Plasmodium falciparum 60S stalk ribosomal acidic protein P2 (PfP2) (PFC0400w), which gets exported to the IE surface for 6–8 hrs during early schizogony, starting around 26–28 hrs post-merozoite invasion. The surface exposure is demonstrated using multiple PfP2-specific monoclonal antibodies, and is confirmed through transfection using PfP2-GFP. The IE surface-exposed PfP2-protein occurs mainly as SDS-resistant P2-homo-tetramers. Treatment with anti-PfP2 monoclonals causes arrest of IEs at the first nuclear division. Upon removal of the antibodies, about 80–85% of synchronized parasites can be released even after 24 hrs of antibody treatment. It has been reported that a tubovesicular network (TVN) is set up in early trophozoites which is used for nutrient import. Anti-P2 monoclonal antibodies cause a complete fragmentation of TVN by 36 hrs, and impairs lipid import in IEs. These may be downstream causes for the cell-cycle arrest. Upon antibody removal, the TVN is reconstituted, and the cell division progresses. Each of the above properties is observed in the rodent malaria parasite species P. yoelii and P. berghei. The translocation of the P2 protein to the IE surface is therefore likely to be of fundamental importance in Plasmodium cell division.Malaria parasites reside inside erythrocytes and the disease manifestations are linked to the growth inside infected erythrocytes (IE). The growth of the parasite is mostly confined to the trophozoite stage during which nuclear division occurs followed by the formation of cell bodies (schizogony). The mechanism and regulation of schizogony are poorly understood. Here we show a novel role for a  Plasmodium in erythrocytes occurs through the peculiar process of schizogony where the nucleus undergoes several divisions followed by cell body formation. Inside a red blood cell (RBC), how does the parasite know when to start the cell division? Is there an influence from the external milieu? Here we report a novel involvement of a Plasmodium ribosomal protein P2 in this process. P2 gets trafficked to the infected erythrocyte surface at the onset of cell division for 6–8 hrs. Detergent resistant P2-tetramer is observed on erythrocyte surface and such an oligomerization occurs in the parasite just prior to the cell division. Blocking P2 on erythrocyte surface using monoclonal antibodies causes an arrest of parasites at the first nuclear division. Removal of antibodies reverses the arrest and parasites progress through cell division. Earlier reports have documented that a tubovesicular network (TVN), required for nutrient uptake, develops in early trophozoite stage of the parasites. Treatment with anti-P2-antibodies causes disintegration of TVN, resulting in impaired lipid import, which may be the eventual cause of the cell-cycle arrest. A study of this process of P2 oligomerization and elucidating the role of P2 on the infected erythrocyte surface at a specific parasite developmental stage might uncover novel regulations operating at the start of cell division in Plasmodium.Malaria is a serious human health problem and the disease manifestations are caused by erythrocytic stages. The growth of  Plasmodium, is a prevalent tropical infectious disease, which continues to take a large toll on lives . The malarial pathology is largely caused by the cyclic growth of the parasite within the infected erythrocytes (IE). Very little is known about molecular mechanisms of replication of Plasmodium in the erythrocytes. Cell division in Plasmodium occurs largely through a schizogonic process, in which the nuclei divide asynchronously at first into about 16–24 nuclei, followed by the formation of cell bodies Drosophila embryonic syncytial divisions Drosophila embryo. Certain filament forming fungal species also form multinuclear hyphal compartments, in which the nuclear position and cell cycle is only loosely coordinated with septum placement Malaria, caused by the species The entry of a cell into division in eukaryotic animal cells is controlled at two steps, the G0 to G1 transition, as also within the G1stage through a restriction (R) or ‘Start’ stage Plasmodium, or check-point controls in schizogony. Certain cell division components such as cyclin-dependent protein kinases (CDKs) and cyclins have been reported in Plasmodium falciparum and it is apparent that there are important divergences in the composition and properties of these components of cell cycle machinery in PlasmodiumPlasmodium have been controversial as to whether the cells go through the classical G1, S, G2, M phases; however, the asynchrony in nuclear divisions appears to favor the G1 phase followed by alternation between independent S and M phases for each nucleus Virtually nothing is known about the start of the nuclear division in Plasmodium falciparum ribosomal protein PfP0 as a protective protein during the asexual erythrocytic stages Plasmodium contains one gene each for P1 and P2 proteins, which possess a homologous carboxy-terminal domain as is observed in yeast and human P-proteins Neisseria gonorrhoeae P2 orthologue has been shown to exist at the cell surface and play a role in invasion In an earlier differential immunoscreen we have identified Plasmodium, the P-proteins were expressed in E. coli and specific reagents were generated. In this paper we report a novel involvement of the Plasmodium ribosomal P2 protein at the onset of cell division. We document a translocation of P2 protein, but not P0 or P1 proteins, to the infected erythrocyte surface during early cell division. We also observe an unusual cell-cycle arrest of Plasmodium at the first nuclear division, when treated with a panel of anti-P2-specific monoclonal antibodies (anti-P2-mAbs). Upon removal of anti-P2-mAbs, such an arrest was released in about 80% of cells even after 24 hrs of antibody treatment. The anti-P2-mAb treatment causes a disintegration of the tubovesicular network in the parasite-infected cells and results in an impaired lipid import, which may be one of the downstream causes for the cell-cycle arrest.In an attempt to assess the properties of the three P-proteins of P. falciparum P2 protein (PfP2), and certain carboxy-terminal deletion constructs, (P2Cdel20 and P2Cdel40), were expressed as His-tag fusion proteins, and several monoclonal antibodies (mAbs) were generated against these  was expressed as a GST-fusion protein. GST-PfP0C, which contained 80% of C-terminal PfP0 protein Plasmodium P2 protein and reacted specifically with the 27–49 amino-acids (aa) peptide  were tested, and none of these proteins exhibited IE surface localization  concentration was lower prior to 24 hrs, but even that did not vary significantly after 24 hrs PMI  search showed that MAMK motif is not present in any other Plasmodium protein, nor in the P2 protein of any other species. The 65 kDa portion of the uninfected erythrocyte ghost did not show any signature of PfP2. A representative spectrum for the NVLGAVNADVEDEVLNNFIDSLK peptide obtained from the parasite is shown in P. falciparum, was also seen consistently at the 65 kDa position from the parasite extract IP, albeit with a low score of 17 (www.Plasmodb.org). It is not clear as to whether PfP2 forms a complex with this protein. FPLC of asynchronous parasite extracts show large complexes containing PfP2 eluting at void volume (>600 kDa) . However, further analysis is needed to elucidate the role of this protein  in the physiology of PfP2. Since the score of all other peptides at 65 kDa was low, and the mass of this SDS-resistant P2 molecular species fitted a homotetramer, it appears that the 65 kDa protein band largely consists of an SDS-resistant 65 kDa PfP2-homotetramer. The homotetramer was also observed in the IE-ghosts from the two rodent parasite species P. berghei and P. yoelii  analysis . The IE-ed to MS . The peped to MS  and S2. ow score . The MAM protein , and a Pre of 17 . This pe. yoelii . In the . yoelii , but RBCIn order to determine the population of IE showing surface localization/exposure of PfP2, a flow cytometric analysis of solution stained cells was carried out and the fraction of cells, showing staining beyond a cut-off set up on flow cytometer , was meaThese results document that the total amount of protein PfP2 in the parasite, and the processes of oligomerization of PfP2 and its transport to the IE-membrane surface as an SDS-resistant homotetramer, are regulated in a development specific manner. The SDS-resistant P2-oligomerization occurs in the parasite at 24 hrs, just prior to the IE-membrane localization of P2 protein at 30 hrs PMI. The window of IE surface exposure of PfP2 is mainly from 28 to 36 hrs PMI, and the SDS-resistant 65 kDa PfP2 protein, occurring largely in the IE ghost and cytosol, is most likely to be a P2 homotetramer.P. falciparum Pf3D7 strain was episomally transfected with P2-GFP construct using the constitutive hsp86 gene promoter region  stages at 48 hrs  were observed rapidly . A largetibodies . No actiP. falciparum at various stages is shown in A flow cytometric analysis of the P2 surface stained cells showed that the population of cells with surface P2 was maximum at 30–36 hrs PMI in about 25% of cells, and that this population rapidly decreased significantly over the next 6 hrs to 5%, concomitant with an increase in DAPI . A repreP. yoelii IE also exhibited arrest upon incubation with the mAb E2G12 for 12 hrs, and could be rescued upon removal of antibodies  cells (P. yoelii both the SN and DN were predominant after incubation with E2G12 (P. berghei.tibodies . Upon retibodies . HoweverN) cells . Hence ith E2G12 . AntibodP. falciparum IEs can be reversibly arrested at the onset of first nuclear division specifically through the treatment of anti-P2 mAbs. Like P. falciparum, erythrocytes infected with rodent parasites could also be reversibly arrested using anti-P2 mAbs. Since P2 protein is very conserved, these results open up the possibility of targeting IE cells selectively, without affecting uninfected RBCs, for all Plasmodium species using anti-P2 mAbs. These results also indicate an important role of P2 protein on IE surface in Plasmodium. The P2 proteins of eukaryotic cells are deemed non-vital since yeast S. cerevisaea P2 protein can be knocked out, and such cells are viable P. berghei using standard constructs. However, the P2 gene could not be knocked out despite several attempts, although simultaneous knock-outs of Pf16 and MSP7 These results demonstrate that P. falciparum. Therefore it was envisaged that the anti-P2 mAb treatment might be affecting some IE signaling and/or crucial nutrient uptake. It has been documented that the parasite imports several nutrients via a tubovesicular network (TVN) P. berghei and P. falciparum within several hours -4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (FM4-64) Plasmodium plays a novel extra-ribosomal role through its trafficking to the infected erythrocyte surface as a SDS-resistant homo-tetramer at the point of the first nuclear division in the erythrocytic stage of the parasite. Upon treatment with a panel of anti-PfP2-specific mAbs, parasite development was arrested at the onset of nuclear division, and this arrest could be released upon removal of the antibodies. We also demonstrated that one of the downstream event(s) of such antibody-mediated arrest is the disintegration of the TVN, resulting in impairment of lipid uptake by infected erythrocytes.Our results show that the acidic ribosomal protein P2 of The acidic ribosomal P2 protein has been studied extensively mainly in context with the ribosomal functions in eukaryotic systems Plasmodium, since PfP0 protein was detected on parasite surface in vivo, through passive immunization followed by rodent parasite challenge Neisseria gonorrhoeae, the functional orthologue of P2 (L12), is surface localized; and is shown to mimic human chorionic gonadotropin implicating its role in cell invasion Plasmodium P1/P2 proteins do not appear to be absolutely essential for ribosomal functions, since we have shown that ribosomes containing just PfP0 (without any P1/P2 proteins) were capable of synthesizing proteins in a complementation study in Saccharomyces cerevisaePlasmodium P2 protein amino acid stretches (for instance MAMK)  the preliminary observations that we were unable to obtain a P2 knock-out in P. berghei; indicate Plasmodium specific important roles for the Plasmodium P2 protein. The trafficking of P2 protein to the infected red cell surface, during a specific window of parasite development, shows extra-ribosomal functions of this Plasmodium P2 protein. Surface exposure of a parasite protein on IEs would make the protein vulnerable to host-immune responses and conforming to this expectation most of the proteins exported to outer surface of IE are variant immuno-evasive malaria proteins Ribosomal proteins are known to play varied roles besides protein synthesis ce MAMK) ; b) simiP. falciparum proteins get exported to the erythrocyte var genes P. falciparum proteins get exported to the erythrocyte, with the exception of the variant protein PfEMP1 (and perhaps RIFINS and STEVOR family proteins), most remain intra-erythrocytic Plasmodium P2 protein has a putative trans-membrane domain (amino acids 64–84) but does not contain any signal sequence or PEXEL motif. The anti-P2-mAbs, E2G12 and A12D9, stain IE surface and can cause nuclear division arrest, and these map to the 27–49 amino acids-N-terminal and 92–112 amino acids-C-terminal regions of P2, respectively, suggesting that both these domains of P2 are IE-surface exposed. Exposure of both these domains would be unlikely if the 64–84 amino acids domain did act as the transmembrane domain. However, the protein P2 is present on the RBC surface as a homo-tetramer, and that may allow both N- and C- terminal domains to be accessible externally. It is also possible that P2 protein is present as a complex with other proteins, which provide the anchor to the IE-membrane. The comparative ease with which the P2 protein gets solubilized in biochemical studies, suggests that it may have a peripheral association with the IE-membrane.Several Plasmodium P2 protein aid in the export. The oligomerization of the P2 protein may be involved in this process. In the parasite, P2 forms SDS-resistant oligomers in a stage dependent manner, coinciding with the RBC surface translocation , and the G1 phase and DNA synthesis (S-phase) occurs in late trophozoites. Within hours the single trophozoite nucleus begins to divide into two daughter nuclear bodies (the first M phase) and an asynchronous independent nuclear division ensues P. falciparum schizogonic divisions, and it was observed that the first CP duplication occurs between 24 and 26 hrs PMI Very little is known about replication during Plasmodium. Certain structural features have been noted for the MTOC in Plasmodium. Recently, an Aurora-A-related kinase, PfArk1, was identified in P. falciparum and was shown to localize to mitotic spindle poles during schizogony P. falciparum parasites, PfCEN3 localizes to discrete regions of the nuclear membrane that coincide with the mitotic spindle poles Plasmodium is poorly understood. Biochemical, genetic, and genomic studies indicate that Apicomplexan (and in particular Plasmodium) signaling networks for cell division may not rely on all of the components that are conserved in other eukaryotes The CP and mitotic spindles constitute the microtubule organizing centres (MTOCs) in Plasmodium P2 protein gets exported to the IE surface and associates with RBC membrane at about 26–28 hrs PMI  and the parasite membrane. Finally it would need to signal across the nuclear membrane as well, since nuclear membrane appears to be intact during chromosomal division in PlasmodiumPlasmodium, which gets set up in early trophozoite for nutrient uptake We observed that  hrs PMI , at the It is known that serum factors are essential for parasite growth and there have been attempts to dissect out the essential components for parasite growth in the erythrocytic cultures. Earlier reports have shown that fatty acids are mandatory for the parasite for cell division Plasmodium erythrocytic development, since blockage through multiple pathways appear to block at this stage, and not allow parasites to undergo further cell-division. Drugs which interfere with sphingolipid metabolism (such as PPMP); which inhibit depolymerization of microtubules (such as Taxol); which block microtubule polymerization (such as colchicine); as well as anti-P2-mAb treatment - all appear to bring the cells to halt at this stage Plasmodium protein P2, blocking of which causes nuclear division arrest. Since PfP2 export to the IE surface occurs at a specific parasite cell division window, and anti-P2 mAb treatment arrests cell division, it is tempting to speculate that PfP2 protein, possibly in complex with other molecules, may be transported to IE surface to sense the external milieu and to signal whether to proceed, or to halt cell division until conditions are favourable.All these severe perturbations, such as impairment in lipid import or microtubule dysfunction appear to halt the cells at the first nuclear division. In the case of anti-PfP2 mAb treatment, the blocked IE show a spread of nuclei, from enlarged single nucleus up to a dumbbell shaped nuclei, but rarely proceeding beyond that . Thus thPlasmodium, especially since the presence of P2 protein on IE surface is transient, and PfP2 protein is conserved in the vertebrate host. Indeed, preliminary vaccination experiments using P2 protein demonstrates only a partial protection . However, coupling of anti-P2 mAbs to anti-malarials should allow selective targeting of drugs to Plasmodium-infected red cells, which has been difficult so far. Considering very little is known regarding the cell-cycle stages of Plasmodium, arrest of IEs with anti-P2 mAbs and then following them through the release would be extremely useful for determination of the transcriptomic, proteomic and metabolomic states of the parasite through the schizogonic cell division stages.Inhibitors such as PPMP and agents that affect microtubule functions  also arrest the cells, and have been documented as promising anti-malarials Plasmodium plays a novel extra-ribosomal role at the infected erythrocyte surface at the point of the first nuclear division in the erythrocytic stage of the parasite. So far, all P. falciparum IE surface-exposed proteins are documented to be variant antigens, and it has not been possible to selectively target the IEs. The conserved presence of the P2 protein in P. falciparum and in rodent species, and the identical behavior of anti-P2-mAb treated Plasmodium falciparum, yoelii and berghei IE, demonstrates that the mechanism of P2-mediated IE-surface function is fundamental to Plasmodium cell-division. Therefore, anti-malarials coupled to anti-P2 mAbs would allow selective targeting of drugs to Plasmodium-infected red cells to possibly all human Plasmodium species. In addition, synchronization of Plasmodium IEs using anti-P2 mAbs will help in further study of the peculiar schizogonic cell-cycle of Plasmodium. The oligomerization of P2 protein in the parasite, which is timed crucially to the start of cell division in IE, precedes the trafficking of P2 protein to the IE surface. Understanding the process of the oligomerization may elucidate some of the regulations operating on PfP2 protein transport. The major question regarding the role played by the P2 protein at the infected red cell surface remains elusive. Detergent resistant membrane domains are important components of cellular signals, and an analysis of the interactors of P2 protein in the infected RBC membrane may throw some light on the function of the infected-erythrocyte-surface localized P2 protein in Plasmodium.The acidic ribosomal protein P2 of Tata Institute of Fundamental Research (TIFR) Animal House is registered under CPCSEA , ministry of environment and forest, Govt. of India (registration no. 56/1999/CPCSEA) for breeding and experiments on animals. This study was carried out under strict accordance with the guidelines of CPCSEA, India, for the care and use of laboratory animals. The study was approved by the institutional animal ethics committee, TIFR, Mumbai (project no. TIFR/IAEC/2008-1) formulated by CPCSEA.Plasmodium falciparum. The procedure for such collection, details of informed consent and the frequency of samples to be collected were in accordance with a detailed proposal approved by the Institutional Human Ethics Committee (IHEC) of TIFR. The IHEC of TIFR functions as per the guidelines of Indian Council of Medical Research (ICMR), Govt. of India.Human blood was collected from volunteers, after obtaining their written consents, for the in-vitro cultures of + blood group, was collected in acid citrate dextrose as the anticoagulant. After removing the leukocytes, the erythrocytes were washed and suspended in complete RPMI (cRPMI with 0.5% Albumax). Asexual stages of P. falciparum 3D7 strain were maintained at 5% haematocrit in cRPMI at 37°C in a humidified chamber containing 5% CO2.Parasites were maintained in culture as described earlier P. falciparum cultures for each set of experiments.Infected RBCs were injected into male Swiss mice intraperitonially. Parasitemia was monitored on a daily basis and mice were sacrificed at around 10–40% parasitemia. Blood was harvested from the mice as per animal ethics protocol and used the same way as Plasmodium falciparum (3D7) genomic DNA using the following primers containing EcoRI  at the 5′ end and XhoI (NEB) at the 3′ restriction overhang, respectively.P2 gene was PCR amplified from GAATTCATGGCTATGAAATACGTTGCTG-3′5′-CCCC;Forward primer: CTCGAGTTAACCAAATAAGGAAAATCCTAAGTC-3′5′-GGGG.Reverse primer: Both the PCR amplified P2 gene fragment and the pProExHTa vector (Lablife) DNA were digested using EcoRI and XhoI restriction enzymes at 37°C, purified and ligated at 16°C for 16 hrs using T4 DNA ligase . DH5α competent cells were transformed, and positive clones were identified through plasmid purification and restriction digestion. PfP2CΔ20 and PfP2CΔ40 were also cloned in the same vector following the same methodology, excepting that the amplifications were carried out using the following reverse primers:CTCGAGTTATTCTTTCTTATCTTCfTTTCTTAG-3′5′-GGG; PfP2 CΔ40: CTCGAGTTAACCACCTCCAATATTTTG-3′5′-GGGG keeping the forward primer as mentioned above.PfP2 CΔ20: P. falciparum (3D7) cDNA library using the following primers:GST-PfP1 construct was made by cloning PfP1 gene between EcoRI and XhoI sites of the pGEX-4T-3 vector . PfP1, with one postulated intron, was amplified from a GAATTCATGGCATCAATTCCAGCATC-3′5′-CCCC;Forward: CTCGAGACCAAATAAGGAGAAACC-3′5′-GGGG.Reverse: Cloned PfP1gene was transformed in DH5α competent cells. All the constructs have been schematically described in E. coli BL21 DE3 strain and protein expression was induced by 0.5 mM IPTG . Recombinant PfP2 (rPfP2), rPfP2CΔ20 and rPfP2CΔ40 proteins were purified using Ni-NTA beads , while GST-PfP1 and GST-PfP0C proteins were purified using GST-sepharose beads . All the recombinant proteins were fusion proteins, with rPfP2, rPfP2CΔ20 and rPfP2CΔ40 containing additional 30 amino acids (aa) at the N-terminus, including 6-Histidine amino acids, totaling to 142, 122 and 102 aa, respectively. PfP2 was cloned in pQE and pET vectors to obtain non-fusion or a cleavable P2-recombinant protein. However, no stable expression of PfP2 protein could be achieved without these additional 30 amino acids. The native PfP2 protein (112 aa) moved on the SDS-PAGE at 16 kDa  was constructed in pBS-DHFR in which the polylinker sites flank a T. gondii dhfr/ts expression cassette conveying resistance to pyrimethamine. PCR primers PbP2 FP1 (GGGCCCGATATCACAAAATTATATATTAACAC 3′5′CCCC), and PbP2 RP1 (AAGCTT GATAAGCTGCAACGTATTTCATAGCC 3′5′ GGGG) were used to generate a 748 bp fragment 5′ upstream sequence of PbRP2 from wild type genomic DNA, which was inserted into ApaI and HindIII restriction sites upstream of the dhfr/ts cassette of pBS-DHFR. A 492 bp fragment generated with primers PbP2 FP2 (GAATTC GAAGAAGAAGATGATTTAGGATTTTCC 3′5′ CCCC) and PbP2 RP2 (TCTAGA GAACAACTGTATATACAATGTTCC 3′5′GGGG) from the 3′ flanking region of PbRP2 was then inserted downstream of the dhfr/ts cassette using EcoRI and XbaI restriction sites. The linear targeting sequence was released from the plasmid using ApaI/XbaI and transfected into schizont following the protocol The gene k/o strategy was followed as described in All the monoclonal antibodies were generated  using rPfP2 and screened against various constructs . PolycloP. falciparum lines expressing PfP2-GFP chimeric protein, we used a modified plasmid vector pSSPF2/ACP-GFP using hsp86 promoter sequence  and 50 µg of plasmid DNA (prepared using QIAGEN plasmid kit) was added to 400 µl of the RBC suspension. Electroporation was carried out in a 0.2 cm cuvette (BioRad) using a BioRad gene pulser . Two days after electroporation, the antifolate drug WR99210 (Sigma) was added to cultures at 5 nM final concentration to select for transfected parasites.To generate transgenic sequence   with protease inhibitor cocktail , with brief sonication at 4°C. The lysate was centrifuged at 15,000 g at 4°C for 30 min and the supernatant was used for the immunoblots after protein estimation using Bradford reagent (Sigma). Before loading, the protein was mixed with gel loading buffer  and boiled for 10 min. Samples were resolved typically on 10–12% SDS–PAGE and proteins were transferred to methanol-activated Polyvinylidene Fluoride (PVDF) membrane (Millipore) using anode buffer  and Trans Blot Semi Dry Transfer system . Membranes were blocked with 5% non-fat skim milk powder in 1× PBS overnight and probed with specific antibodies. Primary antibody dilution was made in PBST (PBS containing 0.2% Tween-20) and incubated with the membrane for 3 hrs at RT on a rocker. Primary antibody binding was detected by appropriate secondary antibodies conjugated to horseradish peroxidase . Dilution of secondary antibody was made in PBST. After every incubation, membrane was washed with PBST at least 4–5 time and between every 5 mins interval washing buffer was changed. The immunoblots were developed using the ECL (Amersham) also by Super signal West Picochemiluminescent substrate . The antibodies used were E2G12 (1∶100), A12D9 (1∶100), α-PfP1 polyclonal (1∶2000), E5F4 (1∶100) ; anti-MSP1-19(1∶1000) ; anti-β tubulin antibody (1∶1000) ; anti-PCNA (1∶2000) , α-β actin antibody (1∶1000) .The soluble recombinant proteins were harvested from bacteria after IPTG induction and purified using GST-or Ni-NTA column as described earlier. To prepare 2O2. The OD was measured at 405 nm and plotted after the subtraction of negative control value. For each antigen, experiments were done in triplicate. For peptide ELISA, 7 different peptides along P2 protein were generated . These peptides were 1. PfP2 aa 41–70 (H-LNNFI DSLKG KSCHE LITDG LKKLQ NIGGG-OH), 2. PfP2 aa 67–89 (H-IGGGV AAAPA GAAAV ETAEA KKE-OH), 3. PfP2 aa 1–11 (H-MAMKY VAAYL M-OH), 4. PfP2 aa 1–20 (H-MAMKY VAAYL MCVLG GNENP-OH), 5. PfP2 aa 27–49 (H-NVLGA VNADV EDEVL NNFID SLK-OH), 6. PfP2 aa 86–101 (H-AKKED KKEEK KEEEEE-OH), 7.PfP2 aa 15–30 (H-GGNEN PSTKE VKNVL G-OH).In a 96 well plate , 200 ng of recombinant proteins and peptides in PBS were coated and incubated at 37°C for 3–4 hrs. Wells were blocked using 5% skimmed milk in PBS for 1 hr at 37°C. Primary antibodies, anti-PfP2 mAbs  were used in 1∶100 dilutions whereas anti-PfP1 polyclonal antibody was used in 1∶2000 dilutions. Primary antibody was incubated for 3–4 hrs at 37°C. Anti-mouse HRP conjugated  and anti-rabbit HRP conjugated  secondary antibodies were used in 1∶5000 dilutions in PBS. After every incubation, plates were washed with PBS containing Tween-20 (0.05%) for at least 5–6 times with a gap of 5–7 min between each wash. Bound secondary antibody was developed using the substrate ABTS  and HP. falciparum, P. berghei and P. yoelii infected RBC population was performed in solution. Infected blood was centrifuged at 500 g for 5 min, pelleted RBCs were washed twice, and resuspended in complete-RPMI (cRPMI) medium. Cells were fixed using 0.25% glutaraldehyde in PBS for 20 min at 4°C g, washed and treated with appropriate Alexa 488 or 594 conjugated secondary antibody at 1∶500 to 1∶1000 dilutions  for 2 hrs. After washing 3–4 times, cells were incubated for 5 min with DAPI (1 µg/ml) . Tubulin tracker  was also used to stain tubulin of the parasite, but this step was carried out in the absence of glutaraldehyde. For permeabilized IFA, both on slides and in solution, fixation method was followed as described above. Post-glutaraldehyde treatment, cells were permeabilized with 0.05 to 0.1% TritonX-100 in PBS for 20 min at RT, and the media used subsequently was PBS containing 0.05 to 0.1% TritonX-100. Cells were treated with secondary antibody conjugated with either Alexa 488 or 594 . To stain the nucleus of the parasite, DAPI (1 µg/ml) was used. Cells were imaged using confocal microscope Exciter LSM500 from Zeiss (Germany). Acquired IFA images were processed by ImageJ software. The primary antibodies used were anti-PfP2 mAbs E2G12 and A12D9 (1∶50 or 1∶100), α-PfP1 polyclonal (1∶1000), anti-PfP0 mAb E5F4 (1∶50); anti-MSP1-19 (1∶1000) ; anti-β tubulin antibody (1∶1000) ; anti-PCNA (1∶2000) , Tubulin-tracker , α-actin antibody (1∶1000)  .SIFA of g for 5 min, and washed with cRPMI once. RBC pellet was resuspended in 0.15% Saponin and protease inhibitor cocktail  and 1 mM PMSF in PBS, pH 7.4 for15 min at 37°C. Sample was then centrifuged for 10 min at 10,000 g at 4°C to get the parasite pellet, which was sonicated and stored at −80°C. About 60–70% of the opaque supernatant (ghost and cytosol fraction) was gently separated to avoid contamination with the parasite pellet. This supernatant fraction was pelleted at 20,000 g for 2 hrs at 4°C, washed twice with PBS, pH 7.4, and stored at −80°C for use as IE ghost. After ghost precipitation, the supernatant (IE cytosol) was stored at −80°C for subsequent analysis. All buffers contained protease inhibitor cocktail .Infected RBC at about 10% parasitemia was pelleted at 500 P. falciparum (Pf) and P. yoelii (Py) parasite pellets were suspended in 200 µl non-denaturing lysis buffer  in the presence of protease inhibitor cocktail  on ice for 15 min. Cells were then briefly sonicated  for 1 min, and centrifuged at 15,000 g at 4°C. The supernatant was collected, protein was estimated using Bradford reagent , and 100 µg protein was incubated with 10 µl packed protein G-sepharose beads  at 4°C for 1 hr for pre-clearing. Packed Protein G sepharose beads (20 µl) was washed repeatedly and the protein content of the pre-cleared lysate was estimated. For 0.1 mg protein lysate, 0.001 mg of anti-P2 mAb (E2G12) or control pre-immune serum was added to get a final concentration of antibody of 1∶100. The protein-antibody solutions were incubated at 4°C for 6 hrs on a rotary shaker, followed by incubation with 20 µl of packed Protein G-sepharose beads at 4°C for 2 hrs. Subsequently, the beads were centrifuged at 500 g and washed 6 times with lysis buffer. To the beads, SDS-PAGE loading buffer was added and boiled for 5 min followed by centrifugation at 15,000 g for 15 min at room temperature. The supernatant was loaded on SDS-PAGE for immunoblot. Appropriate gel-bands were cut out and processed for MS analysis.SDS-PAGE of appropriate protein preparation was carried out and the gel was stained with Coomassie Brilliant Blue R250  or through silver staining. Gel pieces were cut at the region corresponding to 65 kDa and used for in gel digestion as described below. Gel pieces were washed with 100 mM ammonium bicarbonate for 15 min at 37°C followed by repeated washings with 50% Acetonitrile (ACN) in 100 mM ammonium bicarbonate (buffer A) for 15 min at 37°C, until the gel pieces were completely destained. 10 mM DTT  in 100 mM ammonium bicarbonate was added and incubated at 56°C for 30 min. Gel pieces were then incubated in 55 mM Iodoacetamide  prepared in 100 mM ammonium bicarbonate at room temperature for 35 min in the dark, followed by washing once with100 mM ammonium bicarbonate and twice with buffer A. Digestion was set up by the addition of trypsin  (0.2 ug/ul in 100 mM ammonium bicarbonate) to give a trypsin: protein ratio of 1∶20; incubated at 37°C for 12 hrs. The supernatant was collected without the gel pieces and peptides were extracted in three stages by washing once with 50 mM ammonium bicarbonate and twice with 10% formic acid in 50% ACN. All the extracts were pooled and dried under vacuum. To desalt the peptide preparation, C18 spin columns  were used as per manufacturer protocol. Briefly, C18 column material or resin was activated using 200 µl of buffer A, and centrifuged to separate resin from ACN. Resins were equilibrated with 200 µl of 5% ACN, 0.5% formic acid (buffer B) and centrifuged again to separate from the ACN/formic acid. This was repeated 3 times. Vacuum dried samples were dissolved in 200 µl of sample buffer containing 2% formic acid in 20% ACN. Samples were loaded on to the resins and subsequently centrifuged to get the supernatant. Again the supernatant was loaded on to the column and repeated 4 times to ensure maximum binding of the peptide to the column material. Resins were washed twice with 200 µl of buffer B. Peptides were eluted using 50 µl 70% ACN. The samples were dried completely under vacuum and suspended in 10 µl of 5% ACN in 0.05% formic acid, prior to MS/MS analysis using ESI MS/MS, nano LC, model no. 6510, Agilent Technologies and data was analyzed using Mascot protein database.P. falciparum culture was synchronized using sorbitol P. falciparum culture used was typically 2–5%. At appropriate time points, samples from triplicate wells were collected, washed with cRPMI twice and smears were made on glass slides, and remaining cells were pelleted for SIFA, IFA or parasite/IE protein preparation. Counting was performed by staining the nucleus with DAPI and determining the numbers of single nucleated (SN); di-nucleated (DN); tri-nucleated (TN) and MN  cells. For statistical analysis, each microscopic field was counted and 100 such fields were used for analysis of the percentage of various cell types. Each field consisted of about 100 cells, of which 8–10 cells were infected.P. falciparum 3D7 infected erythrocytes were synchronized and IE from different time points (PMI) were solution stained using anti PfP2 mAb E2G12 (neat culture supernatant) and DAPI (1 µg/ml). Up to 10 million cells were counted using LSR Fortessa  and analyzed by FACS DIVA software. Gating strategy has been shown in BODIPY-TR ceramide  was used to stain tubo-vesicular network (TVN) as described in FM4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)- phenyl) hexatrienyl)pyridinium dibromide)  is a fluorescent lipid marker which was used to monitor endocytic lipid import as described in P. falciparum 3D7 infected RBCs of different erythrocytic stages, starting from 6 hrs to 48 hrs PMI, were incubated with 16 µM of FM4-64 for 15 min at 37°C in cRPMI. Cells were washed twice using cRPMI and stained for the nucleus using DAPI. The uptake of FM4-64 was monitored under live cell conditions. The uptake of FM4-64 was measured under different conditions, such as control (no antibody), in the presence of E2G12 (neat culture supernatant) and after removal (washed) of E2G12. About 3–4 million cells were counted in triplicate and monitored for mean fluorescence intensity of FM4-64.p value<0.05, denoted by *. A p value<0.01 was denoted by ** and a p value<0.001 was denoted by ***.Statistical significance was calculated by two ways ANNOVA with post-test, for all pair of columns. Any pair was considered to be significantly different if they yielded a Figure S1ClustalW analysis of ‘P’ proteins. (A) Clustal W of P2 protein across several Plasmodium species, exhibiting 75% identity. The bar shows the position of putative transmembrane domain as predicted by TMpred and TMHMM2.0 and TopPred transmembrane domain prediction software from expasy.org (B) Amino acid sequence comparison of P. falciparum P1, P2 and P0 proteins. Conserved amino acids have been shown in yellow, identical residues in green, similar residues in cyan.(TIF)Click here for additional data file.Figure S2Immunoblot of parasite, IE ghost and IE cytosol proteins using various antibodies.P. falciparum infected erythrocytes from asynchronous cultures were lysed gently with saponin to separate parasite and the IE components. Immunoblots of 40 µg each of parasite protein lysate (Pf), infected erythrocyte ghost (IE ghost), and infected erythrocyte cytosol (IE cytosol) were probed with α-P2 mAb E2G12, α-β actin mAb and α-MSP1 polyclonal antibody.(TIF)Click here for additional data file.Figure S3Immunoprecipitation (IP) of P. falciparum parasite crude proteins using E2G12. IP of parasite crude protein extract was carried out using anti-P2 mAbs E2G12. (A) Protein complex was separated on a 12% SDS-PAGE and silver-stained. The 65 kDa band (shown by arrow) was subjected to ESI MS/MS analysis. (B) Arrow indicates parent ion and peptide sequence was determined using observed and calculated mass. Data was analyzed using Matrix Science-Mascot database. (C) Immunoblot of (I) P. berghei and (II) P. yoelii infected RBC ghost and RBC cytosol probed using anti-PfP2 mAb E2G12 and anti-spectrin antibody.(TIF)Click here for additional data file.Figure S4Gating strategy of Flow cytometry. (A) shows the FSC vs SSC plot of all the particles drawn in the flow cytometer (BD Fortessa). Out of these we gated out the single cell population as shown. Further analysis was carried out using only this population. (B) Staining pattern of uninfected single cells with DAPI. This was done to set a threshold level of DAPI fluroscence beyond which the cells were considered to be DAPI positive (infected). (C) DAPI staining of RBCs with asynchronous stages containing 2% parasitemia. Note that the infected cells show a DAPI fluroscence beyond the threshold set previously. (D) Solution staining pattern of uninfected red cells with anti-P2 mAb E2G12. This was done to set a threshold level of fluroscence beyond which the cells were considered to be P2 positive. (E) Solution staining of RBCs with asynchronous stages having 2% parasitemia using E2G12. (F) Staining of uninfected red cells with FM4-64 to set a threshold level for FM4-64 fluroscence. (G) FM4-64 staining of RBCs with 2% parasitemia. The uptake of FM4-64 by infected RBCs was strong with a large shift in MFI.(TIF)Click here for additional data file.Figure S5Vector map for P2/pSSPF2. The gene expression of P2-GFP is carried out by two units in the malarial parasite. The first unit is for expressing the recombined gene of interest, P2  between BglII and AvrII sites. The GFP tag  is downstream of the gene of interest between AvrII and XhoI (or PstI) sites. The expression of this recombined gene is under the regulation of P.falciparum heat shock protein 86 promoter region  and the 3′ sequence of P. berghei DHFR-TS gene . The other vital unit for selection of transfectants is the human DHFR gene  which confers resistance under drug pressure with the anti-folate WR99210. P. falciparum calmodulin promoter , and the 3′ sequence of the P. falciparum histidine-rich protein 2 gene  drive the expression of this unit. The two expression units are arranged in head-to-head orientation on either side of the 0.8 kb DNA sequence containing the Rep20 repeats . The arrows indicate the direction of transcription in each expression unit. Unique restriction sites AvrII, BamHI, BglII, EcoRI, HindIII, NotI, PstI and XhoI are indicated. The plasmid backbone was derived from the E. coli vector pGEM (TIF)Click here for additional data file.Figure S6Arrest of P. falciparum infected erythrocytes using anti-P2 mAb (A12D9). (A). Synchronized P. falciparum cells were treated with A12D9 mAb for 24 hrs starting from 12 to 36 hrs PMI. At 36 hrs the arrested cells were washed and split into two flasks and cultured for further 24 hrs with and without A12D9 . The % IE was scored using DAPI at 36 hrs, and after another 24 hrs post washing; corresponding to 60 hrs PMI. (B and C). Representative images for the DAPI stained cells showing control and arrested cells in the presence of A12D9 antibodies. Scale bar indicates 2 µm.(TIF)Click here for additional data file.Figure S7P. falciparum growth inhibition in culture using anti-P2 mAb (E2G12). (A). Synchronized P. falciaprum infected RBCs at 8% parasitemia were treated with anti-P2 mAb (E2G12) or Sp2/O at 1 mg/ml from 12 to 60 hrs. Sp2/O is the hybridoma cell culture supernatant which was ammonium sulfate precipitated the same way as the E2G12 mAb supernatant. Parasitemia was measured through Geimsa staining at 48 hrs and at 60 hrs. **p<0.01. n = 5 (B) Percent parasitemia change in comparison with the starting parasitemia at different time points in parasite development for control (no antibody) and E2G12 treated cells as determined through flow cytometric analysis using DAPI stain. *p<0.05.(TIF)Click here for additional data file.Figure S8Flow Cytometry dot-plots of surface PfP2 and DAPI Staining. Representative flow cytometric data of P. falciparum infected synchronously cultured cells, double stained with E2G12 and DAPI, at various stages of development. The stretch of DAPI positive cell population is in quadrant 4 and P2/DAPI double positive cells are in quadrant 2. The percentages mentioned in Q2 and Q4 are for DAPI positive infected cells only. Panels A–D show dot-plots for control infected RBCs without antibody at A: 12 hrs; B: 30 hrs; C: 36 hrs; and D: 48 hrs in the erythrocytic cycle, while Panels E–G show dot-plots of infected RBCs incubated with anti P2 mAb (E2G12) at E: 30 hrs; F: 36 hrs and G:48 hrs PMI. The mAb was added at 12 hrs PMI. The total number of DAPI positive cells decrease considerably by 48 hrs in the presence of E2G12.(TIF)Click here for additional data file.Figure S9Flow Cytometry histograms of PfP2 Staining. Representative flow cytometric frequency histograms of PfP2 stained P. falciparum infected RBCs at various time points PMI. During the acquisition of such data, only the infected cells were gated out through DAPI staining, and appropriate cutoff was marked for P2 positivity  added at 12 hrs; C with anti-P2 mAb (E2G12) added at 12 hrs and washed off at 36 hrs, monitored at 42 and 48 hrs PMI.(TIF)Click here for additional data file.Figure S10Growth inhibition and synchronization of P. yoelii infected erythrocytes using anti-PfP2 mAb E2G12 in culture.P. yoelii infected erythrocytes (IE) were incubated with E2G12 for 24 hrs; washed thoroughly with cRPMI, split into two sets and cultured further for 11 hrs in cRPMI, with (Ab present) or without (Ab removed) mAb E2G12. (A) Cells were removed at different time points and solution immunofluorescence (SIFA) was performed. (B) DAPI images of IE after 11 hrs of culturing; E2G12 removed (control) and E2G12 continued. (C) Shows the representation of single nucleated (SN), di-nuclear (DN); tri-nuclear (TN) and >3 nuclei (MN) cells in the population, as determined through DAPI staining of infected RBC population up to11 hrs in the absence (C-Upper: Ab removed) and presence of E2G12 (C-Lower: Ab present). (D) Upper panel shows SIFA of live P. yoelii infected RBC using E2G12 (red) and Tubulin Tracker (green) and Lower panel shows IFA of P. yoelii infected RBCs cultured with E2G12 for 32 hrs using E2G12 (green) and antibodies against proliferating cell nuclear antigen (PCNA) and tubulin (red). *P<0.05, **P<0.01. Significance of each data point was calculated with respect to the previous data point. n = 5. Scale bar indicates 2 µm.(TIF)Click here for additional data file.Figure S11BODIPY-ceramide staining of Tubovesicular network (TVN) of P. falciparum and P. berghei infected RBCs in the presence of mouse pre-immune sera, anti-P2 mAbs (A12D9 and E2G12) and after removal of E2G12. Representative images of BODIPY-ceramide staining of P. falciparum and P. berghei infected RBCs after various treatments; P. falciparum infected (A): control cells; (B): cells treated with A12D9. Antibodies were added at 12 hrs and the cells were observed at 24 and 36 hrs. (C):P. berghei and (D):P. falciparum infected cells treated with control (CI) mouse pre-immune sera; (DI) no antibody;  with E2G12 continued and  after removal of E2G12, monitored after 4 hrs post-washing. Scale bar indicates 2 µm.(TIF)Click here for additional data file.Table S1Mass spectrometric data of immunoprecipitated 65 kDa band excised from SDS-PAGE of IE ghost preparation from IEs at 30 hrs PMI. List of peptides of P2 and other proteins was obtained through mascot analysis and is shown with all mass spectrometric parameters.(XLS)Click here for additional data file.Table S2Mass spectrometric data of immuno-precipitated P. falciparum parasite protein using E2G12 . List of peptides of P2 and other proteins was obtained through mascot analysis and is shown with all mass spectrometric parameters.(XLS)Click here for additional data file.Video S13D reconstruction of confocal images of tubovesicular network stained using BODIPY- ceramide in P. falciparum infected RBC showing the mesh like structures.(AVI)Click here for additional data file.Video S23D reconstruction of confocal images of tubovesicular network stained using BODIPY-ceramide in P. falciparum infected RBC treated with anti-PfP2 mAbs E2G12, showing the absence of mesh like structures.(AVI)Click here for additional data file.


Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation. Enhancer of zeste homolog 2 (EZH2) is the catalytic component of polycomb repressive complex 2 (PRC2), an epigenetic regulator of stem cell pluripotency, and expression of tissue-specific genes involved in cellular differentiation and developmental programs ,2,3,4,5.Overexpression of EZH2 has been found in a number of human cancers ,14. IncrBecause of the important role of EZH2 in tumorigenesis, much effort has been put into discovery of inhibitors of EZH2 catalytic activity. Recently, several groups reported novel inhibitors of EZH2 ,30,31,32To better understand the molecular mechanism of EZH2 function, we solved the crystal structure of the C-terminal region of EZH2 containing a novel CXC domain and the catalytic I-SET, SET, and post-SET domains. Although the majority of canonical SET domain characteristics were observed, our structure reveals significant differences between the arrangement of the I-SET and post-SET regions compared to that of other SET-domain containing proteins. An unusual conformation of the post-SET domain suggests a potential mechanism of activation of EZH2 by the other core PRC2 subunits. Our structure also provides insight into the gain-of-function mutations in lymphomas.A DNA fragment encoding residues 520-746 of human EZH2 (Uniprot isoform 1) was sub-cloned from cDNA (GenBank BC010858) into the baculovirus expression vector pFBOH-LIC (GenBank EF456740).  The pFBOH-LIC N-terminal Hexa-His tag was removed by inserting the gene at an upstream Nco1 restriction site and a C-terminal Hexa-His tag introduced by PCR. The resulting expressed protein product is expected to have the AA sequence: MYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPHHHHHH. The protein was expressed in Sf9 cells (Invitrogen). The harvested cells were resuspended in lysis buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazol, 2 mM β-mercaptoethanol, 5% glycerol, 0.6% NP-40, protease inhibitor cocktail (Roche), 3000 U of benzonase (Novagen). Cells were lysed by brief sonication. The clarified lysate was loaded onto a 2 mL TALON column (Clonetech). The column was washed with 50 column volumes of 20 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl, 5% glycerol and 5 mM imidazole. The bound protein was eluted with elution buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5% glycerol, 250 mM imidazole. The eluted protein was further purified to homogeneity on a Superdex200 column , equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 500 mM NaCl.Purified EZH2 (10 mg/mL) was mixed with SAM at 1:10 molar ratio of protein:SAM and crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solution containing 20% PEG 3,350, 0.1 M HEPES, pH 7.5, 0.2 M Li SO4. The crystals were frozen in liquid nitrogen using 10% ethylene glycol as cryo protectant.An initial 2.24 Å diffraction data was collected at the Canadian Light Source (CLS) beamline CMCF 08ID-1 at the Zn absorption edge and used to solve the structure of EZH2 by the single-wavelength anomalous dispersion (SAD) phasing method. A second data set (2.0 Å) collected at beam line 19ID of Advanced Photon Source (APS), Argonne National Laboratory was used to further refine of the structure. All data sets collected at a temperature of 100K and were processed using the HKL-3000 suite . REFMAC 3H-labeled methyl group into a peptide corresponding to residues 21 to 44 of histone H3 [H3(21-44)] using Scintillation Proximity Assay (SPA). The enzymatic reactions were conducted in triplicate at 23 °C with 0.5 hour incubation of 20 µl reaction mixture in 20 mM Tris-HCl, pH 8.0 , 8 µM cold-SAM, 2 µM H3(21-44) peptide) and various concentrations of enzymes. Enzymatic reactions were stopped by adding 7.5 M Guanidine hydrochloride followed by 180 µl of buffer , mixing and then transferring to a 96-well FlashPlate . After mixing, the reaction mixtures in Flash-plates were incubated for 2 hour and the CPM were measured using Topcount plate reader . Methyltransferase activity assays for trimeric EZH2 complex (EZH2-EED-SUZ12) and EZH2 (520-746) were performed by monitoring the incorporation of The kinetic parameters for trimeric EZH2 complex were determined using 20 nM of EZH2 at a fixed concentration of peptide (5 µM) and varying concentrations of SAM (up to 20 μM), or at a fixed concentration of SAM (10 µM) and varying concentrations of peptide (up to 5 μM) and therefore kinetic parameters are considered apparent values at the above mentioned conditions. Assays were performed in 20 mM Tris-HCl pH 8, 0.01% Triton X-100, 5 mM DTT. The reaction mixtures were incubated for 30 min at 23 °C. To stop the reactions, 7.5 M Guanidine hydrochloride was added and mixed. A total of 10 µl of the reaction mixture was spotted onto SAM2® Biotin Capture Membrane  and placed at room temperature for 5 minutes. Membranes were washed with 2 M NaCl and deionized water at least twice each and were dried. Scintillation liquid was added and counts per minute (CPM) were measured. Experiments were performed in triplicate. Purified EZH2 (520-746) was dialyzed for binding assay with SAM and peptide in 50 mM Tris-HCl buffer, pH 8.0 and 250 or 500 mM NaCl respectively. Solutions of 1 mM SAM or 0.62 mM histone peptide H3 (residues 21-44) in dialysis buffer were injected into the sample cell containing approximately 1.4 ml of 0.05 mM protein solution. ITC titrations were performed on a VP-ITC Micro Calorimeter from GE Healthcare at 25 °C by using 10 μl injections with a total of 25 injections.Analysis of crystallographic structures was conducted with ICM . “Distances” between the two edges of the substrate binding groove were calculated as the distance between the backbone carbonyl of the catalytic tyrosine, at the C-terminal extremity of the SET domain, and the backbone nitrogen of the I-SET domain which forms a conserved hydrogen bond with the backbone carbonyl of the substrate lysine in all available ternary SET domain methyltransferase structures. These tWe crystallized and solved the 2.0 Å structure of the C-terminus of EZH2 , PRDM2 (2QPW) , PRMD4 . PRDMs aTogether, these results indicate structural features underlying suboptimal binding or positioning of both cofactor and substrate in isolated EZH2, which are expected to preclude catalysis. To test whether the specific structural features observed in our structure contribute to lack of substrate or cofactor binding we designed several EZH2 mutations that would be expected to disrupt or ‘relieve’ the apparent inactive conformations of the I-SET, post-SET and CXC domains. We mutated post-SET residue Ser729  to an aspartate to destabilize the observed conformation of the post-SET domain. We also mutated I-SET residue Phe667  into a leucine to remove interactions that stabilize the shifted conformation of the I-SET domain . NeitherFinally, given the recently reported potent SAM-competitive inhibitors of EZH2 ,30,31,32The crystal structure presented here reveals that EZH2 adopts a canonical SET domain methyltransferase fold in the absence of binding partners, and that the catalytic site is well formed . We findOur EZH2 structure contributes to the growing evidence for conformational plasticity of the I-SET and post-SET domains of SET domain methyltransferases and hints at potential mechanisms of regulation of catalytic activity. The post-SET domain participates in the formation of both cofactor and substrate binding sites, and is expected to adopt a catalytically competent conformation only when bound to both SAM and peptide Click here for additional data file.Figure S2EZH2, EHMT1 and vSET catalytic sites. The catalytic site of EZH2 is structurally closer to that of the human H3K9 dimethylase EHMT1 than the viral H3K27 trimethylase vSET .(TIF)Click here for additional data file.Figure S3Electrostatic potential. The expected location of the substrate peptide binding site of EZH2, at the interface of the post-SET (blue) and I-SET (cyan) domains, is electronegative. Bottom: electrostatic potential color coding. Blue: electropositive; red: electronegative.(TIF)Click here for additional data file.Figure S4Post-SET domain conformation. The post-SET domain (blue) of cofactor-bound SET domain methyltransferases is structurally diverse but always participates in the formation of the cofactor site. In the EZH2 structure, it projects away from its expected position and the cofactor is absent. When present, cofactor is shown as CPK and substrate is in green.(TIF)Click here for additional data file.Figure S5EZH2’s secondary pocket. A mesh representation of EZH2 (color-coding as in other figures) with the cofactor of a superimposed EHMT1/GLP structure (conserved hydrogen-bonds are highlighted), reveals the existence of a secondary pocket, juxtaposed to the cofactor site.(TIF)Click here for additional data file.Figure S6EZH2’s dimeric state in solution. EZH2 elutes both as a monomer and dimer out of a gel filtration column.(TIF)Click here for additional data file.Figure S7Interactions between the post-SET and I-SET domains. The altered orientation of the post-SET domain, resulting in incomplete formation of the cofactor site, is associated with a buried conformation of Ser 729. The shifted orientation of the I-SET domain, resulting in closure of the substrate-binding groove, is stabilized by a hydrogen-bond between the backbone of N668 and Y726, and orthogonal pi-stacking between Phe 667 and Phe 724. Color coding as in other figures.(TIF)Click here for additional data file.Figure S8Post-SET domain in PRDM structures. The post-SET domain in all human PRDM structures (blue) is oriented away from the putative cofactor site, and the cofactor is absent from all these structures. In a mouse PRDM9 structure crystallized in complex with SAH (green sticks), the post-SET domain (green ribbon) is folded on the cofactor. Mesh representation of human PRDM9 where the post-SET domain was truncated. Post-SET domain of human PRDM1 (PDB code 3DAL), PRDM2 (2QPW Wu 20084102), PRMD4 (3DB5), PRDM9 (4IJD), PRDM10 (3IHX), PRDM11 (3RAY), and PRDM12 (3EP0), and mouse PRDM9 (4C1Q).(TIF)Click here for additional data file.Figure S9Conserved, but incomplete folding of the cofactor-binding site. The cofactor site of EZH2 is in a conformational state that is compatible with the formation of 4 out of 6 hydrogen bonds (black) between the SET domain and the cofactor that are conserved across all available structures of cofactor-bound SET-domain methyltransferases. Preserved hydrogen bonds are shown in cyan. Lost hydrogen bonds are shown in magenta. The EZH2 structure (color coding as in other figures) is superimposed with cofactor-bound EHMT1/GLP (beige - PDB code 2RFI). Top-right: same view, with a mesh representation of EZH2, where the EHMT1/GLP ribbon was removed.(TIF)Click here for additional data file.Figure S10Atypical conformations of the I-SET and post-SET domains. Superimposition of the EZH2 structure  with ternary complexes of EHMT1/GLP (PDB code 2RFI), SETD7 (PDB code 1O9S) and SETD8 (PDB code 1ZKK) bound to cofactor  and substrate (no shown) shows that the I-SET domain of EZH2 is shifted towards the post-SET domain, resulting in hydrogen-bonding between Asn 668 and Tyr 726.(TIF)Click here for additional data file.


Wide area circumferential ablation (WACA) is used to achieve pulmonary vein isolation (PVI) for treatment of AF. AF recurrences associated with pulmonary vein reconnection are common.We assessed the hypothesis that acute PVI results from a combination of irreversible (necrosis) and reversible  tissue damage at the left atrial (LA)-pulmonary vein (PV) junction as evaluated by cardiac magnetic resonance (CMR) imaging.15 patients with paroxysmal atrial fibrillation (PAF) underwent CMR scanning pre and immediately post WACA. 12 patients  had good quality images for delayed enhancement DE (necrosis) Figure  and highTwenty four pairs of pulmonary veins were electrically isolated during the ablation and subsequently divided into no recurrences and recurrences of AF; quantifying the percentage(%) circumferential lesion of DE, high T2-weighted signal and DE + T2. Patients free from AF at follow-up  had mean ± SD of DE and high T2-weighted signal of 73.2±25.4% and 19.3±21.9% respectively. Patients with recurrences  had mean ± SD of DE and high T2-weighted signal of 46.5±25.9% and 47.5±28.3%. Between the two groups, the statistical difference in the T2-weighted means (p=0.01) was twice that of the DE means (p=0.02). No statistical difference was seen in the DE+T2 means. On visual assessment areas of high T2 signal (oedema) not only overlapped with areas of DE but also filled in gaps between areas of DE, producing in combination almost circumferential lesions in all PVs.CMR scans performed post ablation confirms DE (necrosis) and high T2 signal (oedema) forming a near complete ring around the pulmonary veins contributing to electrical isolation. In patients with recurrent atrial fibrillation, more of this ring was composed of high T2-weighted signal than DE. This may provide evidence for immediate post procedural oedema without necrosis being the mechanism of pulmonary vein reconnection and recurrence of AF post catheter ablation during follow-up.


P<0.001) and negatively correlated with vimentin expression and lymph node metastasis (P<0.05). Meanwhile, p120ctn cytoplasmic expression negatively correlated with E-cadherin expression (P<0.001) and positively correlated with vimentin expression and lymph node metastasis (P<0.05). Cells expressing high (H460 and SPC) and low (H1299 and LK2) levels of p120ctn were screen to investigate its impact on EMT. E-cadherin was restricted to the cell membrane in H460 and H1299 cells, whereas it was expressed in the cytoplasm of SPC and LK2 cells. Ablation of endogenous p120ctn isoform 1A in cells expressing high levels of the protein resulted in decreased E-cadherin expression, increased N-cadherin, vimentin and snail expression and enhanced invasiveness in H460 cells. Meanwhile, completely opposite results were observed in SPC cells. Furthermore, transfection of in H1299 cells expressing low p120ctn levels with the p120ctn isoform 1A plasmid resulted in increased E-cadherin expression, decreased N-cadherin, vimentin and snail expression and weakened invasiveness, while LK2 cells showed completely opposite results. Both cell lines expressing low p120ctn levels and transfected with the p120ctn isoform 3A plasmid appeared to have increased E-cadherin expression, decreased N-cadherin, vimentin and snail expression and weakened invasiveness. In conclusion, in cells with membrane E-cadherin, both p120ctn isoforms 1A and 3A inhibited EMT and decreased cell invasiveness. In cells with cytoplasmic E-cadherin, p120ctn isoform 1A promoted EMT and increased cell invasiveness, while p120ctn isoform 3A inhibited the EMT and decreased cell invasiveness.The epithelial mesenchymal transition (EMT) is an important process in tumor development. Despite previous investigations, it remains unclear how p120-catenin (p120ctn) isoforms 1A and 3A affect the EMT of tumor cells. Here we investigated expression of p120ctn, E-cadherin and vimentin in 78 human non-small cell lung cancer (NSCLC) samples by immunohistochemistry and found that p120ctn membrane expression positively correlated with E-cadherin expression ( The epithelial mesenchymal transition (EMT) is a rapid and often reversible change of cell phenotype and plays a particularly important role in tumor development. In the process of EMT, epithelial cells undergo a phenotypic switch to form mesenchymal cells that are similar in appearance to fibroblasts Previous studies on the relationship between p120-catenin (p120ctn) and EMT have been confined to the switch from short to long p120ctn isoforms during the EMT induced by expression of SIP1/ZEB2 Some studies have shown that p120ctn may promote or inhibit tumor growth and invasiveness depending on whether E-cadherin expressed or not The aim of this study was to determine the potential effects and regulatory mechanisms of p120ctn isoforms 1A and 3A on EMT in lung cancer cells. We first revealed that the membrane or cytoplasmic expression of p120ctn correlated with expression of E-cadherin and vimentin or lymph node metastasis by immunohistochemistry. We further detected the expression levels of p120ctn, E-cadherin and vimentin in lung cancer cells by Western blot and screened cell lines expressing both low and high levels of p120ctn and with E-cadherin in the membrane or cytoplasm. Changes in expression of EMT-related molecules and cell invasion were also investigated by knockdown of endogenous p120ctn-1A or overexpression by transfection of p120ctn-1A and 3A plasmids into cells.This study was conducted with the approval of the institutional review board at China Medical University. Written consent was given by the participants for their information to be stored in the hospital database and for their specimens to be used in this study. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Samples were collected from 78 cases of squamous cell lung cancer and lung adenocarcinoma diagnosed at the First Affiliated Hospital of China Medical University . The samples were from 46 male and 32 female patients with an average age of 57 years. The samples were classified according to lung tumor histological criteria (2004) of the World Health Organization (WHO) http://www.bioleaf.com) and stored in the Department of Pathology, Harbin Medical University. Cells were cultured in RPMI 1640  containing 10% fetal calf serum (Invitrogen), 100 IU/ml penicillin  and 100 mg/ml streptomycin (Sigma).Normal human bronchial epithelial (HBE) cells and A549, H1299, H460 and H157 cell lines were obtained from the American Type Culture Collection . The SPC-A-1, LTEP-A-2 and LK2 cell lines were purchased from the Shanghai Cell Bank of Chinese Academy of Science. The human lung ADC Anip973 and AGZY83a cell lines were purchased from Shanghai Bioleaf Biotech Co., Ltd  have been described previously The paraffin embedded samples were cut serially into 4-μm thick sections. Normal bronchial epithelium present in the tumor slides was used as an internal positive control. Immunostaining was performed by the streptavidin-peroxidase (S-P) method. The tissue sections were incubated with a p120ctn mouse monoclonal antibody , E-cadherin rabbit monoclonal antibody  or vimentin rabbit monoclonal antibody  at 4°C overnight. PBS was used as a negative control. Biotinylated goat anti-mouse serum IgG or biotinylated goat anti-rabbit serum IgG  was used as the secondary antibody. After washing, the sections were incubated with streptavidin–biotin conjugated with horseradish peroxidase , and then the peroxidase reaction was developed with 3,3-diaminobenzidine tetrahydrochloride (MaiXin Bio). Light counterstaining was performed with hematoxylin, and then the sections were dehydrated in alcohol before being mounted.Two investigators independently examined all the tumor slides. Five random fields were examined per slide, and 100 cells were observed per high magnification field (400×). The percentage of positive cells was scored as follows: 0 = no staining; 1+ = 0–25%; 2+ = 26–50%; 3+ = 51–75%; and 4+ = 76–100%. The staining intensity was scored as follows: 0 = no staining; 1 = light yellow granules; 2 = dark yellow or brown granules. The labeling score defined by multiplying the percentage of positive cells by the staining intensity was the final score for the section. When the total score was ≥3, the case was defined as positive. When the total score was <3, the case was defined as negative. For scores greater than 3 points, when more than 30% of the tumor cells stained strongly and continuously for p120ctn signal on the cell membrane, the sample was defined as membrane positive. When fewer than 30% of the tumor cells displayed membrane expression but stained strongly and continuously for p120ctn in the cytoplasm, the sample was defined as cytoplasm positive.Fifty micrograms of proteins were separated by SDS-PAGE (10%). After transfer to a polyvinylidene fluoride (PVDF) membrane , the proteins were incubated overnight at 4°C with antibodies to the following: p120ctn , E-cadherin , N-cadherin  , vimentin , snail   and twist . After incubation with anti-mouse  or anti-rabbit  IgG  at 37°C for 2 h, the protein bands were visualized using enhanced chemiluminescence  and quantified using BioImaging Systems . Relative protein levels were calculated in reference to GAPDH as the loading control.Cells grown on glass coverslips were fixed with ice-cold 4% paraformaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 and incubation with normal goat serum for 30 min at 37°C. Cells were then incubated overnight with p120ctn mouse monoclonal antibody  and E-cadherin rabbit polyclonal antibody . Primary antibodies were applied overnight at 4°C, followed by incubation with a rhodamine/fluorescein-5-isothiocyanate (FITC)-labeled secondary antibody goat anti-mouse or TRITC-labeled goat anti-rabbit IgG . The nuclei were counterstained with propidium iodide/4, 6 diamidino-2-phenylin-dole. Epifluorescent microscopy was performed using an inverted Nikon TE300 microscope , and confocal microscopy was performed using a Radiance 2000 laser scanning confocal microscope .5 cells) was added to the upper chamber, while the lower chamber was filled with RPMI 1640 medium containing 10% fetal calf serum. Each upper and lower chamber was separated by a 8-μm porous polycarbonate membrane. The cells were incubated for 24 h at 37°C in a humid atmosphere with 5% CO2. After the medium was discarded, the cells were fixed with methanol for 30 min and stained with hematoxylin (Sigma). For each filter, the numbers of cells that invaded to the lower surface of the porous membrane in five different fields of 400× magnification were counted randomly using a Nikon E200 microscope. The mean was calculated from data obtained from each experiment repeated three times.Matrigel cell invasion assays were performed according to the manufacturer's instructions . A 100-μl cell suspension  for Windows software. The chi-square test was used to analyze immunohistochemistry data. The independent samples T test was used to examine transwell experimental data. P<0.05)  . In otheP<0.05) ; meanwhiP<0.05) .We examined the protein expression levels of p120ctn and E-cadherin in normal HBE cells and nine lung cancer cell lines by Western blot and found that they all expressed mainly isoforms 1A (120 kDa) and 3A (100 kDa) of p120ctn . AlthougP<0.01) (P<0.01)  , whereas(P<0.01) . These rP<0.01) (P<0.01)  . The western-blot analysis demonstrated that overexpression of the p120ctn isoform 1A led to increased E-cadherin expression and decreased N-cadherin, vimentin and snail expression ; on the P<0.01) , while e(P<0.01) .. Overex(P<0.01)  and redu P<0.01)  in both The phenomenon of EMT in tumor cells often leads to decreased cell adhesion and increased mobility, and this transition is accompanied by decreased E-cadherin expression and increased expression of N-cadherin, vimentin and other mesenchymal biomarkers We first found that the p120ctn membrane expression was positively correlated with E-cadherin expression and negatively correlated with vimentin expression and lymph node metastasis, while the cytoplasmic expression of p120ctn was negatively correlated with E-cadherin expression and positively correlated with vimentin expression and lymph node metastasis by immunohistochemistry. Although these results were consistent with previous studies To confirm the different impacts of p120ctn isoforms 1A and 3A on EMT in cells expressing E-cadherin in different locations, we selected H460 and H1299 cells with E-cadherin membrane expression and SPC and LK2 cells with E-cadherin cytoplasmic expression for further analysis. Plasmids expressing the p120ctn isoforms 1A and 3A were constructed, and the full-length p120ctn siRNA was synthesized for these experiments. Since the sequence beyond amino acids 1–101 of p120ctn isoform 1A is similar to that of p120ctn isoform 3A Knockdown of p120ctn isoform 1A in H460 cells destroyed the epithelial cell adhesion complexes. E-cadherin expression was also downregulated due to the loss of its important stabilizing factor, p120ctn isoform 1A, which was consistent with previous studies Previous studies have shown that although p120ctn isoform 1A could bind E-cadherin in the cytoplasm, they could not form effective adhesion complexes on the membrane between epithelial cells It was worth noting that the same result was observed in LK2 and H1299 cells transfected with the p120ctn isoform 3A plasmid, both showing increased E-cadherin expression, decreased N-cadherin, vimentin and snail expression and inhibited cell invasiveness. These results suggested that p120ctn isoform 3A has the function of inhibiting EMT of lung cancer cells, and this function is independent of the cellular E-cadherin localization. Past research had also confirmed a shift from p120ctn isoform 3A to p120ctn isoform 1A expression after the induction of EMT Importantly, we also found that knockdown of p120ctn-1A in SPC cells with cytoplasmic E-cadherin resulted in decreased twist expression . MeanwhiPrevious studies have observed that p120ctn-1A restored the cytoplasmic expression of E-cadherin, whereas p120ctn-3A could not In conclusion, we, for the first time, found that p120ctn isoforms 1A and 3A to have different functions in EMT of lung cancer cells with E-cadherin expressed in different subcellular locations. When E-cadherin was localized on the cell membrane, p120ctn isoforms 1A and 3A both could inhibit EMT and reduce the cell invasiveness phenotype. When E-cadherin was localized in the cytoplasm, p120ctn isoform 1A promoted EMT and enhanced cell invasion, while p120ctn isoform 3A inhibited EMT and reduced cell invasiveness.


Listeria monocytogenes infection using 13C-labelled glucose or glutamine as carbon tracers. The 13C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon  Salmonella enterica, Legionella pneumophila, Mycobacterium tuberculosis, Chlamydia trachomatis, Brucella abortus, or - after escape from the primary phagosome - in the host cell’s cytosol, e.g. Shigella flexneri, Listeria monocytogenes, and Rickettsia prowazekiAdaptation of the bacterial metabolism to their host cells is a crucial step in the replication cycle of intracellular bacterial pathogens. This important aspect of pathogenesis of intracellular bacteria has long been neglected, but has recently received increased attention  are cancer cells which carry out a significantly altered metabolism. Most normal cells use the tricarboxylic acid (TCA) cycle to produce ATP in the presence of oxygen by oxidative phosphorylation (OXPHO). Although OXPHO occurring in mitochondria provides more ATP than glycolysis, the glycolytic pathway can produce ATP at a higher rate However, a possible flaw of these studies is that most of these data were obtained with established epithelial and macrophage cell lines. In contrast to the differentiated cells infected by the intracellular bacteria during In addition, enhanced glutaminolysis, i.e. uptake and conversion of glutamine to glutamate and further to α-ketoglutarate (α-KG) in the mitochondria, is also frequently observed in cancer cells and probably also in the established cell lines Induction of “core” metabolic host cell genes may occur by the interaction with virtually all bacterial pathogens mainly via NF-κB activation, triggered by pathogen-associated molecular patterns (PAMPs) L. monocytogenes using 13C-isotopologue profiling of amino acids. Primary host cells (like BMM) are of special interest in this context as they resemble more closely the target cells affected by in vivo infections than the commonly used established cell lines (like J774A.1). Recognition of specific metabolic host targets indispensible for the proliferation of intracellular bacterial pathogens may also provide novel approaches for antimicrobial interventions. The data indicate that the expectedly different core carbon metabolism of the two cell types is quite differently affected by intracellular L. monocytogenes. In sharp contrast, the carbon metabolism of the intracellular bacteria is similar in both host cell types.As a first step to evaluate the need and efficiency of redistribution of resources between host cells and intracellular bacterial pathogens, we compared in this study the carbon assimilation of mouse bone marrow derived macrophages (BMM) and of macrophage-like J774A.1 cells without and with infection by 13C-Isotopologue profiling of protein-derived amino acids is a powerful technique to determine active pathways and metabolite fluxes in living cells supplied with appropriate 13C-labelled carbon sources, such as glucose (reviewed in Listeria monocytogenes-infected bone marrow macrophages derived from C57BL/6 mice (in the following abbreviated BMM) and macrophage-like J774A.1 cells derived from a murine reticulum sarcoma Listeria monocytogenes was taken up by both host cell types with similar efficiency (about 5% infected cells) and escape into the cytosol occurred at similar times . The intracellular replication rates during the 8 h incubation period apparently differed considerably (L. monocytogenes-infected J774A.1 cells (and accordingly more intracellular bacteria) in comparison with L. monocytogenes-infected BMMs. Since glucose and glutamine are the most important carbon sources for proliferating mammalian cells, uniformly 13C-labelled glucose ([U-13C6]glucose) or glutamine ([U-13C5]Gln) were used as precursors in experiments with uninfected and infected host cells. Separation of the intracellular bacteria and the host cells was performed as described previously 13C-isotopologue profiles of de novo-synthesized (hence 13C-labelled) amino acids were determined after hydrolyzing protein under acidic conditions. Since the culture medium contained all amino acids, the biosynthesis of amino acids requiring multiple enzymatic steps should be repressed under these conditions. Indeed, 13C-incorporation occurred mainly into Ala, Glu and Asp from the host cells whose production requires only transamination of specific catabolic intermediates, generated by glycolysis (Ala from pyruvate) or by the TCA cycle  and Glu from α-ketoglutarate (α-KG)). Hence, the 13C-enrichments in Ala, Asp, and Glu reflect the cellular concentrations of the respective 13C-labelled catabolic intermediates and therefore allow conclusions concerning the activity of the metabolic pathways leading to these intermediates in the uninfected and infected host cells.iderably . MoreoveL. monocytogenes could be identified. To specifiy the impacts due to live L. monocytogenes, 13C-profiles of host cells treated with UV-inactivated L. monocytogenes or interferon-gamma (IFN-γ) Comparing the labelling profiles of amino acids from infected and uninfected host cells, metabolic changes caused by the intracellular cytosolically replicating 13C-incorporation occurred mainly into Glu and Ala, when fed with [U-13C6]glucose for 8 h , which is further degraded by pyruvate dehydrogenase (PDH) to 13CO2 and [13C2]-acetyl-CoA. The latter metabolite is channeled into the TCA cycle and linked to unlabelled oxaloacetate (OAA – for example derived from unlabelled Gln - see below) yielding [13C2]-α-KG, the precursor of [13C2]-Glu (flux 1 in 13C1]-isotopologues can be explained by carbon flux via pyruvate carboxylase (PYC) involving 13CO2 as a substrate  result in the formation of the [13C1]-, [13C3]-, [13C4]-, and [13C5]-α-KG precursors (flux 2 in Infection with live (and at a lower extent by UV-inactivated)  and Glu , but als amounts . The 13C13C1]-, [13C2]-, and [13C3]-Asp isotopologues were formed. The [13C1]- and [13C3]-Asp isotopologues require as precursors [13C1]- and [13C3]-OAA, respectively, which are generated by ATP-dependent pyruvate carboxylase (PYC) either from unlabelled pyruvate (derived from unlabelled Gln - see below) and 13CO2 or from [13C3]-pyruvate and unlabelled CO2 (flux 2 in 13CO2 could originate either from the 13C-labelled pyruvate by PDH or from decarboxylation of [13C6]-6-phosphogluconate in the pentose phosphate pathway. The [13C2]-OAA precursor for [13C2]-Asp is formed in the TCA cycle by linking [13C2]-acetyl-CoA to unlabelled OAA (again derived from Gln - see below)  enhance glycolysis leading to an increased level of labelled pyruvate, which is channeled at increased rates into the TCA cycle via the PDH- and PYC-generated intermediates.Together, the data show that BMM cells exhibit low metabolic activity, but viable 13C6]glucose showed much higher 13C-incorporation into Glu, Ala and Asp (up to 5-fold depending on the amino acid) than BMM  indicates a high metabolite flux of the added [U-13C6]glucose through the glycolytic pathway in J774A.1 cells. The low amount of 13C-labelled Ser and Gly apparently derived from the glycolytic intermediate [13C3]-3-phosphoglycerate. These data again show that biosynthesis of amino acids that requires more than only transamination of a glycolytic or TCA intermediate is repressed when the corresponding amino acid are externally supplied, regardless whether the carbon metabolism is low (as in BMM) or high (as in J774A.1).Other than in BMM, the isotopologue distribution of J774A.1 amino acids did not significantly change upon infection with live or killed MM cells , arguingMM cells . Thus, t 13C-incorporation into the above mentioned amino acids was observed when IFN-γ activated J774A.1 cells were used, indicating that IFN-γ activation has a different effect on carbon metabolism in the J774A.1 tumor cell line.Additionally and in contrast to the experiments with BMMs, lowerL. monocytogenes infection hardly alters these metabolic steps.In summary, the results show that the glycolytic pathway and at least the α-KG- and OAA-producing steps of the TCA cycle which are also essential for anabolic processes  are highly active in J774A.1 cells. L. monocytogenes were supplemented with 2 mM [U-13C5]Gln. In both host cell types, the major amount of 13C-label was found - as expected - in [U-13C5]Glu, indicating that most of the supplied [U-13C5]Gln was directly incorporated into protein  and Asp ([13C4]), suggesting that it is mainly used in BMM and J774A.1 cells. The fluxes 2, 3 and 4 in  13C isotopologues of Glu and Asp. Of special interest is the generation of [13C3]-Asp, which is the second major 13C-Asp isotopologue in J774A.1, but not in BMM. This Asp isotopologue can be mainly generated by flux 2 shown in Obviously, glucose  were ]glucose . Even thpatterns  were sim.1 cells .13C5]-Gln, L. monocytogenes replicating in BMM incorporated substantial amounts of [13C5]-Glu/Gln into bacterial protein  and the established murine macrophage cell line J774A.1. The activities in the core carbon metabolism of the two cell types turned out to be quite different. Glycolytic and glutaminolytic activities were low in BMMs, but high in neoplastic J774A.1 macrophages  causing reduced viability of these host cells Interestingly, the metabolic patterns in J774A.1 were hardly altered by infection with live (or killed) L. monocytogenes-treated BMM population (see above), the increment in metabolic induction caused by the intracellular L. monocytogenes should be even higher than expressed by the reported experimental numbers. Activation by IFN-γ or UV-killed L. monocytogenes induced a similar flux modulation, albeit at a considerably lower level.In sharp contrast, infected BMM cells showed a strongly increased glycolytic activity , but also release of the bacteria into the host cell’s cytosol (which occurs with live but not with UV-inactivated L. monocytogenes). The thereby triggered induction of host cell glycolysis could be caused by HIF-1 activation (via stabilization of the HIF-1α subunit) and is indeed frequently observed during infections with extra- and intracellular bacterial pathogens de novo synthesis of Glu and Asp in BMM (particularly when infected by live L. monocytogenes) requires active pyruvate dehydrogenase (PDH), pyruvate carboxylase (PYC), citrate synthase (CS), isocitrate dehydrogenase (probably IDH1 and IDH2), ATP-dependent citrate lyase (ACL) and malic enzyme (ME). Hence, PDH must be active in the infected BMMs which becomes particularly evident by the increased production of [13C2]-Glu as major 13C-Glu isotopologue species in the infected BMM cells when cultured in the presence of [U-13C6]glucose. Generation of this Glu isotopologue requires [13C2]-acetyl-CoA that can only be produced by PDH from [U-13C6]glucose-derived [13C3]-pyruvate. Thus, HIF-1 is probably not responsible for the highly induced glycolysis in BMMs when infected with live L. monocytogenes and it remains an intriguing question which host cell regulator is responsible for this induction and which listerial factor triggers it. Listeriolysin might be a candidate on the listerial side The IFN-γ-mediated relatively low induction may be due to enhanced expression of inducible nitric oxide synthase (iNOS or NOS2) L. monocytogenes, deduced from the 13C-profiles of the labelled amino acids by [U-13C6]glucose, was similar in BMM and J774A.1. Substantial 13C-incorporation did not only occur into bacterial Ala, Glu and Asp, but also into Ser, Gly, Pro, Thr and Lys, indicating that the intracellular listeriae sense the obviously low level of these latter amino acids in the host cell cytosol, whereas the host cells sense, as expected, the high external amino acid concentrations. The bacterial carbon metabolism is apparently not influenced by the highly different efficiency of glutamine uptake and consumption by the two host cell types. This may be due to the missing α-ketoglutarate dehydrogenase L. monocytogenes, rendering its catabolism insensitive to glutamine. As shown previously L. monocytogenes seem to prefer glycerol and glucose 6-phosphate as carbon sources provided by the host cell. Indeed, the availability of these carbon compounds depends only on the glycolytic but not on the glutaminolytic activity of host cells.Notably, the carbon metabolism of the intracellular L. monocytogenes infections modifies the metabolism of primary host cells (at least of BMM) with an a priori inadequate metabolism for listerial replication in such a way that it becomes appropriate for the intracellular metabolism of these bacteria.In summary, the data show that L. monocytogenes strain EGDe and the used murine macrophage-like cell line J774A.1 were described before http://conventions.coe.int/Treaty/en/Treaties/Html/123). All animal experiments were in compliance with the German animal protection law and were approved (permit no. 03-001/08) by the responsible Federal State authority and ethics committee. The isolation and use of mouse macrophages were approved by the responsible Federal State authority (AZ 55.2-1-54-2531.6-14-02).L. monocytogenes was grown in BHI at 37°C with aeritation to an OD600 ≈ 1. Then, the culture was centrifuged at 7,000 × g and washed two times with ice-cold PBS. Aliquots resuspended in PBS containing 20% (v/v) glycerol were frozen at -80°C and used for later infection. The differentiated mouse primary macrophages (BMM) and the J774A.1 cells were seeded in 24 hole plates at cell densities of 2×105 and 1×105 cells per hole, respectively, and incubated overnight to allow adherence of the macrophages. BMM and J774A.1 cells were infected with L. monocytogenes EGDe (5 bacteria per host cell) resuspended in RPMI medium with 10% FCS and 10 µM recombinant human insulin to enhance glucose uptake by BMM g and 37°C for 5 min. After 60 min of incubation, the medium was replaced by RPMI containing additionally 50 µg/ml gentamicin to kill extracellular bacteria. This was defined as time point 0 post infection (pi). Under these conditions, up to 5% of the BMM and J774A.1 cells contained intracellular bacteria. Intracellular bacterial growth was measured over a period of 8 h by lysing the cells in distilled water and counting the released viable bacteria by plating them on BHI agar plates.6 BMM and 1×106 J774A.1 cells, respectively, were seeded in culture plates (Ø 10 cm), containing medium supplemented with unlabelled substrates, 20 ng/ml interferon-gamma (IFN-γ) where indicated, and incubated for 24 h. Infection was carried out as described above with L. monocytogenes EGDe at a MOI of 5. As controls, an equal number of host cells remained uninfected or were infected with UV-inactivated L. monocytogenes (5 bacteria per host cell). After 30 min of gentamicin-treatment, the medium was replaced by fresh RPMI now containing additionally 10 mM [U-13C6]glucose  or 2 mM [U-13C5]glutamine . At 8.5 h pi, cells were washed with PBS buffer and lysed in 1 ml ice-cold distilled water containing 0.1% TritonX-100, 10 mM sodium azide, 5 µg/ml tetracycline and 50 µg/ml chloramphenicol for 10 min. The lysate was vigorously vortexed and the bacteria were pelleted at 25,000 × g. The bacteria-free supernatant was frozen (in liquid nitrogen). The bacterial pellet was washed with RIPA buffer 2×10+-form, 200–400 mesh, 5×100 mm), washed with water and developed with 4 M ammonium hydroxide. The eluate was dried under nitrogen, and the residue dissolved in 50 µl water-free acetonitrile. A mixture of 50 µl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide containing 1% tert-butyldimethylsilylchloride (Sigma) was added. The derivatization took place at 70°C for 30 min. The resulting N-(tert-butyldimethylsilyl) (TBDMS)-amino acids were then measured via GC/MS. The yields of TBDMS-Arg and TBDMS-Met were low. Therefore, isotopologue data of theses amino acids are typically not listed.Protein-containing samples  were heated in 6 M HCl for 24 h at 105°C. Under these conditions, protein-derived Gln and Asn were converted into Glu and Asp, respectively. Trp and Cys were destroyed by this treatment. HCl was evaporated under nitrogen and the pellet was dissolved in 50% acetic acid. Separation and purification of the protein-derived amino acids was performed via a cation exchange column of Dowex 50Wx8  equipped with a fused silica capillary column  and a quadrupol QP2010 plus detector (Shimadzu) working with electron impact ionization at 70 eV. The derivatized sample was injected in a 1∶10 split mode at an interface temperature of 260°C and a helium inlet pressure of 70 kPa. The GC-column was developed at 150°C for 3 min and then with a temperature gradient of 7°C/min to a final temperature of 280°C which was held for 3 min. With a sampling rate of 0.5 s, selected ion monitoring was used. Data collection proceeded via GC/MS Solution software (Shimadzu). All samples were measured three times. Figure S1Reconstructed metabolic fluxes13C-labelled amino acid isotopologues deriving from [U-13C6]glucose. Flux 1based on the  describes the formation of [13C3]-Ala, [13C2]-Asp and [13C2]-Glu isotopologues by glycolysis and the canonical TCA cycle using unlabeled OAA (deriving from glutamine) and [13C2]-Ac-CoA from [U-13C6]-glucose. Flux 2 describes the formation of the [13C1]-, [13C2]- and [13C3]-Glu isotopologues and of the [13C1]-, [13C3]-, and [13C4]-Asp isotopologues via glycolysis, PYC-derived 13C-labeled OAA intermediates and unlabeled Ac-CoA (from glutamine-derived pyruvate (Pyr). The intermediates deriving from citrate are converted to the corresponding 13C-Asp and 13C3-Glu isotopologues by ACL and ICD-1 and ICD-2, respectively. Intermediates predominantly deriving from 13C-glucose are written in red letters, while those predominantly deriving from unlabeled glutamine are in black letters and those deriving from 13C-glucose and unlabeled glutamine in green letters. The red dots indicate the number of 13C-atoms in the respective compound.(TIF)Click here for additional data file.Figure S213C-labelled amino acid isotopologues deriving from [U-13C5]glutamine. Flux 1Reconstructed metabolic fluxes based on the  shows the formation of [13C5]-, [13C3]-Glu and [13C4]-Asp via glutaminolysis, TCA cycle reactions, and ACL-catalyzed reaction to [13C4]-OAA. Intermediates deriving predominantly from [U-13C5]glutamine are written in blue letters, while those deriving predominantly from unlabelled glucose are in black letters and those deriving from [U-13C5]glutamine and unlabelled glucose in green letters. Flux 2 shows the formation of [13C5]-Glu and [13C3]-Asp isotopologues via glutaminolysis, citrate production by isocitrate dehydrogenase (ICD2)-dependent carboxylation of α-KG and citrate conversion to α-KG and OAA by cytosolic ACL and ICD1. Intermediates deriving predominantly from [U-13C5]glutamine are written in blue letters, while CO2 as well as the intermediates deriving predominantly from catabolism of unlabelled glucose are in black letters and those deriving from [U-13C5]glutamine and unlabelled CO2 are in green letters. Flux 3 describes the formation of [13C2]-Glu, [13C2]- and [13C4]-Asp isotopologues via glutaminolysis, canonical TCA cycle reactions to [13C4]-malate, conversion to [13C3]-pyruvate and 13CO2 generation of [13C2]-Ac-CoA by PDH, condensation to unlabelled OAA (from unlabelled glucose) yielding [13C2]-citrate which is further converted by ACL to [13C2]-OAA and by ICD1 to [13C2]-α-KG. Flux 4 describes the formation of [13C1]-Glu and [13C1]-Asp isotopomers via glutaminolysis, canonical TCA reactions yielding (among other intermediates) 13CO2 which is used for PYC-catalyzed carboxylation of unlabelled pyruvate to [13C1]-OAA, giving rise together with unlabelled Ac-CoA (from unlabelled Glc) to [13C1]-citrate and further to [13C1]-OAA and [13C1]-α-KG. The blue dots indicate the number of 13C-atoms in the respective compound. For further details and abbreviations, see main text.(TIF)Click here for additional data file.Table S113C-Isotopologue abundance in mol% of protein derived amino acids from experiments with uninfected and Listeria monocytogenes13C6]glucose and with or without IFN-γ.-infected BMM and J774A.1 macrophages with 11 mM glutamine and with or without IFN-γ.-infected BMM and J774A.1 macrophages with 2 mM [U- (I) and (II) represent two biological experiments. Isotopologues are described by an extended binary code: 1 represents a 13C-atom, 0 stands for 12C, X is unknown. Y is unknown, but for a given number (outside the brackets) it represents a 13C-atom. Data from host cells represent mean values of three measurements (with S.D.) of cell lysate. Data from intracellular Listeria monocytogenes represent calculated values of the bacterial fraction (spill-over factor of host cell pellet was determined as described under (PDF)Click here for additional data file.