Glioblastoma multiforme is characterized by rapid proliferation, aggressive metastatic potential, and resistance to radio- and chemotherapy. The matricellular protein CYR61 regulates cellular proliferation and migration and is highly expressed in Glioblastomas. MicroRNAs are 22-nucleotides long RNAs that regulate gene expression post-transcriptionally. Here, we utilized the LN229 glioblastoma cell line and found that CYR61 is a target of miR-136, miR-155, and miR-634. Over-expression of miR-136 and miR-634 miRNAs negatively affected proliferation, but not migration, while expression of miR-155 reduced migration but did not affect the proliferation of LN229 cells. Investigation of the molecular mechanisms affected by expression of miR-634 revealed an increased phosphorylation of p70S6 kinase, suggesting an induction of the mammalian target of rapamycin (mTOR) complex 1 pathway. Additionally, in miR-634 overexpressing cells, TSC2, a negative regulator of mTOR signaling, was found to be decreased. Altogether, our study provides insights on the differential roles of miRs-136, -155, and -634 in regulating glioblastoma cell growth and migration, and how microRNAs could be manipulated to decrease the aggressiveness and metastatic potential of tumor cells.  Glioblastoma multiforme (GBM) is a devastating malignancy with a median survival of approximately 15 months ,4,5,6,7.Cysteine-rich 61 (CYR61) is a member of the CCN  family of matricellular proteins . The CCNThe mTOR signaling pathway controls numerous aspects of cellular biology, including proliferation, cell cycle progression, translation, and cell size. mTOR is a serine/threonine kinase that can be found in two distinct complexes, mTORC1 and mTORC2. The complex mTORC1 can be inhibited by rapamycin and is responsible for controlling protein synthesis and the phosphorylation of downstream targets such as p70S6 kinase . SignaliMicroRNAs (miRNAs) are 19-25 nucleotide species of non-translated RNA that bind to the 3’UTR of target mRNAs with incomplete complementarity . The binRenilla and firefly luciferase reporter sequences. The CYR61 3’UTR sequence contains one putative binding site for each microRNA  as predicted by TargetScan microRNA gene target prediction database . A 20% decrease in the Renilla/firefly luciferase ratios was observed when the psiCHECK vector was co-transfected with miRNA-expressing plasmids, as compared to empty plasmid control. This result was repeated in LN229 cells with miR-155 and miR-634 under the control of a doxycycline-inducible vector system. Here, a 50% reduction in the Renilla/firefly ratio was observed upon induction of microRNA expression by doxycycline . Cells were transiently transfected, sorted for GFP expression into 12-well plates, and counted at 24 h, 48 h, and 72 h. Cell growth is indicated as the number of viable cells counted by flow cytometry at each time point using the Guava ViaCount assay. In order to examine the effect of these miRNAs on the invasive potential of Glioblastoma, we conducted cell migration assays with transiently transfected LN229 cells. Overexpression of miRs-136 and -634 did not significantly affect cell migration, as compared to that of cells transfected with vector alone. However, overexpression of miR-155 did significantly reduce cell migration across the membrane B. This rDespite a dramatic reduction in CYR61 expression, miR-155 transfected cells do not show deficits in proliferation, suggesting that CYR61 is not strongly involved in controlling cell growth. This argument is reinforced by the fact that cells expressing miR-136 and miR-634, in which CYR61 is still highly detectable, grow less efficiently than the miR-155 transfected cells.0), which were not stimulated with 10% FBS, show no significant phosphorylation of p70S6 kinase. Importantly, densitometric analysis revealed that the stable miR-136, -155, and -634 expressing cell lines showed 3.6 (+/− 1.3), 3.7 (+/− 0.1), and 2.6 (+/− 0.4) respective fold increases in p70S6 kinase phosphorylation as compared to vector control. This result is in agreement with that found under conditions of transient overexpression of the miRNAs  was conducted using lysates incubated with LY294002 and rapamycin C. As expIn addition to targeting the 3’UTR of CYR61, we sought to determine whether down-regulation of this protein by the three selected miRNAs was dependent on the activation of ERKs, AKT, or mTOR pathways. Results indicated no significant changes in CYR61 levels upon inhibition of any of these kinases (data not shown). The protein TSC2 negatively regulates the mTOR pathway by binding and blocking the GTPase activity of Rheb, a positive regulator of mTORC1 activity . PreviouFenofibrate is a member of a class of drugs known to stimulate the activity of the transcription factor, peroxisome proliferator activated receptor α (PPARα). This drug is used clinically to lower levels of cholesterol, triglycerides, low-density lipoprotein, and very low-density lipoprotein levels in patients. However, fenofibrate also possesses antitumor activities, which have been demonstrated in multiple cancers, including glioma, medulloblastoma, endometrial, and melanoma, among others ,36,37,38The effect of fenofibrate treatment on gene expression in LN229 cells was evaluated by mRNA microarray analysis. Since CYR61 3’UTR is predicted to be a target of miR-136, miR-155, and miR-634, we next sought to investigate the role of our selected microRNAs in the fenofibrate-mediated down-regulation of CYR61. Expression levels of CYR61-targeting miRNAs  in LN229 cells treated with fenofibrate were measured by quantitative real-time PCR (qRT-PCR) as described in the Although changes in the expression of our selected miRNAs were not detected in fenofibrate treated cells, we still reasoned that they could enhance the fenofibrate inhibitory effect on CYR61, perhaps sensitizing cells to fenofibrate treatment. Experiments were carried out to determine if clonogenic growth in the presence of fenofibrate is affected by miRNA expression. Cells stably expressing the indicated miRNAs were plated at very low density in 35 mm dishes . After 24 h, fresh medium or medium containing 50 µM fenofibrate was added to the plates, and replaced every three days thereafter. After 14 days, cells were fixed and stained with crystal violet. The clonogenic density in the presence of fenofibrate was then normalized to clonogenic density in the absence of fenofibrate for each miRNA . The res2. Fenofibrate was obtained from Sigma . Rapamycin was purchased from EMD Millipore . LY294002 and U0126 were from Enzo Life Sciences . Recombinant CYR61 protein was purchased from Abnova .LN229 and HeLa cells were obtained from the American Type Culture Collection . Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin  and incubated at 37°C in 5% CO6 cells were resuspended in 100 µL of Nucleofector Solution T, mixed with 2 µg of plasmid DNA, and nucleofected using Nucleofector Program X-01 . For the experiments shown in 5 cells/60 mm dish and transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Transfection using Lipofectamine 2000 was followed by cell sorting for enrichment of GFP+ cells. Cell sorting was performed using a FACSAria . In some experiments, cells were washed twice with PBS and serum starved overnight in DMEM. After 24 h, fresh serum-free DMEM with or without the presence of inhibitors was added to plates. After 1 h, cells were stimulated with 10% FBS with or without the presence of inhibitors for 1 h at which time cells were collected by scraping.For experiments requiring transient expression of miRNAs, 0.5 × 102, 1 mM EGTA, pH 8.4, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM sodium orthovanadate, phosphatase inhibitor and protease inhibitor cocktails (Sigma)). Whole-cell lysates (30 to 70 µg) were separated on a 4%–15% SDS-PAGE gel . CYR61 antibody (MAB4055) was purchased from R&D Systems . Phospho-p70S6 kinase (T389) (#9234), total p70S6 kinase (#2708), phospho-ERK1/2 (T202/Y204) (#9106), total ERK1/2 (#9102), phospho-AKT (S473) (#4060), total AKT (9272) and TSC2 (#4308) antibodies were purchased from Cell Signaling Technology . Phospho-TSC2 (S664) antibody (ab133465) was obtained from Abcam . Monoclonal antibodies specific to PP2A catalytic α subunit (610555) and GRB2 (610112) were from BD Transduction Laboratories . 14-3-3 antibody (sc-1019) was obtained from Santa Cruz Biotechnology .LN229 cells were collected by scraping the plates in the presence of PBS, followed by centrifugation and disruption of the cell pellet in the appropriate volume of lysis buffer .LN229 Glioblastoma cells were treated with 50 µM fenofibrate for 24 h and 48 h. RNA was isolated from the cells at the indicated time points and subjected to nanodrop analysis to verify the quality of the RNA. mRNA microarray analysis was performed using a HumanWG-6 Expression BeadChip  covering over 37,000 genes, including control gene sequences. Bioinformatics analysis for miR predicted gene targets was performed using GenGo software and miRWalk database . RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit containing TaqMan® primers specific for each respective miRNA . Quantitative real-time PCR for detection of mature miRNAs  or control U6 was performed using TaqMan® RT-PCR primers (Applied Biosystems). For each sample containing cDNA template, RT-PCR was performed in duplicate using a LightCycler 480 Real-Time PCR System from Roche . Each sample was normalized using U6 control and relative quantification of miRNA expression was calculated using the comparative Ct (ΔΔCt) method, as described in our previous publications ,41,42.The mature hsa-miR-136 sequence used corresponds to miRBase acession #: MIMAT0000448. The following oligos were used to clone hsa-miR-136 into the Block-iT Pol II miR RNAi Expression Vector (Invitrogen): forward, 5’-TGCTGACTCCATTTGTTTTGATGATGGAGTTTTGGCCACT-GACTGACTCCATCATAAACAAATGGA and reverse, 5’-CCTGTCCATTTGTTTATGATGG-AGTCAGTCAGTGGCCAAAACTCCATCATCAAAACAAATGGAGTC. For hsa-miR-155, the mature sequence (miRBase accession #: MIMAT0000646) was cloned using the oligos: forward, 5’-TGCTGTTAATGCTAATCGTGATAGGGGTGTTTTGGCCACTGACTGACACCCCTATCGATTAGCATT and reverse, 5’-CCTGAATGCTAATCGATAGGGGTGTCAGTCAGTGGCCAAAACA-CCCCTATCACGATTAGCATTAAC. For the cloning of mature hsa-miR-634 (miRBase accession #: MIMAT0003304), the oligos used were: forward, 5’-TGCTGAACCAGCACCCCAACTTT-GGACGTTTTGGCCACTGACTGACGTCCAAAGGGGGTGCTGGT and reverse, 5’-CCTGA-CCAGCACCCCCTTTGGACGTCAGTCAGTGGCCAAAACGTCCAAAGTTGGGGTGCTGGTTC. All oligos were cloned into the Block-iT Pol II miR RNAi Vector (Invitrogen) according to the manufacturer’s protocol.Renilla luciferase reporter gene in the PsiCHECK2 vector . This vector also contains a firefly luciferase reporter sequence, which allows for normalization of transfection efficiency. The primers used were: forward, 5’-CTACTCGAGTTTCCAGGGCACACCTAGAC, and reverse, 5’-CTACTCGAGGCTTAATTCACTGCTGTATCC, containing the XhoI restriction site (underlined). The genomic sequence corresponding to the 3’UTR of CYR61 was PCR amplified from LN229 cells. This PCR product was ligated into the multiple cloning site downstream of the 4 cells/well in a 12-well plate. After 24 h, cells were harvested and lysates were assayed for luciferase activity with the Dual-Luciferase Reporter Assay System (Promega) using a Synergy 2 microplate reader . Relative units of Renilla luciferase activity were normalized to firefly luciferase internal control for each sample. Experiments were performed in duplicate.HeLa cells were nucleofected with 2 µg of plasmid DNA according to the manufacturer’s protocol . Nucleofected cells were plated at a density of 8 × 104 cells/well in a 12-well plate. The next day, cells were transfected with 0.6 µg of DNA/well using Lipofectamine reagent (Life Technologies) according to manufacturer’s protocol. pcDNA3 was used to maintain a constant amount of DNA in each transfection. After 24 h, cells were harvested and assays were performed as described above.MicroRNA target validation was also carried out in LN229 cells with miRNA expression under control of the Tet-on inducible vector system (Clontech). LN229 cells were plated at a concentration of 8 × 10LN229 cells were transiently transfected with empty or miR-expressing Block-iT plasmids and sorted into 12-well plates at a density of 10,000 cells/well. At 24 h, 48 h, and 72 h after plating, cells were trypsinized and resuspended in 200 µL of PBS. Cells were then diluted 10-fold into ViaCount reagent  and viable cells were counted by flow cytometry using the Guava easyCyte 8HT instrument (Guava Technologies).Empty plasmid or microRNA-expressing LN229 cells were plated at a density of 25,000 cells into wells in a 24-well plate containing 8 µm filter membranes (BD Biosciences). Cells were allowed to migrate for 16h. Next, the top side of the filter membranes were wiped free of cells and washed with PBS. Cells were fixed, stained with crystal violet, and counted under light microscopy. The experiment was performed in triplicate.Untransfected or miR-expressing LN229 cells were plated at a density of 1000 cells/35-mm dish. After 24 hrs, medium containing 50 µM fenofibrate was added to the dishes. After 14 days, cells were fixed and stained with crystal violet. Relative density of clones was quantified using ImageJ (Version 1.44p). Experiments were performed in duplicate.P-values ≤ 0.05 were considered statistically significant.Data are presented as mean ± SD. Comparison between two experimental groups was performed using the Student’s t-test. Glioblastoma multiforme is a tumor characterized by rapid proliferation, metastatic spread, and resistance to chemo- and radiotherapy. CYR61 appears to be a key regulator of cell migration and proliferation in various types of cancer, including Glioblastoma ,19.Among the various microRNAs that are predicted to target CYR61, miR-155 has been shown to inhibit the 3’UTR of CYR61 , and we In the attempt to characterize the molecular mechanisms implicated in the differential effects of the three miRNAs on proliferation and migration of LN229 cells, we investigated the phosphorylation status of three key factors typically involved in those cellular functions: AKT, ERK1/2 and p70S6K. Our results  were disFenofibrate is an FDA approved PPARα agonist that has previously been shown to block the proliferation and migration of glioblastoma ,39. In tet al. found that increased expression of phospho-mTOR in primary breast tumor samples correlated with a reduced response to tamoxifen [et al. showed that rapamycin blocks doxorubicin chemoresistance in an in vivo murine lymphoma model [et al. found that chemoresistant Intrahepatic cholangiocarcinoma cells showed increased activation of ERK1/2, along with increased AKT activation [Fenofibrate has previously been shown to block the clonogenic growth of medulloblastoma and glioblastoma cells ,39. We tamoxifen . In anotamoxifen . In linema model . In additivation . Anothertivation .In conclusion, our studies demonstrated that overexpression of CYR61-targeting microRNAs impairs the growth (miR-136 and miR-634) and the migration (miR-155) of Glioblastoma cells. Expression of these microRNAs was accompanied by activation of mTOR pathway signaling, which was dependent upon the activity of AKT, ERK1/2, and mTORC1. In clonogenic growth assays miR-634-expressing cells, but not miR-136 or miR-155, were partially desensitized to the inhibitory effects of fenofibrate. Further investigation will be required to better understand the role of miR-634 and subsequent mTOR activation in the resistance of Glioblastoma to fenofibrate and to identify other signaling molecules critical to this process. Finally, in addition to providing valuable information on the differential effects of microRNAs-136, -155, and -634 on cell growth and migration, our studies have generated further mechanistic insights into the activity of fenofibrate against Glioblastoma.


P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1–54 amino acids and the Armadillo repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1–54 amino acid sequence and Armadillo repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin. E-cadherin has been shown previously to participate in multiple aspects of cell processes, including development, morphogenesis and carcinogenesis To date, a number of regulatory mechanisms have been discovered involving carcinogenesis and tumor progression. Among these, increased experimental evidence has demonstrated that cadherin-mediated cell-cell interaction plays a pivotal role in the development and progression of many tumors P120-catenin (p120ctn), a member of the catenin family, can interact directly with the intracellular domain of E-cadherin, and thus, plays important roles in regulating cell-cell adhesion P120ctn has four isoforms (isoforms 1 to 4) resulting from four transcription start sites In the current study, we screened and eventually selected 1 human bronchial epithelial cell line (HBE) and 3 lung cancer cell lines , of which E-cadherin is localized to the cell membrane in 2 and cytoplasm in the other 2 cell lines, respectively, and knocked down p120ctn using small interfering RNA (siRNA). P120ctn isoforms 1A or 3A were then restituted in the cells to investigate the effects on E-cadherin expression and cell invasiveness. In addition, multiple p120ctn isoform 1A deletion mutants were constructed and expressed in the p120ctn depleted cells to test which peptide domains are essential for the different function of p120ctn isoforms 1A and 3A.Human bronchial epithelial cell line HBE and lung adenocarcinoma cell line SPC-A-1 were obtained from the American Type Culture Collection . Human pulmonary giant cell carcinoma cell line NCI-H460 and human lung adenocarcinoma cell line LTEP-α-2 were obtained from the Cell Bank of Chinese Academy of Science . The cells were cultured in RPMI 1640 medium  containing 10% fetal calf serum (Invitrogen), 100 IU/ml penicillin  and 100 µg/ml streptomycin (Sigma).GFP-siRNA-P120ctn plasmids and p120ctn isoforms 1A and 3A cDNA plasmids have been described previously The cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, aliquot cells were then transfected with cDNA plasmids of p120ctn isoform 1A, isoform 3A or each of the five p120ctn isoform 1A deletion mutants M1–M5. The transfection was performed with Lipofectamine 2000  or Attractene Transfection Reagent  according to the manufacturers' instruction.Each protein sample (50 µg) was separated by SDS-PAGE. After transferring to a polyvinylidene fluoride (PVDF) membrane, the membrane was incubated overnight at 4°C with either the mouse monoclonal antibody against p120ctn , E-cadherin  or GFP . After incubating with peroxidase-coupled anti-mouse IgG  at 37°C for 2 hours, the protein bands were visualized using ECL  and detected using the BioImaging Systems (UVP Inc.). The relative protein levels were calculated in reference to the amount of β-actin protein.Cells grown on glass coverslips were fixed with ice-cold 4% paraformaldehyde for 30 minutes, followed by permeabilization with 0.2% Triton X-100. Primary antibodies are as follows: anti-p120ctn mouse monoclonal antibody , anti-E-cadherin rabbit polyclonal antibody  and anti-E-cadherin mouse monoclonal antibody . Primary antibodies were applied overnight at 4°C followed by incubation with secondary antibody conjugated to rhodamine/fluorescein isothiocyanate (FITC)-labeled goat anti-mouse or anti-rabbit antibodies . The nuclei were counterstained with propidium iodide/4, 6 diamidino-2-phenylindole. The cells were examined with an Olympus IX51 fluorescent microscope , and images were captured with a CoolPIX 5400 camera .5 cells were grown in serum-free medium on 8-µm porous polycarbonate membranes , which were coated with Matrigel basement membrane matrix . The lower chambers were filled with RPMI 1640 medium containing 10% fetal calf serum. After incubation for 24 h at 37°C in a humid atmosphere with 5% CO2, the cells migrating through the pores were fixed with methanol for 30 min and stained with hematoxylin (Sigma). For each filter, the numbers of cells in five different fields of 400× magnification were counted visually using a Nikon E200 microscope. Each experiment was performed in triplicate.The assay was performed according to the manufacturer's instructions. In each upper chamber, 5×10All statistical analyses were performed using SPSS for Windows software. Data from experimental groups were compared to those of control groups using the Independent-Samples T test. P values less than 0.05 were considered statistically significant.The localization of E-cadherin and p120ctn in the cells was assessed using immunofluorescence. When cells were grown under conventional culture conditions, expression of E-cadherin and p120ctn were restricted to the cell membrane in HBE and H460 cells. On the other hand, in SPC and LTE cells, E-cadherin and p120ctn had similar distributions and were localized in the cytoplasm . WesternP<0.01; HBE: 15.20±1.15 versus 9.33±1.40; P<0.01)    ; whereas P<0.01) .P<0.01; HBE: 7.47±1.46 versus 15.20±1.15; P<0.01)   , whereas P<0.01) .P<0.01) (P<0.01) (P<0.01) (P<0.01)  , HBE (21(P<0.01) , SPC (22(P<0.01)  and LTE (P<0.01) , respectP<0.01) (P<0.01) . The N-terminal 55–101 amino acids are present in p120ctn isoform 2A. Thereafter, five p120ctn isoform 1A deletion mutants M1 to M5 fused to GFP were constructed as described above  and tranP<0.01) , whereas(P<0.01) .P<0.01) (P<0.01)   and SPC (P<0.01) . The oth(P<0.01)  and 6.Consistent with previous studies It is interesting that restitution of p120ctn isoform 1A restored the E-cadherin levels and its localization on the cell membrane in HBE and H460 cells but did not alter cytoplasmic distribution of E-cadherin in SPC and LTE cells. These data suggest that p120ctn isoform 1A could up-regulate the E-cadherin protein levels, but p120ctn alone could not determine the destination of E-cadherin molecules. The actual localization of E-cadherin may be determined by a combination of factors including other catenin family members as well as non-catenin modulators.Moreover, our data demonstrated that p120ctn isoform 1A contributes to the stabilization of E-cadherin in the cytoplasm. P120ctn may facilitate the trafficking of cadherins towards the cell membrane through its ability to interact with kinesin, and the co-immunoprecipitation data showed that p120ctn isoform 1 was much more strongly associated with kinesin than isoform 3 In the current study, we demonstrated that p120ctn isoform 3A, though having no effects on E-cadherin protein levels, could enhance invasiveness in all four cell lines tested regardless of the expression status of E-cadherin. This effect on invasiveness in the cells independent of E-cadherin suggests a distinct pathway of p120ctn isoform 3A in regulating the progression of cancer cells. We and others have previously shown that Kaiso, which can recognize specific promoter sequences and inhibit the expression of target genes in the nucleus, interacts directly with p120ctn isoform 3A Both p120ctn isoform 1A and isoform 3A contain the central Armadillo repeat domain, which is essential for its interaction with the E-cadherin juxtamembrane domain. A previous study showed that there is no obvious difference in the interaction of p120ctn isoform 1A or 3A with E-cadherin on the cell membrane It should be noted that the molecular weight of GFP is several times as big as the M1, M3 or M5 mutants with the Armadillo repeat domain deleted, and using it as a tag molecule to indicate transfection efficacy might compromise the intracellular localization of these p120ctn mutant isoforms. Nonetheless, the molecular weights of M2 and M4 mutants, both of which contain the armadillo domain, are over 100 kDa, much larger than GFP molecule. A distinct membranous fluorescence (M4-GFP) , IX and In conclusion, this study demonstrated that p120ctn isoform 1A up-regulates E-cadherin protein levels, while isoform 3A does not. Both isoforms showed an effect on cell invasiveness. While p120ctn isoform 1A has two opposing effects on cell invasiveness, corresponding to the distinct subcellular localization of E-cadherin in individual cell lines, p120ctn isoform 3A promotes cell invasiveness by an E-cadherin-independent mechanism. The N-terminal 1–54 amino acid sequence and Armadillo repeat domain in P120ctn isoform 1A are indispensable for regulating E-cadherin expression.Figure S1Effects of p120ctn isoforms on E-cadherin expression and cell invasiveness in HBE cell line. (A) Effect on E-cadherin expression by immunofluorescence staining. Note the absence to marked decrease in E-cadherin after knockdown of p120ctn by si-p120ctn transfection (II), in contrast to the membranous staining of E-cadherin in the cells transfected with vector alone (IV). Also note that restitution of p120ctn isoform 1A restores membranous expression of E-cadherin (VI), while restitution of isoform 3A shows only some cytoplasmic expression of E-cadherin with no significant membranous staining (VIII). Green signal indicates the expression of GFP from the reporter of the constructed plasmids, confirming an effective transfection. (B) Effect on E-cadherin expression by Western blot analysis. Note a markedly decreased E-cadherin level after si-p120ctn transfection and restoration of E-cadherin by restitution of p120ctn isoform 1A but not by isoform 3A. (C) Effect on cell invasiveness by Matrigel invasion assay. Note a marked increase in cell invasiveness after si-p120ctn transfection, corresponding to the knockdown of p120ctn ( p120ctn  and decr p120ctn . Restitu(TIF)Click here for additional data file.Figure S2Effects of p120ctn isoforms on E-cadherin expression and cell invasiveness in LTE cell line. (A) Effect on E-cadherin expression by immunofluorescence staining. Note the absence to marked decrease in E-cadherin after knockdown of p120ctn by si-p120ctn transfection (II), in contrast to the cytoplasmic staining of E-cadherin without significant membranous expression in the cells transfected with vector alone (IV). Also note that restitution of p120ctn isoform 1A restores cytoplasmic expression of E-cadherin (VI), while restitution of isoform 3A shows only faint cytoplasmic staining of E-cadherin (VIII). Green signal indicates the expression of GFP from the reporter of the constructed plasmids, confirming an effective transfection. (B) Effect on E-cadherin expression by Western blot analysis. Note a significantly decreased E-cadherin level after si-p120ctn transfection and restoration of E-cadherin by restitution of p120ctn isoform 1A but no significant change by restitution of isoform 3A. (C) Effect on cell invasiveness by Matrigel invasion assay. Note a marked decrease in cell invasiveness after si-p120ctn transfection, corresponding to the knockdown of p120ctn ( p120ctn  and decr p120ctn . Restitu(TIF)Click here for additional data file.Table S1Effects of p120ctn isoforms on level/subcellular localization of E-cadherin and cell invasiveness in four cell lines. While p120ctn isoform 1A up-regulates E-cadherin expression in all 4 cell lines tested, its effect on cell invasiveness seems to be in two opposing directions, corresponding to the subcellular distribution of E-cadherin in each cell line. In cells with endogenous membranous expression of E-cadherin (HBE and H460), up-regulation of E-cadherin by p120 isoform 1A seems to suppress cell invasiveness, whereas, in those with endogenous cytoplasmic expression (SPC and LTE), it appears to enhance cell invasiveness. P120ctn isoform 3A enhances cell invasiveness with no significant alteration of E-cadherin expression. Its effect on cell invasiveness seems to be independent of E-cadherin. *In comparison with the group transfected with vector alone. **In comparison with the group transfected with GFP-si-p120ctn.(DOC)Click here for additional data file.


Salvia Divinorum, is a non-nitrogenous kappa opioid receptor (KOR) agonist. It has been shown to reduce acute pain and to exert potent antinflammatory effects. This study assesses the effects and the mode of action of SA on formalin-induced persistent pain in mice. Specifically, the SA effects on long-term behavioural dysfuctions and changes in neuronal activity occurring at spinal level, after single peripheral formalin injection, have been investigated. Moreover, the involvement of microglial and glial cells in formalin-induced chronic pain condition and in SA-mediated effects has been evaluated.Salvinorin A (SA), the main active component of . SA significantly reduced the formalin-mediated microglia and astrocytes activation and modulated pro and anti-inflammatory mediators in the spinal cord.Formalin induced a significant decrease of mechanical withdrawal threshold at the injected and contralateral paw as well as an increase in the duration and frequency, and a rapid decrease in the onset of evoked activity of the nociceptive neurons 7 days after formalin injection. SA daily treatment significantly reduced mechanical allodynia in KOR and cannabinoid receptor 1 (CB1R) sensitive manner. SA treatment also normalized the spinal evoked activitySA is effective in reducing formalin-induced mechanical allodynia and spinal neuronal hyperactivity. Our findings suggest that SA reduces glial activation and contributes in the establishment of dysfunctions associated with chronic pain with mechanisms involving KOR and CB1R. SA may provide a new lead compound for developing anti-allodynic agents via KOR and CB1R activation. Salvia Divinorumbenzeneacetamide hydrochloride (U-50488) was purchased from Sigma ; 17,17'-(Dicyclopropylmethyl)-6,6',7,7'-6,6'-imino- 7,7'-binorphinan-3,4',14,14'-tetrol dihydrochloride (nor-BNI), 1--5-(4-iodophenyl)-4-methyl-N-piperidin-1-ylpyrazole-3-carboxamide (AM251) and 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1 H-indol-3-yl](4-methoxyphenyl)methanone (AM630) were purchased from Tocris Cookson . All reagents for western blot analysis were obtained from Sigma , Bio-Rad Laboratories  and Microglass Heim . All drugs used  were dissolved in 0.5% v/v DMSO in saline or aCFS, depending on the administration route.Salvinorin A (purity: 99% by HPLC) was isolated from Behavioural and electrophysiological data were expressed as mean ± S.E.M. (n = 6–8) and analyzed by using the one-way analysis of variance (ANOVA) for repeated measures followed by the Student-Newman Keuls for multiple comparisons to determine statistical significance between different treated groups of mice. Immunohistochemical data were expressed as mean ± S.E.M (n = 3) and analyzed by using the one-way analysis of variance (ANOVA) for repeated measures followed by Tukey post hoc. Biomolecular analysis and protein quantifications were expressed as mean ± S.E.M (n = 3) and analyzed by t-test. P <0.05 was considered as significant.The authors declare that they have no competing interests.FG conceived of the study and drafted the manuscript. SM and VdN participated in the design and coordination of the research. MDC and IM performed the behavioural evaluations. LL performed the immunoistochemistry. LG and SB carried out the electrophysiological recording. GA and RC carried out the bio-molecular evaluations. EP and AI helped to draft the manuscript and performed the statistical analysis. JKZ provided the Salvinorin A. All authors read and approved the final manuscript.Figure S1. Effect of vehicle or SA repeated treatment , alone or in presence of nor-BNI , or AM251  on spinal microglial cells in mice receiving formalin into the hind-paws. Iba-1 immunoreactivity (Iba-1-ir) is shown in the ipsilateral dorsal horn 7 days after formalin (A-D). Quantitative analysis of percentage of activated microglial cells on the total cell number in L4-L6 spinal cord sections is shown in “E”. Data are expressed as mean ± S.E.M of 3 mice per group. * and ○ indicate statistically significant differences vs veh/form and SA/form, respectively. . (PPT 390 kb)Click here for fileFigure S2. Effect of vehicle or SA repeated treatment , alone or in presence of nor-BNI , or AM251  on spinal astrocytes in mice receiving formalin into the hind-paws. GFAP immunoreactivity (GFAP-ir) is shown in the ipsilateral dorsal horn 7 days after formalin (A-D). Quantitative analysis of percentage of activated astrocytes on the total cell number in L4-L6 spinal cord sections is shown in “E”. Data are expressed as mean ± S.E.M of 3 mice per group. * and ○ indicate statistically significant differences vs veh/form and SA/form, respectively. . (PPT 303 kb)Click here for file


Several polarity proteins, including Scribble (Scrb) have been implicated in control of vesicle traffic, and in particular the endocytosis of E-cadherin, but through unknown mechanisms. We now show that depletion of Scrb enhances endocytosis of E-cadherin by weakening the E-cadherin-p120catenin interaction. Unexpectedly, however, the internalized E-cadherin is not degraded but accumulates in the Golgi apparatus. Silencing p120-catenin causes degradation of E-cadherin in lysosomes, but degradation is blocked by the co-depletion of Scrb, which diverts the internalized E-cadherin to the Golgi. Loss of Scrb also enhances E-cadherin binding to retromer components, and retromer is required for Golgi accumulation of Scrb, and E-cadherin stability. These data identify a novel and unanticipated function for Scrb in blocking retromer-mediated diversion of E-cadherin to the Golgi. They provide evidence that polarity proteins can modify the intracellular itinerary for endocytosed membrane proteins. Cell polarity is intimately related to vesicle sorting, which ensures the delivery to and removal from the appropriate membrane proteins to the correct territories on the cell surface, and maintains the distinct properties and functions of these territories. The machineries involved in polarization and vesicle traffic are each understood in molecular detail, but our knowledge of their interactions with one another, and their cross-regulation, remains rudimentary. The PAR proteins, plus several other polarity proteins including Lgl and the Cdc42 GTPase, have been implicated in control both of apical protein traffic and E-cadherin endocytosis from adherens junctions while, in a reciprocal fashion, vesicle sorting proteins, including several Rabs, are required for normal localization of the polarity proteins Drosophila embryonic epithelia Scribble (Scrb) was discovered as an apicobasal polarity protein and tumor suppressor in The homophilic connections between cadherin molecules on adjacent cells are weak; however, clustering of cadherin molecules on the lateral surface of cells strengthens cell-cell adhesion In this study we address the mechanisms by which Scrb promotes E-cadherin adhesive activity. We discovered that Scrb stabilizes the p120catenin-E-cadherin coupling to reduce the rate of E-cadherin internalization and, unexpectedly, blocks retromer-mediated retrieval of E-cadherin to the Golgi, thus implicating Scrb in the control of vesicle sorting.MDCK T23 cells were obtained from Keith Mostov  p120KD: 5′GATCCCCGCCAGAGGTAGTTCGGATATTCAAGAGATATCCGAACTACCTCTGGCTTTTTGGAAA;GATCCCCGAGGAGACTTCGATGAGAATTCAAGAGATTCTCATCGAAGTCTCCTCTTTTTGGAAA;Vps29KD1: 5′ 5′- GATCCCCGAATTATCCAGAACAGAAATTCAAGAGATTTCTGTTCTGGATAATTCTTTTTGGAAA.Vps29KD2: Sense and antisense oligonucleotides for shRNAs were purchased from IDT Technologies, annealed, phosphorylated, and ligated into the BglII and HindIII sites of pSUPER 2, 0.5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml DNase I, 1 mM DTT, and 1 mM PMSF). After 3 rapid freeze/thaw cycles, samples were cleared by centrifugation . Supernatants were assayed for protein concentration using Bradford reagent then boiled in sample buffer. Antibodies used were as follows: anti-Scrb (1∶100), anti-Rap1 (1∶200), anti–β-catenin , anti–E-cadherin , anti-p120catenin ; anti–α-tubulin ; anti-GFP ; anti-GST . HRP-conjugated secondary antibodies were used at a dilution of 1∶5,000–1∶10,000 (Jackson ImmunoResearch Laboratories). Band intensities were quantified using ImageJ.For analysis of total cell extracts by immunoblot, cells were scraped directly into Laemmli sample buffer. After SDS-PAGE, proteins were transferred to nitrocellulose and detected by chemiluminescence. For immunoblots of supernatant fractions, cells were washed with cold PBS and lysed in a detergent-free lysis buffer , E-cadherin (1∶500) β-catenin (1∶500), and GM130 (1∶500) were from BD Biosciences. Antibodies against Numb (1∶25) and Mannose 6-Phosphate receptor (1∶500) were from Abcam. Anti-Rab11a (1∶400) was from Invitrogen and anti-Afadin (1∶1000) and anti-Golgi 58K (1∶100) were from Sigma-Aldrich). Anti-Na/K ATPase (1∶250) was a gift from M. Caplan . Alexa Fluor–conjugated secondary antibodies (Invitrogen) were used at a dilution of 1∶1,000. Alexa 546–conjugated phalloidin (Invitrogen) was used at a dilution of 1∶400; DRAQ5  was used at a dilution of 1∶1000. Cells were mounted in Fluormount-G (SouthernBiotech). Lysotracker Red DND-99 was from Invitrogen.For immunofluorescence, cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and blocked with 1% gelatin in PBS for 1 h before incubation with antibodies. For detection of Na/K-ATPase and E-cadherin, cells were permeabilized in methanol at −20°C for 10 min. Primary antibodies against Scrb (1∶50), p120catenin (1∶500), α2, 1 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM Na3VO4 and1 µg/ml DNase I. In some experiments lysis was performed in a hypotonic buffer lacking detergent and salt. After passing through a sub-Q 261/2 needle, lysates were centrifuged for 15 min at 10,000×g. Supernatants were immunoprecipitated for 2 h using the indicated antibodies and sheep anti-mouse IgG or sheep anti-rabbit IgG conjugated Dynabeads (Invitrogen). Samples were washed 4× in lysis buffer and once in PBS then eluted in sample buffer.For immunoprecipitation experiments, cells were lysed in 50 mM Tris pH 7.5, 0.5% NP-40, 200 mM NaCl, 5 mM MgClConfocal images were captured using an LSM510 Meta microscope , using a 40× (1.4 NA) or 100× (1.3 NA) oil immersion lens. Phase-contrast images were captured on a TE200 widefield microscope  using a 10× lens and an Orca charge-coupled device camera . Images were converted to TIFF format using ImageJ and processed using the Adobe Photoshop CS4 Levels tool  to enhance contrast.For FRAP assays, Ecad-GFP cells GST–Scrb fragments and GST-Ral1GDS were prepared as described previously LucKD or ScrbKD transfected cells were seeded onto 0.4 µm filters for 24 h. After culturing cells in serum-free medium for 1 h, medium containing anti-E-cadherin ectodomain antibody- rr1  at a concentration of 10 µg/ml was added to the bottom chamber of the filter plates. Cells were incubated with antibody for 1 h at 4°C and then rinsed thoroughly to remove unbound antibody. Treated cells were then incubated in culture media at either 4°C or 37°C for 3 h, fixed, permeabilized, and stained with an anti-mouse Alexa 546 secondary to detect the rr1 antibody.Previously, we had shown that Scrb depletion disrupts lateral membrane organization, but had not detected significant internalization of E-cadherin As a biochemical test for vesicular redistribution of E-cadherin, MDCK cells expressing the LucKD or ScrbKD shRNAs were ruptured in hypotonic lysis buffer, and centrifuged to separate cytoskeleton-associated structures, nuclei, and other large organelles, from smaller vesicles. The supernatants were analyzed by immunoblot. Although loss of Scrb does not cause any change in total E-cadherin levels  the bandTo determine whether Scrb depletion causes a general defect in membrane protein traffic, we examined the localization of another lateral membrane-associated protein, the Na/K ATPase. As expected, in control cells Na/K ATPase was associated exclusively with the lateral cell boundaries, but the protein was also on the lateral membranes in ScrbKD cells, and was not detectable in intracellular vesicles . We concWe next asked if the intracellular accumulation of E-cadherin in Scrb-depleted cells is caused by a defect in the delivery of the protein to the surface or because of an increase in the rate of endocytosis. To distinguish these possibilities, cells expressing Ecad-GFP were transfected with Luc or Scrb shRNAs then incubated at 4°C with an antibody specific to the extracellular domains of the canine E-cadherin (rr1) As an independent assay for internalization, we performed FRAP on the internalized Ecad-GFP in Scrb depleted cells. Under conditions blocking de novo protein synthesis (cycloheximide addition), a partial recovery of perinuclear GFP fluorescence occurred after bleaching, within 30 min at 37°C . Since EWe next sought to identify the membrane compartment into which E-cadherin accumulates after Scrb depletion. Partial colocalization was observed with Rab11, a marker of recycling endosomes, and with Golgin-97, which marks the trans-Golgi network (TGN) and late endosomes . PartialCargo is commonly retrieved from the endosomal compartment to the Golgi via retromer complexes, which consist of various members of the sorting nexin (Snx) family plus a trimer of Vps26, Vps29, and Vps35 Does internalization of E-cadherin in Scrb-depleted cells result from a loss of junctional stability? Uncoupling of E-cadherin and p120 in mammalian epithelial cells disrupts adhesion and causes E-cadherin to be rapidly internalized and degraded Moreover, although E-cadherin and p120catenin in control cells colocalize almost completely, silencing of Scrb caused a significant redistribution of p120 into the cytoplasm, as assessed by quantification of the overlap coefficient Loss of p120 has been shown previously to result in the rapid degradation of internalized E-cadherin, mediated both by the proteosome and by transport to lysosomes An important conclusion from our study is that Scrb not only helps to maintain E-cadherin at the cell cortex by promoting interaction with p120, but also plays a key role in blocking retromer binding and retrieval of E-cadherin to the Golgi, ensuring its degradation in lysosomes. A strong prediction from this model is that depletion of Scrb should over-ride the effect of silencing p120 and rescue E-cadherin expression, by switching traffic from the lysosomal pathway to Golgi retrieval. We tested this prediction by performing double knockdowns. As assessed by immunoblot  endogenoDrosophila epithelia The function of Scrb in mammalian cells has remained somewhat mysterious. Although essential for apical/basal polarity in In terms of mechanism, we have discovered that Scrb blocks the association of E-cadherin with a retromer complex containing Vps29, preventing retrieval of the protein to the Golgi apparatus. A consequence of this blockage is that any endocytosed E-cadherin is normally sorted to the lysosomes for degradation rather than being recycled. Importantly, targeting to either compartment is independent of p120 binding to E-cadherin, because the cadherin can enter either pathway in the absence of p120: the lysosomal pathway in the presence of Scrb, or the retrieval pathway in the absence of Scrb. Therefore, the association of Scrb with p120 must be independent of Scrb’s ability to block retrieval . Scrb miDrosophila ectoderm the loss of Par-6, Par-3 or Cdc42 can cause arrest of endocytosed apical proteins or of E-cadherin in early endosomes C. elegans the polarity proteins are required for efficient endocytosis and recycling Our data provide evidence that a polarity protein can select the itinerary for internalized membrane proteins, and the first indication that a polarity protein regulates the retromer pathway. In Figure S1Effects of Scrb depletion on lateral membranes and E-cadherin. (A) Control and Scrb-depleted cells cells were plated on slides for 2 d then fixed and stained for Na/K ATPase and GFP. GFP was used as a transfection marker. (B) FRAP of Ecad-GFP in MDCK cells. Cells nucleofected with shRNA against Scrb were incubated for 3 d then placed at 37°C on the stage of a Zeiss LSM510 Meta confocal microscope. Areas of interest were drawn within individual cells to encircle the Golgi and were bleached using 3 iterations at 100% transmission, 75% laser power with the 25 watt argon laser. Cells were imaged at intervals after bleaching using 3% transmission. Two sets of representative images are shown, pre-bleach, bleach and 30 min post-bleach.(PDF)Click here for additional data file.Figure S2Localization of internalized Ecadherin. Control and Scrb-depleted Ecad-GFP cells were grown on slides for 48 h, fixed and immunostained for (A) Golgin-97, or (B) Rab11. (C) Cells were stained for endogenous E-cadherin and Numb. (D) Cells expressing Ecad-GFP were nucleofected with shRNAs as shown then treated with 1 mM brefeldin dissolved in DMSO, to a final concentration of 10 uM; or with an equivalent amount of DMSO alone as a negative control, for 2 h, and imaged for Ecad-GFP distribution. All images are confocal sections .(PDF)Click here for additional data file.Figure S3Silencing of Vps29 blocks Golgi accumulation of endocytosed E-cadherin. Cells were incubated for 1 hr at 4°C with the RR1 antibody against the extracellular domain of E-cadherin. They were then washed to remove free RR1, and either left at 4°C or switched to 37°C for 4 h. The cells were then fixed, permeabilized and stained for total E-cadherin and for the RR1 antibody. White arrows show the accumulation of E-cadherin and RR1 in perinuclear regions in cells depleted of Scrb alone. All images are confocal sections .(PDF)Click here for additional data file.Figure S4Internalized E-cadherin remains associated with α- and γ-catenin. (A) Cells expressing Ecad-GFP were transfected with shRNA vectors as shown  and after 3d they were fixed and stained for α-catenin or γ-catenin and imaged by confocal microscopy. Representative line scans are shown. In addition, 5 fields per condition were quantified for overlap of the red and green channels and overlap coefficients were calculated using Openlab software. No significant differences were observed between the control and Scrb-depleted cells. (B) Immunoblot of lysates from cells nucleofected with shRNAs targeting Scrb or p120catenin, showing destruction of E-cadherin is promoted by loss of p120catenin but not Scrb. (C) Localization and expression of Ecad-GFP in cells nucleofected as in (B). (D) Control and Scrb-depleted cells were incubated in calcium-free medium overnight, then switched to calcium-containing medium and lysed at indicated times after calcium re-addition. Active Rap1GTPase was precipitated onto glutathione-Sepharose beads bound to GST Ral1-GDS. Samples were analyzed by immunoblot for Rap1 to detect Rap1-GTP. Band intensities were quantified using ImageJ and normalized to total Rap1 in the lysates.(PDF)Click here for additional data file.Figure S5Depletion of p120catenin drives E-cadherin to the lysosomes. MDCK cells expressing Ecad-GFP were nucleofected with control or p120catenin-targeted shRNAs, and grown on chambered coverslips. After 3 d some chambers were treated with 100 µg/ml of leupeptin to inhibit lysosomal proteases. After 2 h, red Lysotracker Red DND-99 was added to a final concentration of 100 nM. The cells were then imaged live, at 37°C, by confocal microscopy. Overlap coefficients were calculated using Openlab software. Boxes on right provide a 3× magnification of the regions outlined in white on the merged images. The green channel was enhanced using Photoshop Levels to stretch the contrast from 0 ->255 to the values shown.(PDF)Click here for additional data file.


The human 90-kDa heat shock protein (HSP90) functions as a dimeric molecular chaperone. HSP90 identified on the cell surface has been found to play a crucial role in cancer invasion and metastasis, and has become a validated anti-cancer target for drug development. It has been shown to self-assemble into oligomers upon heat shock or divalent cations treatment, but the functional role of the oligomeric states in the chaperone cycle is not fully understood.Here we report the crystal structure of a truncated HSP90 that contains the middle segment and the carboxy-terminal domain, termed MC-HSP90. The structure reveals an architecture with triangular bipyramid geometry, in which the building block of the hexameric assembly is a dimer. In solution, MC-HSP90 exists in three major oligomer states, namely dimer, tetramer and hexamer, which were elucidated by size exclusion chromatography and analytical ultracentrifugation. The newly discovered HSP90 isoform HSP90N that lacks the N-terminal ATPase domain also exhibited similar oligomerization states as did MC-HSP90.While lacking the ATPase domain, both MC-HSP90 and HSP90N can self-assemble into a hexameric structure, spontaneously. The crystal structure of MC-HSP90 reveals that, in addition to the C-terminal dimerization domain, the residue W320 in the M domain plays a critical role in its oligomerization. This study not only demonstrates how the human MC-HSP90 forms a hexamer, but also justifies the similar formation of HSP90N by using 3D modeling analysis. Heat shock protein 90 (HSP90) is an ATPase-dependent chaperone and the molecular chaperone functions as a dimer. HSP90 is responsible for managing protein folding and quality control in the crowded environment inside the cell. It participates in activating and stabilizing more than 200 “client” proteins involved in post-translational folding, protein stability, activation and maturation of cellular proteins, which are essential to cell-cycle control and signaling. HSP90, HSP70 and co-chaperones form a dynamic complex known as the HSP90 dynamic machine The HSP90 proteins are highly conserved and five human isoforms have been identified, including two cytosolic isoforms HSP90α and HSP90β, a glucose-regulated protein (GRP94) in the endoplasmic reticulum, a tumor necrosis factor receptor-associated protein 1 (Hsp75/TRAP1) in the mitochondrial matrix, and a newly discovered isoform HSP90N The conserved HSP90 structure consists of three domains: an N-terminal (N) domain that contains the co-chaperone binding motif and an ATP and drug binding site that binds the natural compounds geldanamycin and radicicol; a middle (M) domain that is responsible for binding to co-chaperone and client proteins; and a C-terminal (C) domain that contains a dimerization motif, a second drug-binding site, and a conserved MEEVD pentapeptide at the C-terminus, which is recognized by the co-chaperone HSP70/HSP90 organizing protein (Hop) 2+ ion, demonstrated that the building block for its oligomerization is a dimer. In particular, the hexamer with a cozy nest-shape was obtained by negative staining TEM tomography HSP90 exists as a homodimer, and the dimers tend to associate into tetramers, hexamers, and even higher oligomers. It has been reported that the oligomeric forms of HSP90 are present in the cytosol of mammalian cells C2221, P21 and R32. A fourth crystal form of P6322 was obtained using a modified MC-HSP90-Ct (residues 293–697) in which the C-terminal 35 residues were also truncated. The crystals were obtained using different protein preparations or under different crystallization conditions. For the C2221 and P21 crystals, obtained under similar conditions, the protein solution was pre-incubated with cis-dichloro(ethylenediamine)platinum(II) (cis-DEP) and cisplatin, respectively, before crystallization setup. The R32 crystals were obtained by using an additive solution in the crystallization drops. The P6322 crystals were grown under low pH conditions, also with an additive.  the molecular triad and dyads are further expressed as crystallographic symmetry elements  containing the middle segment and carboxy-terminal domain was crystallized in three different forms, ectively , with thelements . If the C2221 and P21 contain nine independent polypeptide chains in total. The N-termini start at residues 294–297, and the C-termini stop at residues 697–699, depending on the corresponding electron densities for the individual subunits. The residue 293 and the last 33 residues, plus the His-tag, were not observed, probably because they were highly flexible. Other disordered regions were located in residues 349–357, 394–404 and 611–629. Three molecules had an additional disordered region in residues 558–613, which also showed high temperature factors in the other molecules. In an attempt to obtain better crystals, we constructed a C-terminal tail truncated MC-HSP90-Ct, but the resulting P6322 crystals did not show any improvement in the diffraction resolution. The R32 crystal showed additional electron densities for most of these regions, but they could not be modeled with certainty due to the poor diffraction resolution. These two structures could only be refined to 3.5 Å resolution, yielding R values of about 30% and Rfree of about 35%.The refined models of MC-HSP90 in the two different space groups of 10-helix, which is followed by a three-layer architecture of α-β-α sandwich and a helical coil. It encompasses two disordered loops comprising residues 351–358 and 396–404. The SM domain is also folded into an α-β-α sandwich architecture, and contains a sulfate-binding site. The sulfate was hydrogen bonded to the side chains of R510, K513 and H514 within helix α7, which is close to the C domain , a middle segment catalytic loop (residues 395–406), an extended loop (residues 616–630) and the C-terminal tail (residues 698–732). To confirm the presence of disordered loops and C-terminal tail in the crystal, the entified  by our e2 on each protomer is buried by its counter protomer upon dimer formation  distribution suggests that MC-HSP90 exists as three species in solution with sedimentation coefficients of 5.3S, 8.3S and 11.6S  experiments were carried out to determine their molecular size. The elution profile of size-exclusion chromatography for MC-HSP90 shows two major peaks between the protein marker thyroglobulin (669 kDa) and aldolase (158 kDa). It suggests that MC-HSP90 contains at least two oligomeric states . No corrnd 11.6S , with a P21 crystal, the sedimentation coefficient values of MC-HSP90 at dimer and each oligomer states were calculated by using the computer program HYDROPRO Based on the observed 3D structure of MC-HSP90 determined in the As shown above, the residue W320 may play an important role in the hexamer assembly. Here it was mutated into an alanine and the resulting mutant protein W320A of MC-HSP90 was subjected to similar analyses as was the wild-type protein for its molecular size distribution. The size-exclusion chromatography profile showed a large shift of the oligomeric states toward the dimer . The subThe HSP90 isoform HSP90N lacks the N-terminal ATPase domain, which is replaced by a 30-residue segment, but the other related domains are the same as HSP90α. HSP90N has the same N-terminal hydrophilic region (residues 31–100), the middle client protein binding domain, and the C-terminal dimerization domain as does HSP90α. Thus the hexameric structure of MC-HSP90 should somewhat resemble the quaternary structure of HSP90N. To test this hypothesis, size exclusion chromatography and sedimentation velocity experiments were also conducted on HSP90N. The size-exclusion chromatography elution profile showed two broad peaks with molecular weight of about 309 and 473 kDa . When thThe N-terminal extension region of HSP90N comprising of residues 1–100 contains a hydrophobic helix, which was predicted as a transmembrane helix by three web version prediction programs. See . Based oP21, C2221, R32 and P6322, which have six, three or one molecules in their asymmetric unit. The protein molecules were assembled into a virtually identical hexameric structure with triangular bipyramid geometry in all crystal forms. Other similar hexameric architecture had been observed in the crystal structures of N-terminal truncated yeast HSP82 (PDB: 2CGE) and Leishmania major HSP83-1 (PDB: 3HJC), which have rmsd of 1.167 Å for 262 atoms , respectively. However, this kind of hexamer structure was not present in the crystal structure of N-terminal truncated GRP94 (PDB: 2O1T). The triangular-bipyramid hexamer is assembled by using six MC-HSP90 molecules. The C-terminal dimerization and the interactions from the M domain contact make the formation of MC-HSP90 hexamer; each protomer provides the interface of about 1267 Å2 and 531 Å2 for the dimerization of MC-HSP90 monomer and trimerization of MC-HSP90 dimer. Presumably dimerization occurs prior to trimerization for hexamerization. The residues W320 and R346 play the key roles for associating MC-HSP90 dimers into the hexamer. These two residues are conserved in other 90-kDa heat shock proteins, including yeast HSP82 and Leishmania major HSP83-1, but not found in canine GRP94, these two redidues are Ser in GRP94 , and the C-terminal tail truncated MC-HSP90-Ct in the hexagonal crystal also has the open-form dimer (data not shown). The open-form dimer is required for hexamer packing and is responsible for the ATPase-coupled molecular clamp mechanism of HSP90 chaperone HSP90N also shows three oligomer states in solution. It lacks the N-terminal ATPase domain and contains the amino acids 224–732 of HSP90α. But it retains the linker and has an additional hydrophobic segment at its N-terminus. Based on the crystal structure and hydrodynanic properties of MC-HSP90, the three oligomer forms should be the dimer, tetramer and hexamer and have similar quaternary structures to MC-HSP90. The hexameric structure of HSP90N reconstructed by modeling is shown in In summary, we have determined the crystal structures of middle and C-terminal domain (MC-HSP90) of human HSP90α. The structure reveals a hexameric assembly with triangular bipyramid geometry, in which the building block is a dimer. In addition to the four-helix bundle formed by the C-terminal dimerization domain, the residue W320 and the hydrophobic pocket in the middle domain provide the major interactions for the oligomerization. Size exclusion chromatography and analytical ultracentrifugation experiments indicated that MC-HSP90 not only forms the functional dimer, but also exists in tetrameric and hexameric states in solution. HSP90N in the dimer, tetramer and hexamer states were also elucidated here. The hexamer structure of MC-HSP90 presented here illustrates the possible oligomer structures of HSP90N. For cancer therapies, inhibitor/drug development has been focused on inhibition of the N-terminal ATPase domain of HSP90, such as the purine mimetic PU24FC1, geldanamycin (GA) and 17-allylamino-17demethoxygeldanamycin (17-AAG) ATTGAGGGTCGCGCTGCTATGCCTGAGGAAACCC-3′5′-CGCGGATCC ) and GTGATGGTGATGGTGATGGTCTACTTCTTCCATG-3′5′-CCGCTCGAGT TA ), and then cloned into a pGEX-6p-1 vector  at the BamH I and Xho I sites. This full length HSP90 construction (residues 1–732), termed pGEX-6p-1-HSP90, was used as the DNA template for making the plasmids of MC-HSP90 (pGEX-6p-1-MC-HSP90), MC-HSP90-Ct (pGEX-6p-1-MC-HSP90-Ct) and HSP90N (pGEX-6p-1-HSP90N). The gene encoding MC-HSP90 (residues 293–732) and MC-HSP90-Ct (residues 293–697) were cloned using the same forward primer (5′-CGCGGATCCATTGAGGGTCGCGCTGCTACAAAGCCCATCTGG-3′) which also encodes a Factor Xa cleavage site and two extra Ala residues. The HSP90N plasmid (pGEX-6p-1-HSP90N) was constructed by overlap PCR method, using six forward primers  to synthesize the DNA encoding the Factor Xa cleavage site, the extra Ala residues, and the 30 N-terminal amino residues of HSP90N. In both constructs of pGEX-6p-1-MC-HSP90 and pGEX-6p-1-HSP90N, the pGEX-6p-1-HSP90 plasmid was used as the initial template, and a reverse primer, 5′-CCGCTCGAGT TAGTGATGGTGATGGTGATGGTCTACTTCTTCCATG-3′ was used. For pGEX-6p-1-MC-HSP90-Ct a different reverse primer of 5′-CCGCTCGAGTTAATGGTGATGATGGTGATGGTGATGACCCAGACCAAG -3′ was used. Mutagenesis of MC-HSP90 W320A was made using the Quick-change site directed mutagenesis kit (Stratagene) on the recombinant plasmid pGEX-6p-1-MC-HSP90 with the following pair of oligonucleotides: forward, 5′-GAG CTTGACCAATGACGCTGAAGATCACTTGGCAG-3′; reverse, 5′-CTGCCAAGTGATCTTCAGCGTCATTGGTCAAGCTC-3′.For HSP90 production, a pCMV6-XL5 plasmid  containing a full length cDNA of the human heat shock protein 90α gene (NM_005348.2) was used as the initial DNA template. The DNA fragments of HSP90 with an N-terminal Factor Xa cleavage site and a C-terminal His-tag were generated using PCR amplification with primers Escherichia coli BL21 (DE3) cell (Novagen) for protein overexpression. MC-HSP90 protein was pre-purified by chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen) and a glutathione column . The glutathione S-transferase (GST) -tagged protein was digested by Factor Xa protease at 4°C for 24 hours in 150 mM NaCl and 50 mM Tris, pH 8.0, and then the cleaved MC-HSP90 was separated by ion-exchange chromatography on a HiTrap™ 26/10 QFF column . The protein was eluted at 350–400 mM NaCl, 50 mM Tris-HCl, pH 8.0. Uncleaved MC-HSP90 and free GST were eliminated by flowing through a GSTrap FF column, and pure MC-HSP90 was collected in the flow-through fractions. The MC-HSP90-Ct and HSP90N proteins were purified by similar protocols, in which the first gluthathione column was omitted in both cases and the QFF column was replaced by a second Ni-NTA column for MC-HSP90-Ct. The mutant protein W320A was expressed and pufiried as was the wild-type MC-HSP90. The purified proteins were then dialyzed against a solution of 50 mM Tris-HCl, pH 8.0, 1 mM EDTA and 1 mM dithiothreiol (DTT), and then concentrated using a 30 kDa filter  to about 12 mg/ml for stock at −80°C.Each of the HSP90 plasmids  was transformed separately into C2221, P21 and R32 were obtained using different crystallization droplet preparations. For the C2221 and P21 crystals, the protein solutions were pre-incubated with solutions containing the platinum additive cis-dichloro(ethylenediamine)platinum (II) and cisplatin, respectively, at a molar ratio of 1∶2 for one hour at 20°C. The mixed protein-Pt solutions (1.5 µl) were then combined with the reservoir (1.0 µl) for crystallization droplet set-up. The crystallization droplet of R32 crystal were prepared by mixing 1.5 µl of the protein solution with 1 µl reservoir solution and 0.5 µl additive solution, Silver Bullets No. 18 which contains 0.33% w/v 2,6-Naphthalenedisulfonic acid disodium salt, 0.33% w/v 2-Aminobenzenesulfonic acid, 0.33% w/v m-benzenedisulfonic acid disodium salt, and 0.02 M HEPES sodium pH 6.8 (Hampton Research). MC-HSP90-Ct crystals were crystallized in the P6322 space group, with a reservoir solution of 2.6 M ammonium sulfate, 0.1 M Tri-sodium citrate at pH 5.0 and the additive solution of Silver Bullets No. 70 -2-aminoethanesulfonic acid, 0.2% w/v phenylurea, 0.2% w/v β-Alanine, 0.02 M HEPES sodium pH 6.8) (Hampton Research). All crystals were flash-frozen with 13–15% glycerol (vol/vol) as a cryo-protectant and the diffraction patterns were recorded at cryogenic temperatures. The diffraction data of C2221, P21 and R32 crystals of MC-HSP90 were collected at the wavelength of 1.000 Å from the synchrotron beamline NW12 of Photon Factory (PF) in Japan, using an ADSC Quantum-210 charge-coupled diode (CCD) detector. Data of the P6322 crystal of MC-HSP90-Ct were collected at the wavelength of 0.900 Å from the synchrotron beamline BL44XU of SPring-8 in Japan, using an MX-225 CCD detector. All data were processed and scaled using the program HKL2000 The crystals of MC-HSP90 were grown by the sitting-drop vapor diffusion method. A solution of 2.5 M ammonium sulfate, and 0.1 M Tri-sodium citrate at pH 5.4–5.8 was used as the reservoir. Three crystal forms C2221 crystal structure was determined by molecular replacement using the program Molrep of the CCP4 program suite The P21 and R32 crystal structures of MC-HSP90 and the P6322 crystal structure of MC-HSP90-Ct were solved using Molrep, with the partially refined C2221 crystal structure as the search model. The monoclinic crystal form has six monomers in an asymmetric unit, and both rhombohedral and hexagonal crystal forms have one molecule in an asymmetric unit.The Rfree values. Manual modifications of the models were performed using the program XtalView Throughout the refinement, 5% of randomly selected data were set aside for cross validation with C2221 crystal structure was refined for individual atomic positions and temperature factors, and the model yielded Rwork/Rfree values of about 22/27 using the program Crystallography and NMR System (CNS) version 1.2 Rwork/Rfree values of 20.9/25.2.The Rwork/Rfree values of 24/30 for all data at 3.05 resolution. After addition of solvent molecules, NCS restraints were removed and the model was refined for individual atomic positions and temperature factors, yielding Rwork/Rfree values of about 23/28 using the program CNS. The model was further refinemet using Refmac5, including individual isotropic B-factor refinement and TLS refinement, from which the Rwork/Rfree values of 20.4/25.9 were obtained.The monoclinic crystal structure was refined with strong NCS restraints and refined to C2221 and P21 crystal structures have been deposited in the Protein Data Bank with accession codes 3Q6M and 3Q6N, respectively.Data collection and final model statistics are shown in 280 was monitored for the eluted solution. The elution volume corresponding to each oligomer types were then selected for analytical ultracentrifugation analysis. Thyroglobulin (669 kDa), aldolase (158 kDa) and ovalbumin (43 kDa) included in the Gel Filtration Calibration Kits  were used as the markers to estimate the molecular weight.The protein solutions (100 µl) of MC-HSP90 and HSP90N at final concentrations of 7–10 mg/ml were loaded separately onto a Sephacryl S-300 HR  size exclusion chromatography column of 1 cm×120 cm . The column was equilibrated with a buffer solution of 50 mM Tris-HCl, pH 8.0, and eluted at a flow rate of 0.5 ml/min at room temperatures. OD280) in a continuous mode without averaging. No time interval was set between scans. Data were analyzed with a c(s) distribution of the Lamm equation solutions calculated by the program SEDFIT Version 12.1b . The software Sednterp (http://www.jphilo.mailway.com) was used to estimate protein partial specific volume (Vbar), buffer density (0.99966 g/ml) and buffer viscosity (0.010167 poise) at 20°C. The Vbar value of MC-HSP90 is 0.7375 ml/g, and that for HSP90N is 0.7342 ml/g. For the sedimentation coefficient (S) values predictions of MC-HSP90 oligomer states, the atomic coordinate models of MC-HSP90 dimer, tetramer and hexamer were extracted from the P21 crystal structure and were used to calculate the S values by the software HYDROPRO, in which all user adjustable parameters were left at the default values Sedimentation velocity (SV) experiments were performed at 45,000 rpm using a 4-hole AnTi60 rotor at 20°C in a Beckman Optima XL-I AUC equipped with absorbance optics. The MC-HSP90 and HSP90N samples collected from the gel-filtration column were diluted to a final concentration of 0.3 mg/ml using 100 mM Tris-HCl buffer. Standard 12-mm aluminum double-sector centerpieces were filled with protein solution, and the reference cell contained the blank buffer. Quartz windows were used with absorbance optics  in 25 mM ammonium bicarbonate, pH 8.5, at 37°C for 16 hr. Following digestion, tryptic peptides were extracted twice with 50% acetonitrile containing 5% formic acid for 15 mins each time with moderate sonication. The extracted solutions were pooled and evaporated to dryness under vacuum. For MALDI-MS, the samples were pre-mixed 1 ∶ 1 with a matrix solution  and spotted onto a 96-well format MALDI sample plate. The data-directed obtain on the quadrupole time-of-flight (Q-TOF) Ultimat MALDI instrument.http://ncbi.nlm.nih.gov). The web-versions of three different topology prediction methods were used to predict the topology of N-terminal residues 1–100 of the HSP90N protein: HMMTOP (http://www.enzim.hu/hmmtop/), TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/), and TMpred . All these methods were used in the single sequence mode and all user adjustable parameters were left at their default values. Graphics of the 3D models of the hexameric HSP90N was produced with UCSF Chimera P21 crystal as a template.The amino acid sequence for HSP90N (ACCESSION: AAC25497) was downloaded from GenBank  hexamer. The protomers of human MC-HSP90 in the same dimer are colored in blue and orange, and all protomers of yeast HSP82 are colored in green.(MOV)Click here for additional data file.


Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including  Researchers and industry have been seeking new AMPs of natural and non-natural origin, with low toxicity and the longer half-life necessary for drug development.Antimicrobial resistance (AMR) is not a recent phenomenon, but it is a critical health issue today. Over several decades, to varying degrees, bacteria causing common infections have developed resistance to each new antibiotic, and AMR has evolved to become a worldwide health threat. With a dearth of new antibiotics coming to market, the need for action to avert a developing global crisis in health care is increasingly urgent in vivoE. coli cells and a successive optimization phase for biological activity, synthesis and purification procedures in vitro against clinical isolates of several Gram-negative pathogens, including MDR strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and Escherichia coli, while being less active against the Gram-positive pathogen Staphylococcus aureus. The peptide also protected mice lethally infected with multi-resistant clinical isolates of P. aeruginosa and is currently under preclinical characterization for the development of a new drug for bloodstream and lower respiratory tract infections.A few years ago, we observed that short peptides synthesized in oligodendrimeric form S. aureus and coagulase-negative staphylococci, including methicillin-resistant strains, with MIC values comparable to those of many antimicrobial agents used in clinical practice. We also report a study of the mechanism of action of M33-D compared to M33-L. Since M33-D retains strong activity against Gram-negative pathogens, it appears to be an interesting candidate for the development of novel broad-spectrum AMPs.In previous reports P. aeruginosa and the same or a slightly lower (2–4 fold) activity against Enterobacteriaceae. On the other hand, M33-D showed higher antimicrobial activity than M33-L against the Gram-positive bacteria S. aureus and S. epidermidis, including methicillin-resistant and vancomycin-intermediate strains, with MICs 4 to 16-fold lower than those of M33-L. As previously observed with M33-L MICs of M33-L and M33-D were determined against strains of different bacterial species, including major Gram-negative and Gram-positive pathogens . CompareS. aureus and Streptococcus faecalis, and LPS from E. coli, P. aeruginosa and K. pneumoniae were injected at a concentration of 10 µg/ml over immobilized M33-L or M33-D peptides. No significant difference in binding or kinetic rates that could explain such dissimilar antimicrobial activity of the two peptides was observed  and E. coli  To investigate interaction of peptides M33-D and M33-L with the bacterial membrane, including possible perturbation, we used vesicles with two lipid compositions to mimic the membrane of These tests, along with the Biacore analysis described above, revealed that M33-D and M33-L have substantially similar behavior in terms of binding to LPS and LTA and of perturbation of membranes of different phospholipid composition. We deduced that the mechanism used by M33-L and M33-D for interacting with bacterial surfaces and disruption of bacterial membranes was basically the same.S. aureus and P. aeruginosa, respectively. These proteins play a key role in bacterial virulence by breaking down natural HDPs produced by the infected individuals S. aureus aureolysin and P. aeruginosa elastase are members of the family of M4 metallopeptidases (thermolysin family) P. aeruginosa elastase showed moderate peptide stability after 5 h , which is consistent with the difference in MIC of the two isomers for this strain , we compared the in vivo activity of this peptide and the original M33-L in an animal model of infection caused by the highly virulent MRSA strain USA 300, a lineage that has become a dominant cause of community-associated MRSA infections in North America Given the good 6 in the presence of 7% mucin. An LD100 killed mice within 20 hours. Mice were infected with the LD100 of bacteria and treated i.p. with the peptides 30 minutes later. 100% survival after 7 days was obtained with mice treated with M33-D, while mice treated with M33-L showed a mortality overlapping that of controls  after intra-peritoneal (i.p.) injection was 1×10controls , confirmin vivo where M33-D neutralized signs of sepsis due to S. aureus USA300, while M33-L, stable to mouse The M33 peptide, previously reported as active against a broad spectrum of Gram-negative bacteria S. aureus, and elastase, from P. aeruginosa. M33-L was not at all stable to aureolysin and poorly stable to elastase, as confirmed by its activity in vivo against P. aeruginosaM33-D was highly stable to the proteases aureolysin, from S. aureus and retains its antimicrobial activity irrespective of strain-resistance phenotypes and mechanisms. MRSA and S. aureus strains with altered susceptibility to glycopeptides pose a serious clinical threat and major therapeutic challenge. In this context, development of a new broad-spectrum therapeutic agent with no cross-resistance to available drugs would be a major achievement.Concluding, we identified a new form of the peptide M33, which is strongly active against Solid-phase synthesis was carried out by standard Fmoc chemistry on Fmoc4-Lys2-Lys-β-Ala Wang resin with a Syro multiple peptide synthesizer . Side chain protecting groups were 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for R, t-butoxycarbonyl for K and t-butyl for S. M33-L was synthesized using Fmoc-L-aminoacids, and M33-D with Fmoc-D-aminoacids with the exception of the three lysins of the branched core which were Fmoc-L-Lys(Fmoc)-OH (M33-D is consequently a diastereomer). The final products were cleaved from the solid support, deprotected by treatment with TFA containing triisopropylsilane and water (95/2.5/2.5), and precipitated with diethyl ether. Crude peptides were purified by reversed-phase chromatography on a Phenomenex Jupiter C18 column  in linear gradient form for 30 min, using 0.1% TFA/water as eluent A and methanol as eluent B. Purified peptides were obtained as trifluoroacetate salts (TFacetate). The exchange from TFacetate to acetate form was carried out using a quaternary ammonium resin in acetate form . The resin-to-peptide ratio was 2000∶1, resin and peptide were stirred for 1 h, the resin was filtered off, washed extensively and the peptide recovered and freeze-dried. Final peptide purity and identity were confirmed by reversed phase chromatography on a Phenomenex Jupiter C18 analytical column  and by mass spectrometry with a Bruker Daltonics ultraflex MALDI TOF/TOF.4 CFU/well, in a final volume of 100 µl. The tested concentrations ranged from 0.1 µM to 24 µM for both peptides. Results were recorded after 18–20 h of incubation at 37°C.MICs were determined using a standard microdilution assay as recommended by the Clinical and Laboratory Standards Institute. Assays were performed in triplicate using cation-supplemented Mueller-Hinton (MH) broth  and a bacterial inoculum of 5x10Biotinylated peptides were immobilized on SA coated flow cells. M33-L and M33-D peptides, diluted to 10 µg/ml in HBS-EP+ buffer , were injected for 90 sec at a flow rate of 10 µl/min, obtaining 550 RU and 580 RU for M33-L and M33-D respectively.E. coli, K. pneumonia, P. aeruginosa and LTA from S. aureus and S. faecalis, were obtained from Sigma-Aldrich: L-3012, L-4268, L9143, L2515 and L4015, respectively) were diluted in HBS-EP+ buffer at the concentration of 10 µg/ml and injected for 180 sec with a flow rate of 30 µl/min over immobilized peptides. An empty flow cell was used as reference. Regeneration was achieved with a short pulse of SDS 0.05%.LTA and LPS molecules from different species (LPS from L-α-phosphatidylethanolamine (PE), L-α-phosphatidyl-DL-glycerol (PG), cardiolipin (CL), calcein, ammonium thiocyanate and iron (III) chloride hexahydrate and all other chemical (reagent grade) were obtained from Sigma.g for 30 min. For vesicle size homogeneity, the pellet was passed several times through 200 µm polycarbonate membranes in a Mini-extruder apparatus  t are fluorescence before and after addition of detergent and F0 the fluorescence of intact vesicles Calcein-loaded liposomes of two different composition  were prepared as follows. The lipids were dissolved in chloroform (1 ml) and sonicated together with 60 mM calcein solution ; the liposomes were obtained by the reverse phase evaporation method Staphylococcus aureus aureolysin  or Pseudomonas aeruginosa elastase  in 300 µl 20 mM Tris-HCl, 1 mM CaCl2 pH 7.8. At indicated time intervals, 50 µl aliquots were removed, diluted with 950 µl of 0.1% trifluoroacetic acid (TFA)/water and analyzed by HPLC and mass spectrometry. Liquid chromatography was performed on Phenomenex Jupiter C18 analytical column  in a 30 min gradient, using TFA 0.1%/water as solvent A and methanol as solvent B. Mass spectrometry analysis was performed on withdrawn samples and repeated on HPLC-eluted peaks with a Bruker Daltonic ultraflex MALDI TOF/TOF mass spectrometer.Tetrabranched M33-L or M33-D peptides (300 µg) were incubated at 37°C with P. aeruginosa and E. coli) or 37°C (for S. aureus) for 20 h under agitation at 125 rpm. Once biofilms formed, the lids were removed from the plates and the pegs were rinsed twice with phosphate buffered saline (PBS) to remove planktonic cells. The peg-lid was then transferred to a 96-well challenge microtiter plate, each well containing 200 µl of a twofold serial dilution of each peptide in LB medium. The challenge plate was incubated at 37°C for 2 hours. Peptide activity on pre-formed biofilm was evaluated by two independent methods: (i) visual observation of bacterial growth and (ii) counting of living bacterial cells after peptide treatment. In the first case, the peg-lid was removed from the challenge plate, rinsed with PBS and used to cover a 96-well recovery microtiter plate, each well containing 200 µl LB medium. The recovery plate was sealed, incubated at 37°C for 4 hours and then observed for any visible growth of bacteria detached from the peptide-treated biofilm. Growth of bacteria in a particular well indicated regrowth of planktonic cells from surviving biofilm. Minimum biofilm eradication concentration (MBEC) was defined as the minimum peptide concentration preventing regrowth of bacteria from the treated biofilm within 4 hours.Biofilm formation was performed by adapting the procedure described in 10 (99.9% killing) In the second case, to determine viable cell counts of biofilms after peptide treatment, pegs from the challenge microtiter plate were removed and transferred to Eppendorf tubes containing 500 µl PBS. After sonication at room temperature for 15 min to break up the biofilm and remove bacterial cells from the peg, aliquots of bacterial suspension were plated on LB-agar plates for counting. Colony forming units (CFU) were expressed as percentage with respect to control (peptide-untreated biofilms). Minimum bactericidal concentration (MBCb) was defined as the lowest peptide concentration required to reduce the number of viable biofilm cells by ≥3 logAnimal procedures were approved by the Ethical Committee of the Azienda Ospedaliera Universitaria Senese on November 18, 2010. Balb-c mice (20 g) were infected i.p. with lethal amounts of bacteria see  mixed in