cell receptor (TCR) binding and a bystander HLA-DPB1 incompatibility had a significant impact on CTL alloreactivity (p = 0.021). The same HLA-C MM, when recognized by two different responders with the same HLA haplotypes, was recognized differently, emphasizing the role of the T-cell repertoire of responding cells. In conclusion, mismatched HLA-C alleles differing by 10 or more aas in the peptide/TCR-binding region, when occurring together with HLA-DPB1 incompatibilities, should be considered as high-risk MMs in unrelated HSCT.HLA-C locus mismatches (MMs) are the most frequent class I disparities in unrelated hematopoietic stem cell transplantation (HSCT) and have a detrimental impact on clinical outcome. Recently, a few retrospective clinical studies have reported some variability in the immunogenicity of HLA-C incompatibilities. To get better insight into presumably permissive HLA-C MMs, we have developed a one-way  Several studies have recently reported variability in the expression of different HLA-C serotypes, as determined at the cell surface or mRNA steady-state levels (rs9264942) upstream of the HLA-C locus has been reported to correlate with the level of HLA-C expression and with the control of HIV viremia , immunogenicity of HLA-C mismatches (MMs) has been reported to be affected by specific amino acid (aa) residues in the peptide-binding region (PBR). In particular, HLA-C MM involving residue 116 has been reported to be associated with higher risk of aGVHD and mortality –14.in vitro CTLp frequency to single HLA-C MMs. CTLp outcome was reported to correlate both with the expression of the donor and of the recipient mismatched HLA-C antigens  values of anti-HLA-C DT9 antibody binding determined in 200 African-American and in 50 European-American – mostly heterozygous – donors  comparedantigens .Besides HLA-C MMs, HLA-DPB1 MMs are the most frequent disparities in unrelated HSCT with detrimental effects on acute GVHD and other transplantation-related risks –20. Spec+CD56−-activated T lymphocytes in an in vitro mixed lymphocyte reaction (MLR) assay. Our aim was to correlate the immunogenicity of HLA-C MM with HLA-C mRNA expression levels, with disparities in aas known to be relevant for peptide binding and/or T cell receptor (TCR) recognition, and also with concomitant HLA-DPB1 incompatibilities.In this study, we investigated the CTL alloresponse induced by single HLA-C MMs by quantifying CD8in vitro cellular assays (CTLp and MLR) have been performed at LNRH as part of the routine histocompatibility testing for unrelated HSC donor searches.Peripheral blood mononuclear cells (PBMCs) were purified using standard Ficoll procedure from blood collected from patients and donors who have been analyzed by the National Reference Laboratory for Histocompatibility (LNRH) for unrelated HSC donor searches. HLA high resolution typing was performed by reverse PCR-sequence-specific oligonucleotides typing on microbeads arrays , by PCR-sequence-specific primers , and by monoallelic sequencing . This study has been approved by the ethical committee of the Geneva University Hospitals (reference #08-208R). For many years, l-glutamine, penicillin and streptomycin (Gibco), 10% dimethylsulfoxid , and 20% fetal calf serum (Gibco). Responder cells (2 × 106) were stimulated at a ratio of 1:1 with 30 Gy irradiated stimulator cells in RPMI 1640 medium (Gibco) supplemented with l-glutamine, penicillin and streptomycin (Gibco), and 10% human AB serum (own preparation). Twenty units per milliliter rIL-2  were added at days 3, 7, and 11. After 13 days of culture, responding T cells were restimulated overnight with irradiated PKH-2 -labeled PHA blasts obtained by activation of non-irradiated stimulatory PBMCs with 1 μg/ml PHA (Gibco). The percentage of CD137+PKH-2−CD8+CD56− viable T cells was quantified by flow cytometry using APC-labeled anti-human CD8a, PerCP/Cy5.5 anti-human CD56 , and FITC-labeled anti-human CD137  antibodies, as well as APC- and FITC-labeled murine IgG1 isotype controls . As controls, half of the cultures were also restimulated with autologous PHA blasts. The percentage of activated CD8+ T cells referred as Δ%CD137+CD8+ equals the percentage of CD137+CD8+CD56− cells measured in cultures restimulated with stimulator cells minus the percentage of CD137+CD8+CD56− cells measured in cultures restimulated with autologous cells (+CD56− cells using the ACCURI-C6 cytometer (BD) and the CFLOWPLUS analysis software.For allogenic stimulation, one-way MLRs were performed using thawed cryopreserved PBMC, which were cryopreserved in RPMI 1640 medium  supplemented with us cells . Data ac+CD8+ ≥2% was considered as positive, this threshold corresponding to the variability observed in unstimulated cultures. As positive controls to monitor responsiveness of the responder cells and ability of stimulator cells to induce an alloresponse, responder cells were stimulated in parallel cultures with allogeneic stimulatory cells mismatched for several HLA-A, B, and C alleles, and similarly, stimulator cells were used to stimulate allogeneic HLA-A-, B-, and C-mismatched responder cells. In this study, only MLRs with a positive allogenous control were considered. Of the 63 MLRs, 12 could be repeated at different dates, as a way to control for reproducibility of our experiments, because enough cells had been cryopreserved. With two exceptions, all repeated cultures (eight duplicates and two triplicates) allowed the same positive versus negative alloresponse discrimination .An MLR assay with Δ%CD137Total RNA was extracted from thawed stimulator cells prior to irradiation using the RNeasy Micro kit  and according to the manufacturer’s instructions. RNA was reverse transcribed with the M-MLV H-Minus  reverse transcriptase and random oligo dT 15mer-primers in the presence of rRNAsin (Promega). HLA-C cDNA was quantified relative to 18S RNA, as previously described , using Shttp://www.ebi.ac.uk/ipd/imgt/hla/) and used to compute the difference in aa for each allele pair . Each residue was categorized as forming, or not, the pocket-like structures of the PBR, as being involved, or not, in peptide binding according to various biological, chemical, or physical properties . The second approach allowed avoiding too strong assumptions based on an arbitrary cutoff value of alloreactivity. The linear models were validated, whenever appropriate, using diagnostic plots.in vitro MLR assays including 18 different target HLA-C alleles. With two exceptions, all HLA-C MMs differed in the α1/α2 domains. The 18 target alleles included all frequent C allotypes present in populations of European origin except C*08:02. Seven MLR combinations consisted in HLA-C allele MMs within the C*02 (two pairs) and C*03 serotype (five pairs). The C*02 combinations consisted of the C*02:02/02:29 incompatibility that differs at residue 265 in the α3 domain. The C*03 combinations were C*03:03/03:04 MMs differing by a single residue at position 91 in the α1 domain. The remaining 56 pairs consisted in HLA-C antigen MMs .For each of the 18 different HLA-C target alloantigens, 1–9 MLRs were performed with a total of 24 different HLA-A~B~DRB1~DQB1 shared haplotypes. The overall results  or the C*03:04 (three pairs) alleles as target alloantigens . Furthermore, 6/6 C*06:02 and 5/6 C*02:02 MMs were not able to induce a CD8p = 0.637, Figure In order to detect a possible correlation between CTL alloresponses and levels of HLA-C expression, we quantified mRNA steady-state levels of the stimulator PBMCs at day 0 of the MLR. As presented in Figure p = 0.037). The mismatched residues affected predominantly those involved in peptide binding relative to the TCR contact residues. Taking into account both the number of aa differences  and the HLA-DPB1 matching  as predictor variables, only the aa differences appeared to have an impact . However, when the C*03:03/03:04 MM pairs were omitted, the aa differences were not significant anymore, while HLA-DPB1 became borderline significant . Measuring alloreactivity along a continuous axis and testing several predictor variables  or omitting C*03:03/03:04 MM pairs .In order to analyze the impact of functionally relevant aa MMs, we compared the alloresponse with the number of aa differences in the α1/α2 domains. The two pairs with a C*02:02/02:29 MM were excluded because the two alleles have identical sequences in the α1/α2 domains. As determined in 61 pairs, between 1 and 15 aa differences were recorded . On average, the number of mismatched residues was higher in the MLR pairs that resulted in a positive CTL alloresponse  and positive MLRs (19/30). Among aa116 MM pairs, those with HLA-C expression above the mean value (11.23) had a higher rate of positive MLR . MMs at positions 77 and 80 were not associated with T cell alloreactivity (data not shown).p = 0.021, Fisher’s test). It is relevant to note that, as shown in Figure +CD8+ T cells.Of 44 pairs with ≥10 HLA-C aa MMs, 24 induced a positive alloresponse and 20 were negative. A strong correlation was disclosed with the HLA-DPB1-matching status since 87.5% of the positive pairs and 55% of the negative pairs were HLA-DPB1 incompatible   and 2–3 stimulator cells from different donors who have exactly the same 2 HLA-A~B~C~DRB1~DQB1 haplotypes with the same HLA-C MM. As shown in four different mismatched combinations Figure , the sam) Figure E. Furthe) Figure A,C, the ) Figure .in vitro assay. By quantifiying CD137+CD8+CD56− T lymphocytes at day 14, we focused on T cell but not NK cell-mediated alloreactivity. The MLR assay had been developed previously to disclose a silent allele MM in the HLA-B44 serotype . On the other hand, HLA-DPB1-matching status is not the only important parameter because five MLRs showing wide differences in the ratio of CD137+CD8+ T cells , we used the same HLA-C MM cells to stimulate different responder cells that were HLA matched among each other. Since in three of four experiments, a given HLA-C MM was able to induce different alloresponses when tested against two or three different responders with exactly the same HLA haplotypes Figure , this inNevertheless, when both the effects of the number of aa MMs and of TCR repertoire were neutralized by stimulating the same responder cells expressing the same HLA-C MM on identical HLA haplotypes, variable alloresponses were observed Figures A–D. In tin vitro MLR assay can only be a simplified model of the alloreaction occurring in HSCT. Yet, it allowed to confirm the permissiveness of the C*03:03/03:04 MM as well as the higher risk of aGVHD and transplant-related mortality conferred by patient’s C*14:02 MM, which were indeed reported by clinical studies , and possibly on HLA-C expression. However, the complex interplay of these genetic factors is not straightforward. Clearly, neither HLA-C mRNA expression levels nor cell surface expression measured on heterozygous individuals could reliably predict the strength of the alloresponse, as determined by an a effect , 40. TheFB and J-MT conceived and designed the experiments. FB performed the experiments. SB performed the statistical analyses. FB and SB designed the figures. J-MT wrote the first draft of the manuscript, and FB and SB contributed to the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C-methyltransferases possess a high potential to serve as biocatalysts in environmentally benign organic syntheses. Here, we report on the high resolution crystal structure of CouO, a C-methyltransferase from Streptomyces rishiriensis involved in the biosynthesis of the antibiotic coumermycin A1. Through molecular docking calculations, site-directed mutagenesis and the comparison with homologous enzymes we identified His120 and Arg121 as key functional residues for the enzymatic activity of this group of C-methyltransferases. The elucidation of the atomic structure and the insight into the catalytic mechanism provide the basis for the (semi)-rational engineering of the enzyme in order to increase the substrate scope as well as to facilitate the acceptance of SAM-analogues as alternative cofactors.Friedel–Crafts alkylation of aromatic systems is a classic reaction in organic chemistry, for which regiospecific mono-alkylation, however, is generally difficult to achieve. In nature, methyltransferases catalyze the addition of methyl groups to a wide range of biomolecules thereby modulating the physico-chemical properties of these compounds. Specifically, S-adenosyl-L-methionine dependent  S-adenosyl-L-methionine (SAM) as a methyl donor [N2-like nucleophilic substitution mechanism for methyl transfer is proposed yielding S-adenosyl-L-homocysteine (SAH) and the methylated substrate. Natural-product MTases are the functionally most diverse class of MTases and methyl groups are added to S, N, O or C atoms. The proposed catalytic mechanisms include general acid-base catalysis, metal-based catalysis as well as proximity and desolvation effects not requiring catalytic amino acids [Methylation is one of the most essential reactions in all living organisms and plays an important role in the expression, structure, and function of biological molecules such as proteins, DNA/RNA, and small molecules. The methyl groups are selectively introduced by methyltransferases (MTases), a large group of enzymes that can be divided into several subclasses based on their structural features. The most common class of MTases is class I, possessing a Rossmann-like fold and utilizing yl donor . For thiC-methyltransferases, CouO from Streptomyces rishiriensis and NovO from Streptomyces spheroids [1 and novobiocin, i.e. the methylation of the C-8 atom of the coumarin moiety -rational design of CouO and NovO variants for the alkylation of target substrates, which serve as intermediates for various follow-up reactions and applications.Here, we present the high-resolution crystal structure of the N-benzamide, was synthesized as described previously [All commercially available reagents and solvents were used without further purification unless otherwise stated. Demineralized water for HPLC was filtrated through a 0.2 μm cellulose nitrate membrane filter prior to use. The substrate for the activity assay, eviously . MateriaP21 with unit cell dimensions a = 33.1 Å, b = 82.9 Å, c = 76.9 Å and β = 96.9°, respectively [S-adenosyl-l-methionine (SAM)-dependent methyltransferases an initial solution with two molecules in the asymmetric unit was obtained using the program Phaser [free calculations [X-ray diffraction data were collected at DESY, Hamburg to 2.05 Å resolution on a crystal belonging to the monoclinic space group m Phaser . As lessm Phaser  and the m Phaser . The resm Phaser , until tm Phaser  as inclum Phaser . The manulations . The steulations  and the ulations . In the http://www.ebi.ac.uk/msd-srv/ssm) [http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) [http://www.pymol.org). Cavity analyses were performed using the LIGSITE algorithm [Sequences were aligned using the program T-coffee  and the srv/ssm)  were usert.html) . Structurt.html)  as implelgorithm  as implelgorithm  was usedN-benzamide, 3-amino-4,7-dihydroxycoumarin and 4,7-dihydroxycoumarin. For each ligand 25 docking runs were performed without water molecules present. During energy minimization in YASARA water molecules were added automatically.Docking calculations were performed using the program VINA  employinNdeI/HindIII. The mixture was transformed into E. coli TOP10F‘ cells . All constructs were confirmed by sequencing .The mutations were introduced by overlap-extension PCR. The construct pET26b-CouO  was usedE. coli TOP10F‘ cells harboring the different plasmids were grown over night at 37°C in 2xTY (50 mL in 100 mL shaking flask) medium supplemented with 100 mg/L ampicillin (pMS470-constructs). They were used for the inoculation of the main cultures (400 mL 2xTY media in 1 L baffled flasks) to an OD600 of 0.05. The cells were grown to an OD600 of ~0.8 at 37°C and 130 rpm. Protein expression was induced by the addition of 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) and cultivation was continued at 25°C for 20 h and 130 rpm. The cultures were harvested by centrifugation at 3.600 g for 15 minutes at 4°C. The cell pellets were stored at -20°C until further use, when they were resuspended in 25 mL of 50 mM sodium phosphate pH 7.5 and disrupted by sonication  for 6 minutes under continuous cooling. Cell free lysates were obtained by centrifugation . The protein concentrations of the lysates were determined with the Bio-Rad Protein Assay . For gel electrophoresis, NuPAGE® 4–12% Bis-Tris Gels, 1.0 mm,  were used with NuPAGE MES SDS Running Buffer. The percentage of expressed protein in cleared lysates was determined by quantifying the respective band on a Coomassie-stained SDS-PA gel using a G-box HR16 device .The variants CouO-H15A, -H15N, -R24A and -R121L showed the same expression level as soluble protein as CouO-WT. The variants CouO-H117A and -Y216F were slightly less expressed as soluble protein, whereas the R121A variant was slightly better expressed. In the soluble fractions of CouO-H117S and -H120N clearly less target protein was expressed and the band of H120A was hardly detectable in the soluble fraction .et al. [E. coli cells expressing CouO and its variants were incubated in 0.1 ml scale in a thermomixer at 30°C and 1000 rpm for 24 h. The reactions contained 0.5 mM substrate N-benzamide (10 mM stock solution prepared in DMSO), 2 mM SAM- dihydrochloride  in 50 mM sodium phosphate buffer pH 7 and 0.1 mg/ml BSA (in 50 mM sodium phosphate buffer of pH 7)  . The solof pH 7) . Followiof pH 7) . All reaSamples from the activity assays were analyzed using a Shimadzu Nexera HPLC-MS system comprising two Nexera LC-30AD pump modules, Nexera SIL-30AC auto sampler, CTO-20AC prominence column oven, SPD-M20A prominence diode array detector, CBM-20A prominence communications bus module, FCV-20AH2 valve unit and LCMS-2020 quadrupole mass detector. The analyses were carried out on an Agilent Poroshell 120 EC-C18, 100x3 mm, 2.7 μm column using a solvent gradient , which proved to be a challenging task. An important step was truncating the common core-structure to only those parts that were really conserved and obtaining the correct initial solution with two common core-structures in the asymmetric unit. Less than 40% of the total residues were thus used for initial phasing. Still, we continued with the manual rebuilding and refinement by adding only a few extra residues (max. 4) per refinement cycle until the electron density for the whole molecule appeared. After extensive rebuilding and refinement, two CouO molecules in the asymmetric unit could be completely modeled into the residual electron density. In addition, one SAH cofactor molecule was present in each chain . DetaileC-methyltransferase Coq5  [Methanococcus maripaludis , CmoA from E.coli (PDB: 4gek) [Haemophilus influenza (PDB: 1im8) [S-adenosyl-L-methionine (Cx-SAM) from SAM and prephenate [The overall CouO structure exhibits a core with a Rossmann-like α/β-fold typical of class I SAM-dependent methyltransferases and a cap-domain accommodating two α-helices . An analw, 4obx) , the putB: 4gek)  and YecOB: 1im8)  were ideB: 1im8) , 31. CmoS-adenosyl-L-homoselenocysteine) and Cx-SAM. For Rossmann-fold enzymes that use different cofactors, a canonical motif defined by a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu) with a unique geometry has been identified [These structurally very similar enzymes mainly differ in the conformation of the cap-domain and the position of the N-terminal part consisting of a loop and a first α-helix . The cofentified . In CouOStructure examination of CouO located a cavity in the vicinity of the SAH binding site . This cai.e. in line with the methyl group and the sulfur atom of the cofactor, which is required by the classic SN2 reaction mechanism proposed for most other MTases [Docking calculations with the CouO structure using several ligands containing a coumarin scaffold identified His120 and Arg121 as most likely crucial for the enzymatic activity. The lowest energy docking mode places the coumarin moiety in the vicinity of the cofactor, whereas His120 and Arg121 are situated about 3 Å from the hydroxyl-oxygen at C-7 of the substrate . In thisA homology model of NovO was generated using the CouO structure as a template . Both enzymes accept substrates with identical architecture and vary only in respective conversion rates . Most ofvia base-assisted deprotonation of the hydroxyl group followed by a nucleophilic attack of the newly generated resonance stabilized phenolate-ion at C-7 of the substrate on the reactive methyl group of SAM. With His120 playing the role of a general base, the positively charged Arg121 would stabilize the negatively charged intermediate. The C-8 carbon of the coumarin scaffold attacks the methyl group of SAM generating a methylated, non-aromatic intermediate and SAH. Subsequent tautomerization leads to the (re)formation of the aromatic system. A superposition of the structures of CouO and Coq5 places the proposed general bases (His120 in CouO and Arg201 in Coq5) in very similar positions relative to the cofactor  onto the substrate [E. coli also accepts Cx-SAM as its natural cofactor [e.g. the replacement of the more bulky residues Ile6, Phe14, Trp170, Met75, Arg24 and Tyr216 with smaller counterparts–might provide additional means to widen the cofactor and substrate scope of the enzyme.It has been shown that CouO and NovO accept modified cofactors and are able to transfer larger groups Click here for additional data file.S2 Fig12. In the blue trace, peak A corresponds to aggregates, whereas samples from peak B were used for crystallization. A least-squares fit (log(MW) vs. elution volume) yielded a molecular weight of 52.4 kDa for peak B.A) CouO dimer interface formed between the α-helices of the cap-domain that mainly involves hydrophobic interactions as identified by the PDBePISA server. The SAH cofactor is shown in sticks. B) The final step of CouO purification involved size exclusion chromatography , 1 ml/min, Hepes pH 7, 100mM NaCl). Red trace: Bio-Rad gel filtration standard peaks: 1–670 kDa, thyroglobulin, 2–158 kDa, γ-globulin, 3–44k Da, ovalbumin, 4–17 kDa, myoglobin, 5–1.34 kDa, vitamin B(TIF)Click here for additional data file.S3 FigAmino acid residues present in the canonical motif, Asp70, and in the N-terminal tail, Glu6, are shown in a stick representation. The coloring scheme is the same as in (TIF)Click here for additional data file.S4 FigA homology model of NovO was generated using the CouO structure as a template . Selected amino acid residues in and around the active site are shown in a stick representation. Residues 117 and 145 differ in two enzymes. The coloring scheme for CouO is the same as in (TIF)Click here for additional data file.S5 FigThe residues suggested to be involved in catalysis, as well as the SAH/SAM cofactor are shown in sticks representations.(TIF)Click here for additional data file.S6 FigN-benzamide was used as substrate.0.5mM (TIF)Click here for additional data file.S7 Fig1: H15A, 2: H15N, 3: R24A, 4: H117A, 5: H117S, 6: H120A, 7: H120N, 8: R121A, 9: R121L, 10: Y216F, 11: WT, 12: pMS470d8. St: Page Ruler Prestained Protein Ladder (Fermentas). The arrow indicates the location of the target protein (expected MW = 26kDa).(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 TableSequences of restriction sites are underlined, start and stop codon are indicated with boxes. Bold red letters indicate the mutated bases.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Programmed cell death 1 (PD-1) gene may contribute to the development of cancer. In this study, we selected PD-1 rs10204525 T>C, rs2227982 A>G, rs36084323 T>C and rs7421861 A>G polymorphisms and designed a hospital-based case-control study to determine the potential relationship between these functional SNPs in PD-1 gene and esophagogastric junction adenocarcinoma (EGJA) risk. A total of 1,063 EGJA patients and 1,677 controls were enrolled from Eastern Chinese Han population. SNPscanTMgenotyping assay was used to analyze the genotyping of PD-1 polymorphisms. We found that PD-1 rs7421861 A>G polymorphism was associated with the development of EGJA. However, PD-1 rs2227982 A>G polymorphism was a protective factor for EGJA. In addition, PD-1 rs36084323 CC homozygote genotype might be associated with a borderline decreased risk of EGJA. In a subgroup analysis, a decreased risk of EGJA in never drinking and never smoking groups was identified. Haplotype comparison analysis suggested that PD-1 Trs10204525Grs2227982C36084323Ars7421861 haplotype significantly decreased the risk of EGJA. However, Trs10204525Grs2227982C36084323Grs7421861 haplotype in PD-1 gene may confer risk to EGJA. In conclusion, our study highlights rs2227982 A>G, rs36084323 T>C and rs7421861 A>G polymorphisms and haplotypes in PD-1 gene, especially within the intron region, are significantly associated with the risk of EGJA. Further case-control studies with larger sample size and detailed gene-environmental data to replicate these findings in different populations are needed to validate our conclusion.Single nucleotide polymorphisms (SNPs) in  In addition, it is reported that individual's genetic background also plays an important role in pathogenesis of EGJA. Although a number of studies have focused on the etiology of EGJA, it is not well understood. Of late, some studies reported the immune system might be implicated in the etiology of EGJA  was harnessed to construct the haplotypes of PD-1 gene [P < 0.05 (two-tailed). We performed the Power and Sample Size Calculator (http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize) to assess the power value of the study (α = 0.05) [Age was expressed as the mean ± standard deviation (SD). And we used Student's