Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus.in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions.Here, we show that the NS1 protein of the human H3N2 virus subtype interacts NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. Influenza A virus genome consists of eight separate RNA segments, which encode for 12 viral structural and nonstructural proteins .Influenza A virus NS1 is a multi-functional protein that contains an N-terminal dsRNA-binding domain and a C-terminal effector domain. Experiments with the human H3N2 influenza A/Udorn/72 virus demonstrated that the primary role of the NS1 dsRNA-binding activity is to inhibit the activation of IFN-induced 2’-5’ oligo (A) synthetase/RNase L pathway . InsteadEukaryotes have a nucleolus, which is a relatively large, dynamic and highly organized non-membranous subcompartment of the nucleus. The nucleolus is the site for ribosomal RNA synthesis, processing and maturation ,23. ReceThree distinct subsets of nucleolar proteins have been identified. Primarily, fibrillarin is a small nucleolar ribonucleoprotein (snoRNP), which is associated with early pre-rRNA transcripts during elongation and is localizing to DFC. B23/NPM protein is a putative ribosome assembly factor involved in 28S rRNA processing and ribosome assembly that localized in the GC, while the third major nucleolar protein, nucleolin, is involved in the processing of precursor rRNAs .Nucleolar localization signals (NoLSs) that regulate nucleolar localization and retention are usually rich in arginine and lysine residues, which overlap with NLSs. However, there is no obvious consensus NoLS sequence or structure -33. NuclWithin the last few years, increasing evidence has revealed that viruses require the nucleus and in particular the nucleolus for their replication . In addiin vitro-translated B23. Using GFP-NS1 fusion proteins, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy results show that the NS1 protein colocalizes with nucleolin in nucleolus and nucleoplasm and with B23 and fibrillarin in nucleolus in influenza A/Udorn/72 virus-infected A549 cells.Here, we show that the NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. Direct interactions were observed for nucleolin and fibrillarin but not with We have previously shown that the NS1 protein of the H3N2 subtype influenza A viruses contain a C-terminal NLS2/NoLS -NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. It is thus likely that the nucleolar targeting function of NS1 protein plays a role in the pathogenesis of human influenza A viruses that, undoubtedly, deserves further studies.The present study shows that the NS1 protein of the human H3N2 subtype virus interacts Human A549 lung carcinoma cell line  was maintained in a continuous culture in minimum Eagle’s medium-α  supplemented with 0.6 μg/ml penicillin, 60 μg/ml streptomycin and 10% fetal calf serum . Human hepatocellular carcinoma HuH7  cells weReverse genetics in the A/WSN/33 H1N1 background  was perf7 pfu/ml. The multiplicity of infection (MOI) used in the experiments varied from 0.5 to 5 pfu/cell.A549 cells, grown on 6-well plates or on glass coverslips on 24-well plates, were infected with the following viruses: A/Udorn/72 (H3N2), A/WSN/33 (H1N1), A/WSN/33 (A/Brevig Mission/1/18 segm 8) (H1N1), A/Fin/554/09 (H1N1pdm09) and A/mallard/Netherlands/12/2000 (H7N3). Virus stocks were cultivated in eight-day-old embryonated chicken eggs and stored at −70 °C. The hemagglutination titers of the stock viruses ranged from 64 to 256, and the infectivity of the virus stocks in A549 cells was 1-4 × 10In Western blot analysis mouse monoclonal anti-nucleolin , mouse monoclonal anti-B23  and rabbit polyclonal anti-fibrillarin  immunoglobulins were used. Secondary HRP-conjugated goat anti-mouse and goat anti-rabbit immunoglobulins  were used as suggested by the manufacturer. Guinea pig anti-influenza NS1  and rabbE. coli GST  and eukaryotic pcDNA3.1(+)  expression vectors. Wild type A/WSN/33 (H1N1 virus) NS1 gene (GenBank ID: M12597) was modified by PCR to create N- and C-terminal BglII sites for further cloning into the BamHI site of the pcDNA3.1(+) expression vector (Invitrogen). To create point mutations to the A/Udorn/72 and A/WSN/33 NS1 cDNAs, a QuikChangeTM Site-Directed Mutagenesis Kit  was used.Wild type A/Udorn/72 (H3N2 virus) NS1 gene (GenBank ID: V01102) was expressed in BamHI sites for further cloning into the BamHI site of the pcDNA3.1(+) expression vector (Invitrogen). Wild type human laminA/C (GenBank ID: NG_008692) gene was cloned into the pOTB7 vector (Invitrogen).Wild type human nucleolin , B23 , fibrillarin  and HIV-1 Rev  genes weSalI and NheI sites, respectively, for further cloning into the SalI and NheI cloning sites of a pCMX-SAH/Y145F expression vector -labeled  and allowed to bind to Sepharose-immobilized GST or GST-NS1 fusion proteins on ice for 60 min followed by washing. GST-NS1 fusion protein-bound 35S]-labeled proteins were separated on 12% SDS-PAGE. The gels were fixed and treated with Amplify reagent (Amersham Biosciences) as specified by the manufacturer and autoradiographed. GST pull-down experiments from A549 cell extracts were carried out as described [Influenza A virus GST-NS1 fusion proteins were expressed in scribed . In vitrscribed .For indirect immunofluorescence and confocal laser microscopy HuH7 cells, grown on glass coverslips for 24 h, were transfected with GFP, GFP-NS1 or HIV-1-pcDNA3.1(+) expression constructs using FuGENE6 transfection reagent  according to the manufacturer’s instructions. Forty-eight hours after transfection the cells were fixed with 3% paraformaldehyde at RT for 20 min and processed for immunofluorescence microscopy. A549 cells were infected with influenza A/Udorn/72 wt virus for 5 to 8 hours as indicated in the legends for figures, fixed with 3% paraformaldehyde at RT for 20 min, permeabilized with 0.1% Triton X-100 for 5 min and processed for immunofluorescence microscopy. The cells, positive for transiently transfected GFP and GFP-NS1 or viral NS1 proteins, were visualized and photographed on a Leica TCS NT confocal microscope.The authors declare that they have no competing interests.KM participated in the design of the study, performed most of the experiments, analyzed the results and drafted the manuscript. JT and RF participated in the design of the study and carried out some experiments. PR and DH-V provided crucial reagents to carry out the experiments and analyzed the confocal microscopy results. IJ initiated the study, participated in the design and coordination and helped to draft the manuscript. All authors have read and approved the final version of the manuscript.


ING2 (inhibitor of growth family member 2) is a component of a chromatin-regulatory complex that represses gene expression and is implicated in cellular processes that promote tumor suppression. However, few direct genomic targets of ING2 have been identified and the mechanism(s) by which ING2 selectively regulates genes remains unknown. Here we provide evidence that direct association of ING2 with the nuclear phosphoinositide phosphatidylinositol-5-phosphate (PtdIns(5)P) regulates a subset of ING2 targets in response to DNA damage. At these target genes, the binding event between ING2 and PtdIns(5)P is required for ING2 promoter occupancy and ING2-associated gene repression. Moreover, depletion of PtdIns(5)P attenuates ING2-mediated regulation of these targets in the presence of DNA damage. Taken together, these findings support a model in which PtdIns(5)P functions as a sub-nuclear trafficking factor that stabilizes ING2 at discrete genomic sites. Chromatin structure plays a central role in the establishment, maintenance, and propagation of nuclear programs and the dysregulation of gene expression programs occurs in numerous human pathologies, including cancer. A key mechanism regulating chromatin structure involves the reversible covalent post-translational modification (PTM) of histone proteins by chemical moieties such as acetyl- and methyl-groups. Histone PTMs are dynamic, frequently changing in response to external cues1245Inhibitor of Growth family member 2 (ING2) is a tumor suppressor protein9119Phospholipids, including phosphoinositides (PtdInsPs) play a pivotal role in cytoplasmic trafficking of intracellular proteins15171819201722Here we demonstrate that the interaction between ING2 and nuclear PtdIns(5)P (ING2::PtdIns(5)P) directly regulates chromatin-mediated gene expression control. We first identify the DNA damage-associated gene expression network mediated by ING2 and then uncover the subset of these genes that are specifically modulated by association of ING2 with PtdIns(5)P. For this cluster, there is a loss of ING2-associated gene repression when ING2::PtdIns(5)P is disrupted or when nuclear PtdIns(5)P is metabolized. Loss of ING2 promoter occupancy is concomitant with abrogation of DNA damage-induced histone deacetylation resulting in aberrant transcript elevation. Taken together, these findings establish PtdIns(5)P as a new class of nuclear signaling molecules controlling gene expression and provide mechanistic insight into the role of sub-nuclear trafficking in the acute regulation of chromatin structure.To test the role of PtdIns(5)P-binding by ING2 on ING2 gene expression functions, we determined the ING2-mediated DNA damage program using a comprehensive genomic approach coupling ChIP-chip (chromatin immunoprecipitation combined with tiling promoter DNA microarrays) and gene expression microarray analyses. Combining these assays allowed for the identification of gene promoters bound by ING2 as well as the downstream transcriptional changes. Once we established a baseline, we monitored changes to the ING2 expression network resulting from perturbations in PtdIns(5)P signaling (diagrammed in D230A) abrogates ING2::H3K4me3 without ablation of ING2::PtdIns(5)P6K/Rmt) can bind H3K4me3. Additionally we generated and characterized a mutant with the combined amino acid changes in ING2D230A and ING26K/Rmt named ING2D-6K/Rmt. We subjected the panel of ING2 mutants to in vitro histone peptide (6K/Rmt which fails to bind to PtdIns(5)P-containing liposomes, binds to H3K4me3 comparable to wild-type. In contrast, ING2D-6K/Rmt is deficient in binding to both H3K4me3 and PtdIns(5)P (6K/Rmt and the other ING2 mutants can be used to isolate the impact of the ING2::PtdIns(5)P on ING2 gene-regulatory functions. Next we generated human fibrosarcoma cells (HT1080) stably-expressing epitope-tagged, full-length wild-type ING2 (ING2WT) or ING26K/Rmut. Note that endogenous ING2 protein levels are depleted in response to ectopic expression of wild-type and mutant ING2, thus the majority of ING2 in the reconstituted cells is exogenous (For this study it was necessary to identify a form of ING2 that does not bind PtdIns(5)P, but is not deficient in H3K4me3 binding. The C-terminus of ING2  contains peptide  and lipo peptide . As showdIns(5)P . Thus INWT was determined. DNA that co-purified with FLAG-ING2WT in vehicle-treated and etoposide-treated HT1080 cells was hybridized to Nimblegen whole genome human promoter arrays and peaks were detected using Nimblescan software. Significant ING2 peaks (see Methods) were identified at over 200 unique promoters in etoposide-treated cells, whereas no significant ING2 peaks were detected in vehicle-treated cells  represent a statistically-significant  population of genes repressed in response to etoposide  and PCGF2 (a representative from the chromosome organization cluster), but not at ACTA, a negative control P results in impairment of etoposide-induced nuclear programs P P for repression in response to etoposide). Quantitative PCR transcript (qPCR) analyses were performed on several ING2ET-BOUND targets to confirm the 5PRESS expression signature  are elevated over 5-fold in cells expressing ING26K/Rmt compared to cells expressing ING2WT. To summarize, transcript analyses indicate that inhibition of the ING2-PtdIns(5)P interaction results in aberrant DDR-induced gene repression.Based on the observations that (1) ING2 dynamically associates with chromatin in response to etoposide to repress transcription, and (2) nuclear PtdIns(5)P levels increase in response to etoposide-treatment, we postulated that DDR-induced ING2-PtdIns(5)P interactions would regulate ING2 occupancy and/or activity at target genes. To test this we performed global gene expression analyses on cells expressing ING2programs . Since IdIns(5)P . HeatmapdIns(5)P  uncoveredIns(5)P ; more spignature . In cell6K/Rmt is that this mutant has decreased association with HDAC activity. To investigate this possibility, a panel of ING2 complexes were purified then tested for in vitro HDAC activity  activity (on H3K9 and H3K27) comparable to ING2WT  are ING2ET-BOUND, indicating that ING2 is selectively promoter-bound in the presence of etoposide , a putative oncogene that promotes anchorage-independent growthTo focus functionally on the role of PtdIns(5)P in selective gene repression we narrowed our attention to genes directly bound by ING2 that require ING2::PtdIns(5)P for repression  havOL-BOUND .This groOL-BOUND . Intrigu5PRESS-BOUND representative, CIP2A, as well as an ING2CONTROL-BOUND representative, UBE2N, in which ING2 promoter occupancy should not be regulated by PtdIns(5)P , which is consistent with the critical role of H3K4me3::ING2 interaction in stabilizing ING2 at target promoters. These data support the hypothesis that ING2::PtdIns(5)P is required for promoter occupancy at discrete ING2 chromatin targets.Our working model was that select ING2 targets require stable association of ING2 with PtdIns(5)P to properly traffic ING2 to the promoter . To testdIns(5)P . ING26K/2175PRESS-BOUND representative, CIP2A, but not with the ING2CONTROL-BOUND representative, UBE2N P signaling is depleted due to over-expression of the human enzyme phosphatidylinostiol-4-phosphate kinase II beta (PIP4Kbeta), which utilizes PtdIns(5)P as a substrate to generate PtdInsPe, UBE2N  top. Furpromoter  bottom.  by ING2 . Thus, dAn understanding of the gene network controlled by ING2 is critical to unravel the molecular mechanism by which ING2 functions under physiological conditions as a tumor suppressor protein. Here, we uncovered several key features of the nuclear program mediated by ING2 in response to DNA damage. We identified more than 200 novel gene promoters directly bound by ING2 in response to genotoxic stress. Consistent with tumor suppressive functions, ING2 regulates several key cellular processes including chromatin structure, cell cycle progression, and cell proliferation. We focused on a group of ING2 targets that are dependent on the nuclear lipid PtdIns(5)P for proper regulation. The genes controlled by PtdIns(5)P are dysregulated when either (1) the binding event between ING2 and PtdIns(5)P is abrogated or (2) PtdIns(5)P levels are depleted due to PIP4Kbeta over-expression. Together, our data suggest that PtdIns(5)P acts to localize ING2 to target gene promoters which is instrumental in focusing ING2-associated HDAC activity. PtdIns(5)P was discovered nearly two decades agoCIP2A. Our data favor a model in which the ING2::PtdIns(5)P interaction stabilizes ING2 at discrete sites within the nucleus, where ING2 can then bind to H3K4me3 and anchor ING2-HDAC1-SIN3 complexes at gene promoters. While this study identifies a specialized role for ING2::PtdIns(5)P in gene expression control, it also highlights that ING2::H3K4me3 may have a more general role in gene regulatory functions. In 2Chromatin and phospholipid signaling networks impact gene expression and dysregulation of either one of these networks can contribute to human pathological states including cancer. ING2 is of great interest because it is a direct integrator of chromatin and nuclear phosphoinositide signaling networks and here we report that these pathways converge to regulate the putative oncogene Biotinylated H3 peptides and histone-peptide binding assays have been described previouslyHT1080 cells were treated with Etoposide (100 uM) or vehicle (DMSO) for 1 hr, then prepared as described previously for chromatin immunoprecipitation (ChIP)ING2 and derivative constructs were previously described99RNA was isolated using RNeasy plus kits (Qiagen). For gene expression microarrays, RNA was processed for use with Illumina Bead Arrays per manufacturer's instructions. Probe-level data were processed using Genespring software and all subsequent analyses were performed using MeV3234293T and HT1080 cell lines (ATCC) were cultured in Advanced DMEM (Invitrogen) supplemented with penicillin-streptomycin (Invitrogen), glutamine (Invitrogen), and 10% newborn calf serum (Atlanta). Transient transfection, retroviral transduction, and histone deacetylation (HDAC) reactions were performed as previously describedConceived and designed the experiments: D.J.B., G.M., O.B., O.G. Performed the experiments: D.J.B., G.M., O.B. Analyzed the data: D.J.B., G.M., O.B., O.G. Wrote the main manuscript text: D.J.B., O.G. Reviewed the manuscript: D.J.B., G.M., O.B., O.G.Supplementary Info File #1


S100A14 is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effects on different types of cells. However, exact extracellular roles of S100A14 have not been clarified yet. Here we investigated the effects of S100A14 on esophageal squamous cell carcinoma (ESCC) cell lines. Results demonstrated that low doses of extracellular S100A14 stimulate cell proliferation and promote survival in KYSE180 cells through activating ERK1/2 MAPK and NF-κB signaling pathways. Immunoprecipitation assay showed that S100A14 binds to receptor for advanced glycation end products (RAGE) in KYSE180 cells. Inhibition of RAGE signaling by different approaches including siRNA for RAGE, overexpression of a dominant-negative RAGE construct or a RAGE antagonist peptide (AmphP) significantly blocked S100A14-induced effects, suggesting that S100A14 acts via RAGE ligation. Furthermore, mutation of the N-EF hand of S100A14  virtually reduced 10 µg/ml S100A14-induced cell proliferation and ERK1/2 activation. However, high dose (80 µg/ml) of S100A14 causes apoptosis via the mitochondrial pathway with activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase. High dose S100A14 induces cell apoptosis is partially in a RAGE-dependent manner. This is the first study to demonstrate that S100A14 binds to RAGE and stimulates RAGE-dependent signaling cascades, promoting cell proliferation or triggering cell apoptosis at different doses. S100 proteins are small calcium-binding proteins of the EF hand motif which can function as both intra- and extracellular signaling molecules. They exert a broad range of intracellular functions through the modulation of their subcellular localization and interacting with specific target proteins, responsible for cell growth, differentiation, motility, and cell cycle regulation RAGE is a multiligand receptor of the immunoglobulin superfamily and is constitutively expressed during embryonic development, but its expression is down-regulated in adult life in physiological states S100A14 gene was found useful for detection of circulating tumor cells (CTCs) in peripheral blood of advanced cancer patients S100A14 gene was regulated by p53 and was associated with esophageal squamous cell carcinoma in vivoS100A14 is a member of the S100 family of calcium-binding protein, whose biological function is largely unknown by now. It is differentially expressed in a wide variety of cell types and is up-regulated in certain types of tumors, such as lung, breast, and uterus, but down-regulated in some other tumors, such as colon, kidney, and rectal tumors in vitro; therefore, exogenous S100A14 stimulates cell proliferation or induces apoptosis at different concentrations via RAGE ligation.In the present study, we investigated the function of exogenous S100A14 on ESCC cell lines. We found that low doses of exogenous S100A14 activate ERK1/2 and NF-κB signaling, stimulating cell proliferation or promoting cell survival via RAGE ligation; while high dose of S100A14 triggers apoptosis and increases the production of ROS also in a RAGE-dependent manner. Blocking the interaction of S100A14 and RAGE could inhibit the effects of S100A14 Escherichia coli. To ensure that the observed effects were caused by S100A14, we used Myo117 as a negative control, which is a protein of similar size, bearing the same His-tag and produced in an identical manner with S100A14 To investigate the putative effect of extracellular S100A14 on cell function, we produced purified S100A14 as a histidine-tagged fusion protein in Extracellular S100A14 also increased the survival of KYSE180 cells exposed to the cytotoxic agent Doxorubicin (Dox). The addition of S100A14 protected cells from injuries induced by Dox  in a concentration-dependent manner with significant protection obtained at 10 µg/ml . FurtherWe next determined whether the effects of S100A14 on cell proliferation and survival were related with the common signal pathways. Consistent with cell growth, exogenous S100A14 induced ERK1/2 activation in a concentration- and time-dependent manner, with significant effect observed at 1 µg/ml and maximal effect achieved at 10 µg/ml with an incubation of 30 min . With thMoreover, NF-κB activation is an important event mediating cell growth and survival, thus we analyzed NF-κB activity in KYSE180 cells transfected with NF-κB reporter plasmid which then treated with low doses of S100A14 for 8 h. As shown in We also found that constitutive activation of ERK or NF-κB could regulate cell proliferation as low doses of S100A14 in KYSE180 cells by BrdU assays . Taken tRAGE ligation can activate multiple signaling pathways. Previous studies revealed that several S100 family members can function extracellularly through RAGE ligation We subsequently constructed KYSE180 cell clones stably expressing either full-length RAGE or dominant-negative mutant of RAGE which were designated as KYSE180-RAGE or KYSE180-RAGEΔcyto, respectively . MeanwhiTo explore whether RAGE was involved in cell proliferation and the signaling pathways driven by S100A14, we utilized several methods to inhibit RAGE activation. Overexpression of RAGEΔcyto or addition of AmphP or pretreatment with RAGE-siRNA or incubation with U0126, all these treatments significantly inhibited 10 µg/ml S100A14-induced KYSE180 cell proliferation . MeanwhiFurther studies indicated that key amino acid mutations of N-EF hand  significantly attenuated the effects of S100A14. Cell viability assays indicated that incubation with mS100A14-N protein  did not stimulate KYSE180 cell proliferation compared to those with S100A14 . As expeIn addition to cell proliferation, S100A14 was able to trigger cell injuries at relatively high dose. We found that 80 µg/ml S100A14 could remarkably induce cytotoxicity by evaluating the cell viability using MTT assay . To inveN-acetyl-L-cysteine (Nac) was capable of conferring protection against 80 µg/ml S100A14-induced cell injuries in a dose-dependent manner  was measured. As shown in t manner , which cMore attention has been paid to the extracellular functions of S100 proteins recent years. So far, the S100 family members S100A4, S100A6, S100A8/9, S100A12, S100A13, S100B and S100P have been shown to interact with RAGE, resulting in activation of signal pathways and involving in numerous pathologic situations S100A14 transfected EC9706 stable clones and KYSE180 cells, and extracellular S100A14 might regulate its endogenous expression, creating a positive feedback loop. However, it is unclear whether S100A14 exerts extracellular effects. In the present study, using human ESCC cell lines, we found for the first time that low doses of S100A14 promote cell proliferation in a concentration- and time-dependent manner. S100A14 also protected KYSE180 cells from injuries induced by chemotherapeutic agent Dox. At the intracellular level, at least two signal pathways ERK and NF-κB were involved. Low doses of S100A14 stimulate ERK1/2 phosphorylation in a concentration- and time-dependent manner. In contrast, the phosphorylation of JNK or p38 was unchanged which has been classically linked to cell stress and induction of apoptosis We have found that S100A14 was secreted into the extracellular medium from More importantly, we have confirmed the direct interaction of S100A14 and RAGE by pull-down and co-immunoprecipitation assays, and further shown that S100A14 stimulated KYSE180 cell proliferation via interaction with RAGE, since the addition of siRNA for RAGE, AmphP, or RAGEΔcyto expression were all able to clearly block the proliferation effect in a concerted manner. The effects were similar with that of S100P on NIH3T3 cells, which may due to considerable sequence homology within S100 protein family members Another aspect of the cellular function of S100 proteins is related with cell death. For example, S100B was found to exert a cytotoxic effect via stimulation of ROS production in myoblast Furthermore, the cytotoxic activity of high dose S100A14 is also RAGE-dependent since KYSE180 cells transfected with RAGE siRNA or expressing RAGEΔcyto both remarkably inhibited its toxic effects, and KYSE180-RAGEΔcyto obviously protected cells from the decrease of mitochondrial membrane potential. RAGE knockdown, at least in part, reversed the injury of high dose of S100A14, which was different from S100A8/9 whose bimodal function is mediated by two distinct receptors and signaling pathways in vitro and in vivo. Moreover, investigation of S100A14 signaling, regulation of S100A14 expression and secretion as well as interacting proteins may help unveil the complicated biological process. Given the prominent role of S100A14 on cell proliferation and apoptosis, therapeutic intervention targeting the S100A14 and RAGE signaling pathway may provide a novel approach for cancer therapy.In summary, the work presented in this paper revealed important dual effects of S100A14 on KYSE180 cells. We have for the first time demonstrated that S100A14 can interact with RAGE, activating ERK1/2 and NF-κB signaling, promoting cell proliferation and survival at relatively low doses; whereas high dose of S100A14 induced apoptosis in a RAGE-mediated and oxidant-dependent manner. Our data are in line with the function of S100B with trophic or toxic effects on neurons or neuroblastoma cells depending on its concentration via RAGE ligation E. coli was performed as described Expression and purification of the human recombinant S100A14 protein in S100A14 cDNA to convert calcium-coordinating amino acids in the loop of the N-EF hand E39, E45 to A using site-directed mutagenesis by PCR (Beijing SBS), which was designated as mS100A14-N. The plasmid pET-28a-S100A14 was used as template. The nucleotide sequences of the mutant cDNA were checked by DNA sequencing (Applied Biosystems).Point mutations were introduced into the 2 humidified atmosphere. After plating, cells were cultivated for 24 h and then serum-starved for an additional 24 h before treatment with indicated doses of S100A14 in serum-free medium. The target siRNA for RAGE (sc-36374) and a negative-control siRNA (sc-37007) with an irrelevant sequence were purchased from Santa Cruz Biotechnology (Santa Cruz). Cells were grown to 60% confluence and then transfected with the siRNA duplex (50 nM) using HiperFect (Qiagene) according to the manufacturer's instructions. The transfected cells (72 h post-transfection) were then treated with S100A14 for indicated periods.Human ESCC cell lines KYSE180 and KYSE450 were gifts from Dr. Y. Shimada of Kyoto University  Cells were seeded at 35-mm-diameter plate and transfected using Lipofectamine 2000 (Invitrogen) with pcDNA3.0/RAGE and pcDNA3.0/RAGEΔcyto plasmids Different concentrations of S100A14 were added to the cells for indicated time intervals, and cell proliferation was determined by MTT assay as instructed by the manufacturer (Roche Applied Science). Percentage of viability was determined by comparing the number of viable cells in treated culture to that in control culture treated in parallel with Myo117 protein. For survival test, cells were treated with 0.5 µM Dox as well as S100A14 protein for 48 h, cell viability was then estimated by MTT assay.Incorporation of 5-bromo-2′-deoxyuridine (BrdU) was performed as another cell proliferation assay according to the manufacturer's protocol (Roche Applied Science).Protein extraction and Western blot were performed as described For S100A14-RAGE pull-down experiments, purified S100A14 protein (10 µg) was incubated with His-resin beads at 4°C for 1 h and then incubated with KYSE180 cell lysates overnight at 4°C. In some experiments, cells were pretreated with AmphP for 1 h before incubated with KYSE180 lysates. Bound proteins were eluted with 2×SDS sample buffer and subjected to SDS-PAGE, followed by immunoblotting using a RAGE polyclonal antibody.For coimmunoprecipitation experiments, KYSE180 cell lysates were immunoprecipitated with anti-S100A14 antibody at 4°C overnight and RAGE was identified in the immunoprecipitates by Western blotting.Paraffin-embedded forty-one ESCC samples were preserved in our lab Cells were seeded onto 35-mm plastic dishes for 24 h, and cultivated for 48 h in the presence of different doses of protein. Standard PI staining was used for cell cycle analysis and the content of sub-G1 was regarded as apoptotic cells. Annexin V-PI staining was used for the apoptosis assay 4) were seeded in a 96-well plate and transfected with the NF-κB reporter plasmid using Lipofectamine 2000 (Invitrogen). 24 h after transfection, indicated doses of S100A14 were added to the medium. Cells were harvested 8 h later and assayed for luciferase activity using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's manual. The luciferase activities were normalized to the Renilla luciferase activity of the internal control. NF-κB activity is expressed as relative luciferase activity as compared to control cells.Cells .Intracellular ROS production was evaluated by using indirect ROS inhibitory kit  according to the manufacturer's protocol. The clear ability of superoxide is high in untreated control cells considering as 100%, while increased intracellular ROS caused decreased clear capacity.Data were presented as mean ± SD and one-way analysis of variance (ANOVA) analysis was performed using SPSS 13.0 software. The correlation between S100A14 and Ki67 was assessed using Spearman correlation analysis. P<0.05 was considered as significant.Figure S1Western blot analysis for secretion of S100A14 from EC9706 or KYSE180 cells. (A) Western blot was performed to detect S100A14 in the culture media from EC9706 cells stably transfected with HA-tag S100A14 full-length plasmid. Stable EC9706 transfectants with empty vector were used as a negative control (Mock). (B) S100A14 was secreted extracellularly from KYSE180 cells whereas no S100A14 secretion could be detected in EC9706 cells.(DOC)Click here for additional data file.Table S1Immunoreactivity for S100A14 and Ki67 in 41 ESCC specimens. For each case, an immunostaining score was given based on the percentage of cells showing definitive staining regardless of the staining intensity: 0 =  no staining, 1 =  less than 10%, 2 = 10–25%, 3 = 26–50%, 4 = >50% of cells stained.(DOC)Click here for additional data file.Table S2The pathological characteristics of the specimens.(DOC)Click here for additional data file.


Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD+-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.Glycerol is an important osmotically compatible solute in  Dunaliella salina, one member of the genus Dunaliella , is an extremely halotolerant, unicellular, green, and motile algae. The genus Dunaliella is unique in its remarkable ability to survive in the media with a wide range of NaCl concentrations, from about 0.05 M to saturation (around 5.5 M), while maintaining a relatively low intracellular sodium concentration de novo synthesis of the compatible solute, the glycerol, following salt stress D. salina an obvious applicable value as a model organism in studying the mechanism of osmoregulation under salt environment conditions. In addition, under high salt stress, D. salina could accumulate large amounts of β-carotene in cells, which makes it one of the best sources of natural β-carotene Dunaliella under salt stress ella by changing intracellular glycerol content. The glycerol is synthesized rapidly when the concentration of saline increases, and the glycerol transforms to starch when the concentration of saline drops D. salina accumulates massive amounts of glycerol and the level of intracellular glycerol is proportional and osmotically equivalent to the external NaCl concentration, reaching about 8 M or 55% (w/v) of the cell weight at saturated NaCl concentrations Dunaliella tertiolecta could also adapt the different concentration of saline by synthesizing or eliminating the intracellular glycerol to balance the osmotic potential of intracellular and extracellular +)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) plays a major role in the osmoregulation process in DunaliellaGlycerol is an important osmotically compatible solute in D. salina, and these isozymes respectively take effects in different salinities and play important roles in glycerol metabolism +-dependent G3PDH from D. salina, which is one isozyme with highly homology of previously isolated G3PDH in this alga Dunaliella.It was found that there are five isozymes of G3PDH in D. salina strain 435 (UTEX 200) conserved in our laboratory were cultivated in the culture medium according to Chen et al Cells of D. salina cells grown at the late log phase with using E.Z.N.A. Total RNA Kit II (OMEGA) according to the manufacture’s instruction. Subsequently, the total RNA was treated by DNase I (RNase Free) (TaKaRa) and was dissolved in 0.1% (v/v) diethyl pyrocarbonate solution (TaKaRa) The total RNA was prepared from 10 mL of D. salina G3PDH cDNA by using Premix Ex Taq (TaKaRa) following the manufacturer’s instructions. The PCR procedure is as the following: 1 cycle of 94°C, 5 min; 30 cycles of 94°C, 30 s, 51°C, 30 s, and 72°C, 1 min; and 1 cycle of 72°C, 10 min; used primers listed in The first strand of cDNA was synthesized from the total RNA using PrimeScript ™ RT-PCR kit (TAKARA) according to the manufacturer’s instructions Based on the obtained conserved cDNA fragment sequence, gene specific primer Dsgpdh3’F was designed and 3′ RACE was conducted with oligo dT-Adaptor primers using RNA PCR Kit (AMV) Ver.3.0 (TaKaRa). The first-strand cDNA was amplified by LA Taq (TaKaRa) with the parameters set as follows: 94°C, 4 min; 30 cycles of 94°C, 30 s, 46°C, 30 s, and 72°C, 1 min with a final extension at 72°C for 10 min.The 5′ RACE operation was accomplished with SMART™ MMLV Reverse Transcriptase (Clontech) and synthesized primers SMARTAO and 5′-RACE CDS. The second 5′RACE was conducted using the Dsgpdh5’F2primer designed according to the fragment obtained from the first 5′RACE reaction with Dsgpdh5’F1 primer. Other handlings including touchdown PCR were employed according to the SMARTer™ RACE cDNA Amplifcation Kit User Manual, with the exception of LA Taq DNA polymerase (TaKaRa) for touchdown PCR, rather than Advantage 2 Polymerase Mix.The full-length G3PDH cDNA was obtained with specific primes  agar gels, cloned in the pMD18-T vector (TaKaRa) and sequenced before the further experiments. Plasmid preparations, transformations, and other standard molecular biology techniques were carried out as described previously http://blast.ncbi.nlm.nih.gov/). Component analysis of G3PDH was calculated using DNAStar software 7.1.0 (Lasergene). Physical and chemical characteristics of G3PDH were analyzed by ProtParam tool (http://www.expasy.ch/tools/protparam.html). Multiple alignments among similar enzymes were conducted using Clustal X 1.83 . Phylogenetic and molecular evolutionary analysis of the amino acid sequences of different G3PDHs were conducted using the Neighbor Joining method and the molecular evolution genetics analysis (MEGA) software, version 4.0.2. The conserved structural domain of G3PDH was predicted via National Center for Biotechnology Information conserved domain database (CDD) in NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), and the protein conserved module of G3PDH was analyzed via the Pfam database (http://pfam.sanger.ac.uk/) to search its domain combinations. The amino acid sequence was subjected to TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/) for transmembrane analysis and SignalP 3.0 Server  for the prediction of protein signal sequence. Subcellular localization presumption was performed using WoLF PSORT (http://wolfpsort.org/). Secondary structure was predicted via the NPS@ service (http://npsa-pbil.ibcp.fr/) and PredictProtein (https://www.predictprotein.org/); 3D structure was constructed using 3D-JIGSAW (http://bmm.cancerresearchuk.org/~3djigsaw/).Sequence analysis was performed using Blast Software  into the BamH I and Xho I  restriction sites of expression vector pET-32a(+) . The G3PDH cDNA fragment was used as templates to synthesize the G3PDH ORF by using PrimSTAR HS DNA Taq  with forward primer ORF-F and reverse primer ORF-R, which will introduce the BamH I and Xho I site into the 5′ and 3′ end of the ORF, respectively. The PCR procedure is as the following: 1 cycle of 94°C, 30 s; 30 cycles of 94°C, 30 s, 60°C, 30 s, and 72°C, 2 min; and 1 cycle of 72°C, 10 min. PCR product was separated by electrophoresis in 1.5% (w/v) agar gels, cloned in the pMD18-T vector and transformed into E. coli JM109, which was verified by double digestion of BamH I and Xho I. Both plasmids pET-32a and pMD18-T-JM109-G3pdh were prepared from E. coli BL21 and JM109, then digested by BamH I and Xho I at 30°C for 45 min or 2 h. The digested products were separated by electrophoresis in 1% (w/v) and 1.5% (w/v) agar gels, and the ORF and pET-32a fragments were recycled using E.Z.N.A. kit . T4 DNA ligase  was then added, and samples were incubated at 16°C for 12–16 h. 5 µL of the ligation mixture was used to transform electronically competent E. coli (100 µL of BL21(DE3)). The correct strain of E. coli BL21 (DE3)/pET-32a-G3pdh was verified by double digestion of BamH I and Xho I.Plasmid pET-32a-G3pdh was constructed by insertion of the E. coli strains BL21 (DE3) were grown in LB medium at 37°C in darkness on a platform shaker at 230 cycles min−1. Ampicillin (100 µg mL−1) was used for selection or maintenance of plasmids. To induce the expression of the D. salina G3PDH, a final concentration of 1.0 mmol L−1 isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the E. coli culture when the optical density (OD) value reached 0.4–0.6, and the culture was allowed to continue growing for 3–4 h before harvesting by centrifugation.−1 phenylmethysulfonyl fluoride (PMSF). The supernatants were collected by centrifugation at 12,000 g for 30 min at 4°C. Protein concentration was estimated as described The transformed cells with plasmid pET-32a-G3pdh or pET-32a was disrupted by ultrasonication in PBS buffer containing 1 mmol LD. salina on the basis of the G3PDH gene sequences of D. salina, Chlamydomonas reinhardii, Arabidopsis thaliana, Pichia stipitis, zygosaccha and four predicted G3PDH gene sequences. When using total RNA from D. salina cells as RT substrate, an expected 583 bp fragment amplified with primers Dsgdph1-F/R was cloned and sequenced , 83 acidic amino acids , 266 hydrophobic amino acids  and 144 polar amino acids . Analysis by ProtParam tool revealed that the molecular weight of this peptide was 76.6 kDa, the isoelectric point was 6.49.Nucleotide sequence analysis showed that the D. salina G3PDH with NAD+ as coenzyme (AY845323.1), 83% and 78% with D. viridis G3PDH1 (EU624406.1), 76% and 72% with D. viridis G3PDH2 (EU624407.1), which indicated that the protein encoded by the obtained cDNA in this study might belong to the G3PDH family with NAD+ as coenzyme.A complete homologous search by BLAST demonstrated that the nucleotide and putative protein sequence had, respectively, sequence identities of 91% and 95% with the published D. salina contained three conserved regions including phosphoserine phosphatase (SerB) domain, N- and C- terminals binding to NAD+ (+-dependent G3PDH (EC 1.1.1.8). Eukaryotic neural network (NN) search by SignalP 3.0 showed that this polypeptide had no signal peptide. It was also predicted to be non-secretory protein by Markov models (HMM) of SignalP 3.0. TMHMM Server v. 2.0 predicted that the G3PDH had no transmembrane domain. Presumption of subcellular localization performed using WoLF PSORT showed that D. salina G3PDH may be situated in the chloroplast.The Conserved Domain Database (CDD) provided by NCBI was employed to predict the structural and functional region , which m to NAD+ . Using PNPS@service PHD, GOR1, SOPMA and PREDATOR methods. On account of the distinct emphases of various methods, the predicted results were also different. So these methods were made a comparison and PredictProtein was adopted to analyze online , D. viridis G3PDH (GI 189187651), D. viridis G3PDH (GI 189187649), Chlamydomonas reinhardtii predicted G3PDH (GI 159463132), Chlorella variabilis hypothetical G3PDH (GI 307107298), Volvox carteri hypothetical G3PDH (GI 302833661), Hordeum vulgare predicted G3PDH (GI 326525148), Sorghum bicolor hypothetical G3PDH (GI 242060059), Zea mays G3PDH (GI 226494897), Oryza sativa Japonica Group G3PDH (GI 115442511), Cuphea lanceolata G3PDH (GI 840731), Vitis vinifera hypothetical G3PDH (GI 147796339), Arabidopsis thaliana putative G3PDH (GI 19424089), Populus trichocarpa predicted G3PDH (GI 224140865), Ricinus communis putative G3PDH (GI 255572030), Selaginella moellendorffii hypothetical G3PDH (GI 302820075), Physcomitrella patens subsp. patens predicted G3PDH (GI 168031121). The results indicated the general conservatism of botanical and algal G3PDH genes  expression vector in the BamH I/Xho I sites. The constructed prokaryotic expression vector pET-32a-G3pdh was transformed into E. coli strain BL21 (DE3) and IPTG was used for induction. The G3PDH in transgenic strains were analyzed by SDS-PAGE. As shown by The G3PDH ORF sequence of 2100 bp was amplified by PCR with Dunaliella  functioned by varying the intracellular concentration of a compatible solute glycerol to balance the osmotic pressure inside and outside of cells. When subjected to hyperosmotic shock D. salina cells rapidly shrink followed by synthesis of glycerol to increases the internal osmolarity for resuming original volume of cells. Under hypoosmotic shock, it was found rapid swells followed by a decrease in internal glycerol and volume resumption D. salina involved the key enzymes G3PDH, which convert DHAP to glycerol-3-phosphate. Some studies have been performed to investigate the G3PDH for elucidating the mechanism of osmotic stress tolerance by glycerol. In a study, a novel G3PDH (NAD+) (EC1.1.1.8) gene (PfGPD) was cloned from halotolerant yeast Pichia farinosa, and the PfGPD gene was induced by salt stress +-dependent G3PDH from D. salina, and the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively As is known, the osmotic adjustment response of D. salina acts with NAD+ as coenzyme, and the comparative study of conservative regions discovered NAD+ and 3-phosphoglycerate binding sites in the G3PDH protein, theoretically testifying the cloned cDNA encodes G3PDH of osmosis-adjusting type. The G3PDH protein deduced from G3PDH cDNA contains 699 amino acids, of which the molecular weight is 76.6 kDa and the isoelectric point is 6.49, with −3.33 charge in pH 7.0. This G3PDH gene and protein had 91% and 95% identity with the putative G3PDH gene sequence (AY845323.1) and protein (AAX56341.1) + as coenzyme. It was reported that three isoforms of G3PDH have been separated from D. tertiolectaD. salina FAD-G3PDH, which situated in the mitochondrial. The expression of FAD-G3PDH was enhanced by salt treatment. Its catalytic site facing toward the cytosol, combined action of this enzyme with the cytosolic NAD+-dependent G3PDH forms the glycerol-3-phosphate shuttle +-dependent G3PDH oxidizes cytosolic NADH to NAD+, and catalyzes the reduction of DHAP to glycerol-3-phosphate. Subsequently glycerol-3-phosphate passes the outer mitochondrial membrane and is oxidized to DHAP by FAD-G3PDH, simultaneously delivers its electrons to the respiratory chain In the present study, the cloned 2100 bp G3PDH cDNA from DsGPDH2 in D. salina and DvGPDH1 and DvGPDH2 in D. viridis all encoded unique bi-domain proteins with C-terminal G3PDH domains and additional N-terminal SerB domains Debaryomyces hansenii, Candida glycerinogenes and CandidamagnoliaeAccording to the analysis of conservative region of the G3PDH in the present study, it could be speculated that the G3PDH functional domain originate at the 331 amino acid of the amino sequence and the first 330 amino acids are potentially correlated with other properties of the protein. Namely, G3PDH protein has two independent functional domains, SerB and G3PDH domains. SerB (EC 3.1.3.3) and glycerol-3-phosphate phosphatase (EC 3.1.3.21) are attributed to the same type of hydrolase, and two enzymes have similar functions due to their active centers of similar size. Therefore, it was speculated the G3PDH might also have glycerol-3-phosphate phosphatase activity and can catalyze DHAP to glycerol directly without glycerol-3-phosphate phosphatase. Similarly, in the studies by He et al and He et al, protein domain analysis revealed that +-G3PDH are cloned from D. salina. These genes have been transferred into bacteria or plants to increase the salt-tolerance of these species. Zhang et al transferred the DsALDP gene into E. coli cultured in media with different NaCl concentration to analyze its expression Agrobacterium tumefaciens, and DsALDP gene was expressed effectively in transgenic tobacco, which exhibited a higher salt tolerance D. salina has been transferred into led discs cells of tobacco. RT-PCR analysis showed that G3PDH gene integrated into tobacco genome has produced mRNA E. coli strain BL21 (DE3). The analysis by SDS-PAGE showed that the G3PDH protein was expressed successfully in transgenic strains, and the further work would emphasize on transforming this G3PDH gene into other higher plants to improve their salt tolerance.Some key enzyme genes related to glycerol metabolism, such as the cDNA of fructose-1, 6-diphosphate aldolase (DsALDP) and NAD+-G3PDH was successfully isolated from D. salina. The cDNA was 2100 bp long, which encoded a deduced protein sequence of 699 amino acids with an estimated molecular weight of 76.6 kDa. Protein domain analysis revealed that G3PDH protein has two independent functional domains, SerB and G3PDH domains. The D. salina G3PDH was a nonsecretory protein that may be located in the chloroplast. The D. salina G3PDH had a closer relationship with Dunaliella G3PDHs than with those of other species in the phylogenetic analysis. The secondary and three-dimensional structure of the D. salina G3PDH is predicted. In addition, the prokaryotic expression vector pET-32a-G3pdh was constructed and transferred into E. coli, in which G3PDH protein was expressed successfully. To fully understand glycerol metabolism and osmotic adjustment based on glycerol in Dunaliella, future investigation should focus on the gene clone of some enzymes related to glycerol metabolism and the application of transgenic technology to increase salt-tolerance of other plants by transferring these cloned genes.In conclusion, in the present research the cDNA of a NAD


Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisiae.Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote  S. cerevisiae consists of two subunits, eEF1Balpha which interacts directly with eEF1A and eEF1Bgamma. A third subunit, eEF1Bdelta, which also binds to eEF1A has been identified in other organisms such as Artemia salina, rabbit and human fetal cells  is a G-protein which delivers aminoacyl-tRNAs (aa-tRNAs) to the ribosomal A-site and allows for proper codon-anticodon mediated deciphering of the genetic code. This reaction requires hydrolysis of GTP to GDP and is assisted by eEF1B, a guanine nucleotide exchange factor that regenerates GTP-bound eEF1A and in T. brucei, only domain III of eEF1A is EPG-modified (residue E362) despite the presence of a second potential EPG modification site in domain II (residue E289) T. brucei as a model organism have shown that the ethanolamine moiety in EPG originates from the phospholipid phosphatidylethanolamine T. brucei eEF1A is strictly required for attachment of EPG, indicating that the enzyme-mediated attachment of EPG (or its precursor molecule) is highly specific for glutamic acid at this position T. brucei parasites expressing EPG-deficient eEF1A show no detectable growth defect demonstrating that, at least in cell culture, EPG attachment is not essential for eEF1A function in T. bruceiS. cerevisiae eEF1A may lack EPG: analysis of the peptide containing the postulated EPG attachment site by amino acid sequencing showed no evidence for the presence of EPG-modified glutamate S. cerevisiae eEF1A is not EPG-modified in T. brucei. Furthermore, we found that despite highly conserved protein sequences and similar predicted 3D-structures of yeast, T. brucei and mammalian eEF1A, orthologs cannot replace the endogenous protein. However, using plasmid shuffling techniques in S. cerevisiae, we were able to show that inter-species chimeric forms of eEF1A are able to functionally complement in vivo.Being one of the most abundant proteins in eukaryotes , several other functions besides its canonical role in translation elongation have been assigned to eEF1A  was purchased from American Radiolabeled Chemicals Inc. . Kodak MBX films were from Kodak SA .Unless otherwise specified, all reagents were of analytical grade and were from Merck , Sigma-Aldrich  or ICN Biomedicals . DNA polymerase was obtained from Invitrogen . Restriction enzymes were purchased from Thermo Scientific  or New England Biolabs . Etn for 18 h Trypanosomes were labeled during exponential growth , soaked in Amplify , dried, and exposed to Kodak MBX films at −70°C. Semi-dry blotting was performed as previously described Extracted proteins were separated by glycine-SDS-PAGE under reducing conditions using 12% polyacrylamide gels. For fluorography of 6x-tagged ScEF1A was purified from yeast cell extracts essentially as described His6x was transferred to a PVDF-membrane (BIO-RAD) and mass spectrometric analysis was carried out after on-membrane tryptic digestion. Tryptic peptides were analyzed by liquid mass spectrometry (nano-LC-MS/MS) as described previously.Immunoprecipitated HA-eEF1A proteins were analyzed by SDS-PAGE and immunoblotting as described above. After staining with Coomassie Brilliant Blue, HA-eEF1A proteins were cut out, cut into small pieces and exposed to on-membrane reductive alkylation and trypsin digestion + . Detection of polycistronic TbEF1A mRNA using a 32P-labeled probe made by random priming of the PCR product of TbEF1A intergenic region 1, subsequent hybridization and analysis by autoradiography using Bio-Max MS film and a TransScreen-HE intensifying screen was performed as described previously Total RNA for Northern blotting was isolated using a SV Total RNA Isolation System  and 10 μg of the total RNA was separated on formaldehyde-agarose gels propanesulfonic acid). To control for equal loading, rRNA was visualized by ethidium bromide staining. Afterwards, RNA was transferred from the gel to positively charged nylon membrane, Hybond-NS. cerevisiae (ScTEF1_RT_f/ScTEF1_RT_r), H. sapiens (HsTEF1_RT_f/HsTEF1_RT_r), L. major (Lm080_RT (f)/Lm080_RT (r)) or T. brucei . Total RNA extract was used as a negative control and PCR products were analyzed by electrophoresis using 0.8% agarose gel.To demonstrate the presence of transcripts of Hs-EF1A, Sc-EF1A, Lm-EF1A, and Tb-EF1A reverse transcription-PCR (RT-PCR) was performed. Total RNA was extracted from trypanosomes 72 h after induction with tetracycline. Complementary DNA (cDNA) was produced from single-strand RNA according to the manufacturer’s guidelines (SuperScript® II Reverse Transcriptase and Oligo(dT)20 Primer, Invitrogen) using 1 μg of extracted total RNA. Amplification of cDNA was done in 30 cycles using primer pairs , specifiwww.pdb.org). Subsequently structural models have been drawn with PyMOL Molecular Graphics System (DeLano WL).To build three-dimensional models of eEF1A proteins, pdb formats were generated as previously described T. brucei, L. major, H. sapiens were PCR amplified from plasmids pRS314_HA-TbTEF1 (E. coli strain #1873) and pMC4_HsTEF1 (E. coli strain #2056), pMC4_LmTEF1 (E. coli strain #2057) respectively. C. albicans eEF1A (CaTEF1) and A. thaliana eEF1A (AtTEF1) domain III were PCR amplified from a C. albicans cDNA library  and an A. thaliana cDNA library . 5′BamHI and 3′SpeI restriction sites – or 5′BamHI and 3′SacI restriction sites for CaTEF1– flanking the ends of ORFs were introduced with the help of oligonucleotide primers during PCR .eEF1A proteins from homology  and theH. sapiens eEF1A (HA-HsEF1A) and HA-tagged S. cerevisiae eEF1A (HA-ScEF1A) were expressed from ectopic copies in the EP/GPEET locus of T. brucei Δprocyclin#1 cells. Immunoblotting experiments demonstrated that HA-tagged eEF1A from both organisms were expressed in T. brucei and could be detected using α-HA antibodies . Manual interpretation of the fragment spectra of both parental ions identified E374 as the site of EPG attachment (results not shown). In contrast, analysis of the HA-ScEF1A revealed a tryptic fragment with m/z 893.467 and 447.237, representing the [M+H]+ and [M+2H]2+ ions of the unmodified heptapeptide FDE*LLEK  (m/z 447.237 confirmed the absence of EPG (results not shown). The tryptic peptides SVEMHHEALSE*ALPGDNVGFNV (with E* representing E301 in HA-HsEF1A) and SVEMHHEQLE*QGVPGDNVGFNVK (with E* representing E298 in HA-ScEF1A) in domains II of the respective proteins were not modified with EPG either (see + and [M+2H]2+ ions). Together, these results demonstrate that despite the structural conservation of eEF1A proteins from different organisms, only HA-HsEF1A but not HA-ScEF1A could serve as substrate for EPG attachment in T. brucei.Mass spectrometry analysis of eEF1A proteins expressed in te E372) . MS/MS aS. cerevisiae eEF1A 6x-tagged form of eEF1A in a S. cerevisiae lacking endogenous eEF1A and analyzed the immunoprecipitated protein by LC-MS/MS. The results gained on the tryptic peptides FDELLEK and FDELLEKNDR showed that residue E372 was not modified either (S. cerevisiae eEF1A does not become EPG-modified in any of both tested systems (yeasts and trypanosomes).To re-visit an earlier report showing that EPG is absent in d either . We concT. brucei eEF1A (TbEF1A) function. In a previous study, we generated a tetracycline-inducible T. brucei cell line (named C5) in which expression of TbEF1A was ablated using RNAi, resulting in growth arrest of the parasites in T. brucei. The addition of tetracycline to the culture medium allowed us to simultanously down-regulate endogenous TbEF1A and induce expression of HsEF1A or ScEF1A. The results showed that neither HsEF1A nor ScEF1A were able to complement the growth defect of TbEF1A-depleted T. brucei, whereas TbEF1A fully restored growth (L. major (LmEF1A) in the C5 cell line, which is highly homologous to TbEF1A, differing in only 25 amino acids  had no effect on revisiae . Growth revisiae .In addition, we observed that ScI_HsII_ScIII [construct 8] was able to complement endogenous yeast eEF1A. Although the amino acid sequence differs in only few residues from wild type ScEF1A, the construct produced a temperature sensitive growth phenotype . InteresFinally, all constructs lacking yeast domain I such as HsI_ScII_ScIII [construct 11], HsI_ScII_HsIII [construct 9], HsI_HsII_ScIII [construct 3] and TbI_TbII_ScIII [construct 5] were not able to support growth of TKY102 on 5-FOA plates .T. brucei procyclic forms in culture. The attachment site corresponds to the same residue that is modified in HsEF1A in mammalian cells T. brucei. Thus, the enzymatic machinery of T. brucei EPG-modified HsEF1A the same way as endogenous TbEF1A, which is modified in domain II but not in domain III T. brucei, which later during evolution developed the capacity to modify domain II of eEF1A in mammalian and plant cells, or if two different EPG modifying enzyme systems exists in these multicellular organisms.Our results using in vivo labeling and mass spectrometry demonstrate that EPG is attached to E374 of HA-tagged HsEF1A expressed in S. cerevisiae represents so far the only eukaryote lacking EPG modification of eEF1A  EPG modification site on the surface of a β-sheet in domain III is conserved between yeast and T. brucei. In a previous report we have shown that replacement of the primary sequence around the EPG attachment site of T. brucei eEF1A  by the yeast sequence  didn’t affect EPG modification in T. brucei. Thus, the different amino acid sequence around the EPG attachment site of S. cerevisiae eEF1A per se did not prevent EPG attachment Interestingly, T. brucei in which the endogenous (T. brucei) eEF1A is down-regulated by RNAi and the depletion phenotype is dependent on the expression of an eEF1A homolog from related (L. major) or unrelated organisms . The results showed that none of the conditionally expressed eEF1A proteins could rescue TbEF1A depletion in T. brucei. These findings were confirmed in eEF1A-depleted S. cerevisiae cells, where none of the expressed eEF1A proteins  could rescue the depletion phenotype. The lack of complementation cannot be due to missing EPG modifications as human eEF1A is properly expressed and EPG-modified in T. brucei. Furthermore, it has been recently shown that a mutated version of T. brucei eEF1A lacking EPG is able to complement endogenous eEF1A in cell cultures Since EPG was only attached to human but not to yeast eEF1A despite structural similarity, we decided to perform complementation assays in in vivo or in vitro in an eIF4A-depleted yeast cell system Together, these results demonstrate that the essential component of protein translation eEF1A, despite high sequence conservation and overall similarity in 3D-structure is unable to functionally complement across species. Interestingly, functional complementation across species has been reported for other translation factors such as eIF4E S. cerevisiae TKY102, we found that the carboxy terminus of certain eEF1A proteins (comprising domain II or domain III) can be exchanged without loss of viability. Domain III from A. thaliana was not able to complement in conjunction with yeast eEF1A domains I and II as a chimeric construct. This came as a surprise, as plants are assumed to be evolutionary closer related to yeast than human or trypanosome genes and demonstrated that there are also constraints to exchanging the essential domain III of yeast eEF1A.By constructing a series of inter-species eEF1A chimera and expressing them in the conditionally lethal strain S. cerevisiae. Similar findings have been reported for eubacterial EFTu, where recombinant chimeric elongation factor containing domain I of aEF1A from archea Sulfolobus solfataricus and domains II and III from Escherichia coli EF-Tu did not sustain poly(Phe) synthesis in either a S. solfataricus or a E. coli assay system In contrast, the amino terminus (comprising domain I) is essential for function in 2+ ion in domain I (switch regions 1 and 2). The GTPase activity of eEF1A is affected by ribosomes that trigger the hydrolysis of GTP in eukaryotes and similarly, they enhance by more than100-fold the intrinsic GTPase activity of EF-Tu in bacteria The essentiality of domain I may be explained as follows: (i) Domain I is crucial for interactions with macromolecules involved in the process of protein elongation such as ribosomal proteins and other interaction partners like eEF1B. eEF1A is a well-known member of the superfamily of GTPases and carries regions essential for binding of GTP and the MgFigure S1ClustalW Multiple sequence alignment of eEF1A sequences from different sources. Yellow indicates conserved and semi-conserved substitutions within a column of residues. Unconserved changes are black boxed. eEF1A domain I is shown underlined in blue, domain II underlined in orange and domain III underlined in red. Protein accession numbers for eEF1A sequences: T. brucei, P86934; L. major, Q4QEI8; S. cerevisiae, P50522; C. albicans, Q59K68; H. sapiens, P68104; A. thaliana, P1390.(DOC)Click here for additional data file.Table S1Primers used for experiments with T. Brucei.in vivo complementation assay in T. brucei. Underlined nucleotides indicate mutated triplets. T. brucei RNAi cell line C5.(DOC)Click here for additional data file.Table S2Primers used for experiments with S. Cerevisiae.His6X and amplification of eEF1A genes from different eukaryotic sources. Underlined nucleotides indicate sequences of restriction sites, letters in bold prints the His6x-tag sequence. (DOC)Click here for additional data file.Table S3Plasmids used for experiments with S. Cerevisiae.(DOC)Click here for additional data file.Table S4Genotype of yeast strains used in this work.(DOC)Click here for additional data file.


Tumor suppressor p53 is mutated in a wide variety of human cancers and plays a critical role in anoikis, which is essential for preventing tumorigenesis. Recently, we found that a nucleolar protein, Myb-binding protein 1a (MYBBP1A), was involved in p53 activation. However, the function of MYBBP1A in cancer prevention has not been elucidated.MYBBP1A expression levels and breast cancer progression were examined using patient microarray databases and tissue microarrays. Colony formation, xenograft, and anoikis assays were conducted using cells in which MYBBP1A was either knocked down or overexpressed. p53 activation and interactions between p53 and MYBBP1A were assessed by immunoprecipitation and western blot.Relationships between MYBBP1A expression was negatively correlated with breast cancer tumorigenesis. In vivo and in vitro experiments using the breast cancer cell lines MCF-7 and ZR-75-1, which expresses wild type p53, showed that tumorigenesis, colony formation, and anoikis resistance were significantly enhanced by MYBBP1A knockdown. We also found that MYBBP1A binds to p53 and enhances p53 target gene transcription under anoikis conditions.These results suggest that MYBBP1A is required for p53 activation during anoikis; therefore, it is involved in suppressing colony formation and the tumorigenesis of breast cancer cells. Collectively, our results suggest that MYBBP1A plays a role in tumor prevention in the context of p53 activation. Breast cancer is the most commonly occurring cancer in women worldwide. It has been estimated that more than 1.6 million new cases of breast cancer occurred in 2010 . The extracted proteins were immunoprecipitated with antibody-coated protein G Sepharose beads . Bound proteins were separated by SDS–PAGE, transferred to polyvinylidene difluoride membranes , and detected with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized using an enhanced chemiluminescence (ECL) western blot detection system .ChIP assay was performed according to the published procedures . The priCells were fixed in 3.7% formaldehyde in PBS for 10 min. After rinsing twice with PBS, the cells were permeabilized in 0.1% Triton X-100 in PBS and later blocked with TBS-T buffer containing 0.5% bovine serum albumin and 10% goat serum for 1 h at room temperature. Subsequently, the cells were incubated with anti-MYBBP1A and anti-p53 antibodies for 1 h, stained with Alexa Fluor-conjugated secondary antibodies  for 1 h, and mounted with Vectashield . Immunofluorescent images were obtained by Biozero immunofluorescence microscopy .MYBBP1A expression and breast cancer progression, we examined the MYBBP1A expression profiles in breast carcinomas compared to those of normal tissue using the Oncomine database, which provides publicly available datasets of gene expression in cancer. Of the 12 datasets, 11 contained gene chip profiles classified as normal or breast carcinoma tissues, which indicated that MYBBP1A mRNA levels were significantly lower in breast carcinomas than in normal tissues. Three representative results from independent datasets characterized by large population sizes are shown in Figure MYBBP1A expression levels in normal vs. breast carcinoma; P = 2.95E-25, 2.10E-6, and 7.17E-4). Next, we used IHC to assess MYBBP1A expression in non-neoplastic and breast cancer tissues using a human breast cancer tissue microarray. As shown in Figure We previously reported that MYBBBP1A was involved in activating p53 function. Therefore, we assumed that MYBBP1A would have a cancer prevention function via p53. To examine the relationship between MYBBP1A . Conversely, MYBBP1A overexpression decreased the number of these colonies (Figure To investigate a possible relationship between MYBBP1A and breast cancer cell growth, we generated MCF-7 cells that had stably knocked down or overexpressed A Figure A and 2B.MYBBP1A expression on tumorigenicity in vivo. MYBBP1A knockdown cells formed tumors that were significantly larger than those of control cells. In contrast, MYBBP1A overexpressing cells formed smaller tumors than control cells (Figure Next, we performed xenograft experiments to test the effect of s Figure E, and 2FMYBBP1A knockdown (Figure p53 knockdown, while they decreased when p53 was overexpressed. Similarly, MYBBP1A knockdown increased and MYBBP1A overexpression decreased the numbers of viable cells under detached conditions (Figure To examine how MYBBP1A could suppress tumor formation we focused on anoikis, because p53 plays a critical role in anoikis -12. Thusp53 knockdown and MYBBP1A overexpression in anoikis assay using MCF-7 cells (Figure MYBBP1A overexpression decreased the number of viable cells (compare lanes 1 and 2 in Figure MYBBP1A overexpression in p53 knocked-down cells did not show any significant effects (compare lanes 3 and 4 in Figure To confirm that MYBBP1A was involved in anoikis in context of p53 activation, we tested the combination of s Figure A. Based p53 and MYBBP1A knockdown and increased when these genes were overexpressed (Figure MYBBP1A overexpression in p53 knocked-down cells did not result in any significant effects (Figure To further examine the role of MYBBP1A in anoikis, we examined cellular apoptosis as determined by Annexin V–FITC/PI staining, followed by flow cytometric analysis. In accordance with the results shown in Figures MYBBP1A knockdown on the induction of p53-target genes were examined. The mRNA levels of Bax, PUMA, and p21 were increased under detached conditions, whereas the increases in these mRNA levels were suppressed when MYBBP1A was knocked down using siRNA in MCF-7 cells (Figure Next, the effects of s Figure A. Similas Figure B. These MYBBP1A was knocked down using siRNA (Figure The acetylation levels of p53 are increased in response to stress and correlate well with p53 activation and stabilization -28. AccuA Figure A lane 4.Bax promoter was significantly enhanced under detached conditions, while p53 recruitment was abrogated by MYBBP1A knockdown (Figure Consistent with these results, p53 recruitment to the n Figure B.A previous report showed that MYBBP1A activates p53 by facilitating its direct interaction with p53 in response to stress when MYBBP1A translocated from the nucleolus to the nucleoplasm . TherefoIn this study, we revealed the physiological significance of MYBBP1A in p53 activation for prevention of cancer. MYBBP1A was originally identified as a protein that interacted with the negative regulatory domain of c-Myb . HoweverWe previously reported that MYBBP1A interacts with p53 and activates its transcriptional capacity. MYBBP1A localizes predominantly in the nucleolus; however, it translocates from the nucleolus to the nucleoplasm in response to cellular stress and activates p53 ,14,35. SThe regulation of MYBBP1A localization under detached conditions is still to be elucidated. When cells are exposed to UV light, MYBBP1A translocation is accompanied by nucleolar segregation . BecauseNumerous reports have shown that other nucleolar proteins can activate p53 similar to that by MYBBP1A. RPL11 directly bind to HDM2 and inhibit HDM2-mediated p53 ubiquitination -41. Simiet al. recently reported that MYBBP1A regulates the proliferation and migration of head and neck squamous cell carcinoma cells [In addition, Sanhueza ma cells . HoweverMYBBP1A expression was associated with breast cancer tumorigenesis. In vivo and in vitro experiments using the breast cancer cell lines revealed that tumorigenesis, colony formation, and anoikis resistance were significantly enhanced by MYBBP1A knockdown. Co-immunoprecipitation experiments revealed that MYBBP1A binds to p53 under detached conditions and enhances p53 target gene transcription (Figure To determine the role of MYBBP1A in cancer, we conducted an extensive analysis of the Oncomine database and IHC studies, and showed that n Figure . These rp53: Protein 53; MYBBP1A: Myb-binding protein 1a; ECM: Extracellular matrix; p21: Protein 21; Bax: Bcl-2–associated X protein; PUMA: p53 upregulated modulator of apoptosis; p300: Protein 300; IHC: Immunohistochemistry; FACS: Fluorescence-activated cell sorting; FITC: Fluorescein isothiocyanate; RT-qPCR: Reverse transcription quantitative polymerase chain reaction; DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; PCR: Polymerase chain reaction; shRNA: Short hairpin RNA; siRNA: Small interfering RNA; RT-PCR: Reverse transcription polymerase chain reaction; PMSF: Phenylmethylsulfonyl fluoride; SDS-PAGE: Sodium dodecyl sulfate-polyaclylamidegel electrophoresis; ECL: Enhanced chemiluminescence; ChIP: Chromatin immunoprecipitation; PBS: Phosphate buffered saline; TBS-T: Tris-buffered saline and tween 20; TMA: Tissue microarray; OE: Overexpression; CBP: CREB-binding protein; qPCR: Quantitativepolymerase chain reaction; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; CRY1: Cryptochrome 1.The authors declare that they have no conflicts of interests concerning this work.KA participated at the design, execution and interpretation of the experiments, as well as writing up of the manuscript. WO participated at the immunoblotting experiments and the presentation of the manuscript. YH participated at the FACS analysis. HK participated at the interpretation of the data and the presentation of the manuscript. JY participated at the design and interpretation of the experiments, as well as writing up of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/65/prepub


Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3K499A) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3K499A strain. The ATPase activity of Mcm2-73K499A was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 , we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the  In order for DNA replication to occur, the DNA duplex strands need to be separated by a replicative helicase Sulfolobus solfataricus (Sso) MCM  helicase from bovine papillomavirus (E1) and mini-chromosome maintenance (MCM) from archaeal species, provide insight into how DNA translocation is achieved Sso) MCM . MutatioIn eukaryotic cells, the replicative helicase is comprised of six paralogous proteins of the AAA+ family, termed Mcm2-7. Each of the six subunits is essential for DNA replication in cells from yeast to mammals Saccharomyces cerevisiae Mcm2-7 subunits to alanine and examined the effect of the mutations. Interestingly, only the PS1 hairpin of Mcm3 is essential for viability. Mutation of the PS1 hairpin in Mcm7 resulted in growth related phenotypes, and strains with pairwise mutations in the remaining PS1 hairpins displayed synthetic slow or lethal interactions. Consistent with the observed loss of viability, Mcm2-7 complexes containing Mcm3 bearing the PS1 mutation (Mcm3K499A) show decreased DNA unwinding in vitro. The Mcm3K499A-containing Mcm2-7 has reduced binding to single-stranded DNA in an electrophoretic mobility shift assay, and analysis of Mcm3K499A in yeast cell extracts revealed differences in its molecular associations. Together our results indicate the importance of the PS1 hairpins in Mcm2-7 function, and identify an essential function of Mcm3.Here, we have mutated the conserved lysine residue in the PS1 hairpin of each of the MCM genes, two plasmids for each wild-type gene were constructed  in the URA3-containing centromeric plasmid YCplac33 MCM. Oligonucleotide pairs MD81/MD82, MD83/MD84, MD85/MD86, MD87/MD88, MD89/MD90, and MD90/MD91 were used to amplify MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, respectively. The amplified MCM was then ligated into YCplac33 using the restriction enzyme cut sites indicated with the primers in LEU2-CEN; MCM3 through MCM6 these were constructed in a two-step process. The promoter region was amplified by PCR using a UTR primer and a start primer, then inserted into YCplac111 using the restriction sites in BlpI linker was then inserted into the polylinker SmaI site of the vector for MCM4 and MCM5 (pMD242 and 237). The remaining coding region was then inserted from a pET expression plasmids NdeI-SacI for MCM3 (pMD235), NdeI-BlpI for MCM4 and MCM5 (pMD379 and 378), and NdeI-BamHI for MCM6 (pMD239). For MCM7, the promoter region plus coding sequence up to the SalI site at 251 base pairs was amplified by PCR using oligonucleotides (MD91 and 113) and cloned into YCplac111 (pMD241). pMD241 was digested with SmaI and a BlpI linker inserted to generate pMD260. The remaining coding sequence of MCM7 was inserted from a pET expression plasmid SalI and BlpI to give pMD261. To generate K499Amcm3- , K658Amcm4- , K506Amcm5- , K665Amcm6- , K499Rmcm3- , K499Qmcm3- , and K499Nmcm3-  YCplac111, the Stratagene QuikChange site-directed mutagenesis kit was used with the indicated primer pairs. Isolated clones were sequenced.Oligonucleotides used to construct plasmids are listed in MCM3 (pMD562) and K499Amcm3 (pMD563) into YEplac181, a SphI-SacI fragment of pMD235 and pMD386 was ligated into the same sites of YEplac181. A fragment of SphI-SacI from pMD386 was also ligated to the same sites in YIplac211 to generate K499Amcm3-YIplac211 (pMD561). For cloning K658Amcm4 into YIplac211, we first amplified K658Amcm4 by PCR using oligonucleotides MD85, MD274, and K658Amcm4-YCplac111 as template. This product was cloned into YIplac211 using PstI and BamHI to give pMD438. For K506Amcm5, a BlpI site was introduced into YIplac211 at the SmaI site. K506Amcm5 was introduced into this plasmid from K506Amcm5-YCplac111 as a SphI-BlpI fragment (pMD439). pMD440 was constructed by inserting a SphI-BamHI fragment of K665Amcm6-YCplac111 into YIplac211. To generate MCM3 and K499Amcm3 myc9 N-terminally tagged expression plasmids, MCM3 and K499Amcm3 were amplified by PCR using oligonucleotides MD84, MD556, and cloned using NotI and SacI into a derivative of YCplac111 where the DED1 promoter drives expression of a myc9 N-terminally tagged protein K499Amcm3 was cloned by cutting K499Amcm3-YCplac111 with NdeI and SacI and ligating into the same sites of pET24a.To clone 3K499AMCM3 and mcm Flag3-tagged expression plasmids, a GAL10 promoter containing YCplac111 plasmid (pMD407) was linearized with NdeI and SacI. Oligonucleotides MD659/MD660 were annealed and ligated into linearized pMD407. MCM3 and mcm3K499A isolated from pMD502 and pMD503 were then inserted as NotI-SacI fragments to give pMD560 and pMD554.To construct mcm gene were obtained from Open Biosystems. The mcm2::his3 disruption strain (MDY54) was a derivative of YMD33 MCM-YCplac33 plasmid and sporulated to give MDY16, 17, 40, 41, 70, and 100. mcm deletion haploid strains containing their corresponding MCM-YCplac33 were transformed with a KAmcm-YCplac111 or MCM-YCplac111. The transformed strains were grown in YPD, then plated on 5-FOA-containing media to select for cells that lost the MCM-YCplac33 Diploid heterozygous strains containing a KanMX deletion of a K633Amcm2 (MDY225 and 226), MCM3/K499Amcm3 (MDY411), mcm4K658A (MDY220), K506Amcm5 (MDY221 and 222), K665Amcm6 (MDY258 and 259), K550Amcm7 (MDY253 and 254). Each mutation incorporated a unique restriction site (MscI (mcm3), AgeI, (mcm4) BspEI (mcm5), MscI (mcm6), or BamHI (mcm7) and transformed into BY4743. URA3 positive colonies were grown in YPD liquid media, and then selected on 5-FOA-containing media. PCR amplification of the MCM loci was performed and restriction mapping used to confirm integration of KAmcm. The heterozygous diploid strains were sporulated and haploid KAmcm mutants isolated.All yeast strains are listed in ion site  for idenGAL10-MCM3 or YCplac111-K499AGAL10-mcm3 was grown in minimal media lacking leucine supplemented with 2% galactose overnight. The overnight cultures were diluted to 106 cells/mL with minimal media lacking leucine supplemented with 2% galactose. After two hours cells were imaged under bright field using a Nikon Eclipse Ti microscope. Measurements were taken using NIS Elements Imaging Software.BY4741 transformed with YCplac111-Escherichia coli and reconstituted into Mcm2-7 as described The recombinant Mcm subunits were purified from K499Amcm3 pET24a plasmid was transformed into BL21 DE3 Codon+. Twelve liters of transformed cells were grown in LB media with 100 mg/L of ampicillin, and 25 mg/L of chloramphenicol to a density of A600 = 0.6. Cells were cooled to 15°C and isopropyl β-D-1-thiogalactopyranoside added to a final concentration of 1 mM. Cells were incubated at 15°C for 20 hours prior to collecting the cell pellet. The cell pellet was resuspended in 250 mL of Buffer H  and lysed at 15000 psi in an Emulsiflex-C3 high pressure homogenizer. Debris was pelleted by centrifugation at 15000 g for 25 minutes and the supernatant decanted. Ammonium sulfate was added to the supernatant (0.25 g/mL) with stirring at 4°C. Ammonium sulfate precipitate was collected by centrifugation, and resuspended with 150 mL of Buffer H. The solution was dialyzed overnight at 4°C in 4 L of Buffer H with stirring and then loaded onto a Fast flow Q Sepharose column equilibrated with Buffer H and washed with 7 column volumes of Buffer H containing 50 mM NaCl. Mcm3K499A was eluted over 10 column volumes in a gradient of 0–500 mM NaCl in Buffer H. Fractions containing Mcm3K499A were collected and dialyzed overnight in 750 mL of Buffer H at 4°C. A single-stranded (ss) DNA-Sepharose column was made by coupling boiled and sonicated salmon sperm DNA to cyanogen bromide-activated Sepharose 4B (GE Life Sciences). The dialyzed solution was loaded onto the ssDNA-Sepharose column equilibrated with Buffer H, washed with 10 column volumes and protein eluted with 10 column volumes of a 0–500 mM NaCl gradient in Buffer H. The fractions containing Mcm3K499A were pooled and ammonium sulfate added (0.3 g/mL) with stirring at 4°C. The precipitate was collected by centrifugation, resuspended and dialyzed against 400 mL of Buffer A . The solution was loaded onto a MonoQ (GE Life Sciences) column equilibrated with Buffer A containing 50 mM NaCl, and the protein eluted over 20 column volumes of a 0–500 mM NaCl gradient in Buffer A. Fractions containing Mcm3K499A were pooled and dialyzed in one liter of Buffer H with stirring, overnight at 4°C. The dialyzed solution was loaded onto a MonoS (GE Life Sciences) column equilibrated with Buffer H containing 50 mM NaCl, and the protein eluted over 20 column volumes of a 0–500 mM NaCl gradient in Buffer H. Fractions were collected and frozen at −80°C.The et alWestern blotting was performed using polyvinylidene difluoride membranes and anti-myc (Sigma-Aldrich) as described by Mutiu et al32P]ATP , 20 mM Tris–HCl (pH 7.5), 10 mM magnesium acetate, and 2 mM DTT, and 200 nM Mcm2-7. At the indicated times, 2 µL of each reaction was removed and quenched with 2 µL of 50 mM EDTA (pH 8). One microliter was spotted onto a polyethyleneimine cellulose sheet (EM Science), developed in 0.6 M potassium phosphate (pH 3.4), dried, exposed to a PhosphorStorage screen, and scanned with a Storm 860 scanner . DNA unwinding measurements were performed with a DNA substrate containing 30 nucleotides of duplex, with 60 nucleotides of single-stranded DNA on one strand and a 5′ biotin on the other strand. Each reaction (6 µL) contained 20 mM Tris–HCl (pH 7.5), 10 mM magnesium acetate, 100 µM EDTA, 5 mM DTT, 5 mM ATP, 67 nM streptavidin, 1 nM substrate with 100 nM, 200 nM or 400 nM Mcm2-7. Samples were analyzed by native PAGE using an 8% gel in Tris–borate–EDTA buffer.DNA unwinding and ATPase assays were performed essentially as described by Stead K499A purification). Five micrograms of Mcm2-7 complex were applied to the column in buffer H containing 5 mM ATP and 50 mM NaCl and eluted with buffer containing 5 mM ATP and either 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500 mM NaCl. Each elution was performed twice with one column volume. 24 µL of each fraction was separated by SDS-PAGE (6%). The polyacrylamide gels were stained with colloidal blue stain, and then washed with deionized water to destain the gels for imaging. The destained gels were then silver stained according to protocol provided in Pierce Silver Stain Kit (Thermo Scientific) to detect protein in the column fractions.The single-stranded DNA affinity chromatography was performed with a 200 µL single-stranded DNA Sepharose column 60) at 37°C in buffer containing 20 mM Tris–HCl (pH 7.5), 10 mM magnesium acetate, 100 µM EDTA, 5 mM DTT, and 5 mM ATP for 10 minutes. One microgram of anti-Mcm7 antibody (Santa Cruz Biotech) was added to another set of Mcm2-7 samples prior to incubation with radiolabeled oligonucleotide to disrupt Mcm2-7 binding. Four microliters of 12.5% glycerol was added to each reaction and then resolved in a 5% native (Tris–borate–EDTA) polyacrylamide gel  containing 5% glycerol, 0.1% NP-40 and 10 mM Mg(CH3COO)2 at 30 mA for 2.5 hours. The gel was dried, and exposed to film.The electrophoretic mobility shift assay was adapted from Stead et alK449A were resolved by SDS-PAGE and proteins detected by western blotting.Proteins extracts were prepared cryogenically as described by Saleh Solfolobus solfataricus Mcm , which is toxic to URA3-expressing strains. Of the six subunits, only a mutation in the Mcm3 PS1 hairpin (K499Amcm3) resulted in a loss of viability on 5-FOA  hairpin adjacent to the sensor 1 motif of the AAA+ domain . To deteon 5-FOA . Slow grd period . The preK499Amcm3 strain was not due to a failure to maintain the plasmid, we integrated the K499Amcm3 mutation into the diploid yeast strain BY4743 and analyzed the viability of spore colonies after sporulation and tetrad dissection. As shown in K499Amcm3 to support growth. We also addressed whether K499Amcm3 would support growth when overexpressed on a 2-micron plasmid by plasmid shuffling   . ConsistS. solfataricus Mcm (SsoMcm) accommodates several subunits with disruptions in catalytic elements and still maintains significant helicase activity K658Amcm4 and K506Amcm5 were viable (K658Amcm4 or K506Amcm5 and were more sensitive to hydroxyurea and MMS (Our plasmid shuffling experiments indicate that a single mutation of the conserved PS1 lysine (K499) residue in Mcm3 results in loss of viability. In contrast, the homo-hexameric e viable . These g and MMS .K499Rmcm3 supported viability , and serial dilutions plated onto media containing glucose, raffinose, or galactose. In the presence of glucose or raffinose, where the GAL10 promoter is transcriptionally repressed or not induced respectively, there was no effect on growth, whereas in galactose-containing media induction of K499Amcm3 expression resulted in a slow growth phenotype  indicating that the Mcm2-73K499A is capable of binding single stranded DNA. Lack of binding by the peptidyl prolyl isomerase Pin-1 (lower panel), a relatively basic protein with a pI of 9.4, indicated that the binding by the Mcm complexes was specific for DNA and not simply due to charge interactions. We next used an electrophoretic mobility shift assay in an attempt to detect more subtle differences in DNA binding. A 5′ radiolabeled oligonucleotide of 90 bases was used as the substrate. As shown in 3K499A was assayed (lanes 11–14), the amount of Mcm2-73K499A-DNA complex was reduced suggesting that Mcm2-73K499A is less able to bind the 90 base single-stranded DNA or that the binding is unstable under the electrophoresis conditions.The loss of helicase activity in Mcm2-7K499Amcm3 strain. To investigate the effects of the Mcm3 hairpin mutation in cells, we analyzed myc9-Mcm3K499A expressed in yeast to determine if its ability to associate with other components required for replication differs from the wild type protein. Whole cell extracts containing myc9-tagged Mcm3 or Mcm3K499A were prepared from cells grown to mid-log phase in YPD media and analyzed by gel filtration chromatography on a Superose 6 column. As shown in 9-Mcm3K499A resembled that of the wild type in that it eluted as two peaks, but with significant differences for the high- and low- molecular weight complexes. In the high molecular weight complex, Mcm3 appears as a single band that migrates with a mass of 135 kDa, while in the K499Amcm3 strain, Mcm3 appears as a 135 kDa band but there are also two prominent bands at approximately 150 kDa and 175 kDa. These were not detected in the wild-type Mcm3 cells, even after prolonged exposure of the film. The reduced mobility forms of Mcm3 are likely the result of protein modification, but the nature of this modification is unclear. The second difference between wild-type and K499Amcm3 cells is that the smaller complex from wild-type cells elutes with a peak at 200 kDa . Since the purified Mcm2-73K499A hexamer assembles and behaves similarly to the wild-type complex in vitro, these results suggest that in cells the altered activities of the Mcm2-73K499A complex result in changes in its molecular associations. Mcm3 has a unique role in the initiation process in that it is able to recruit its neighboring subunits, Mcm5 and Mcm7, to the origin recognition complex (ORC) independent of Cdt1 K499A, leading to the differences we observe in Mcm3K499A-containing complexes.The loss of helicase activity is the most pronounced functional effect of the Mcm3 hairpin mutation, and may explain the inviability of the Methanobacterium thermoautotrophicum MCM (mtMCM) was then used to assemble each of Mcm 2 through 7 into the hexameric Mcm2-7 complex . The transformed yeast were grown overnight at 30°C in YPD media, serially diluted, and then spotted onto a YPD plate or a plate containing 5-FOA. The plates were incubated at 30°C for the number of days indicated.(TIF)Click here for additional data file.Figure S2Expression levels of Mcm 2 and 3. Yeast strains MDY70 (MCM2), MDY71 (K633Amcm2), MDY405 (9-MCM3DED1-myc) and MDY406 (9-mcm3K499ADED1-myc) were grown to mid-log phase; yeast extracts were prepared by grinding with glass beads, and 10, 20 or 40 µg of total protein separated by SDS-PAGE. Blots of these gels were probed with anti-Mcm2, (Santa Cruz Biotech) or anti-myc (Sigma-Aldrich) antibody to assess the level of Mcm subunit. We note that for Mcm3 detection the plasmids were transformed into BY4741 and thus contain wild-type Mcm3.(TIF)Click here for additional data file.