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S2 Cells (s2 + cell)
Kinds of S2 Cells Selected AbstractsRegulation of calpain B from Drosophila melanogaster by phosphorylationFEBS JOURNAL, Issue 17 2009László Kovács Calpain B is one of the two catalytically competent calpain (calcium-activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal-regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF-hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca2+ sensitivity of the protease. The kinetic analysis of phosphorylation mimicking Thr747Glu and Ser845Glu calpain B mutants confirmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epidermal growth factor-stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies. Structured digital abstract ,,MINT-7214239: ERK1 (uniprotkb:P40417) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214216, MINT-7214228: PKA (uniprotkb:P12370) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214325: CalpainB (uniprotkb:Q9VT65) cleaves (MI:0194) MAP2C (uniprotkb:P11137) by protease assay (MI:0435) ,,MINT-7214275: ERK2 (uniprotkb:P40417-2) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214319: CalpainB (uniprotkb:Q9VT65) and CalpainB (uniprotkb:Q9VT65) cleave (MI:0194) by protease assay (MI:0435) [source] Transcription factor NF-Y is involved in regulation of the JNK pathway during Drosophila thorax developmentGENES TO CELLS, Issue 2 2008Yasuhide Yoshioka The CCAAT motif-binding factor, nuclear factor Y (NF-Y) consists of three different subunits, NF-YA, NF-YB and NF-YC. Knockdown of Drosophila NF-YA (dNF-YA) in the notum compartment of wing discs by a pannir -GAL4 and UAS- dNF-YAIR mainly resulted in a thorax disclosed phenotype. Reduction of the Drosophila c-Jun N-terminal kinase (JNK) basket (bsk) gene dose enhanced the knockdown of dNF-YA-induced phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc) -LacZ enhancer trap line revealed reduction in the level of the JNK reporter, puc-LacZ signals, in dNF-YA RNAi clones. In addition, expression of wild-type Bsk effectively suppressed the phenotype induced by knockdown of dNF-YA. The bsk gene promoter contains a CCAAT motif and this motif plays a positive role in the promoter activity. We performed chromatin immunoprecipitation (ChIP) assays in S2 cells with anti-dNF-YA IgG and quantitative real-time PCR. The bsk gene promoter region containing the CCAAT boxes was effectively amplified in the immunoprecipitates by PCR. However, this region was not amplified in the immunoprecipitates from dNF-YA knockdown cells. Furthermore, the level of endogenous bsk mRNA is reduced in the dNF-YA knockdown larvae. These results suggest that dNF-Y is necessary for proper bsk expression and activity of JNK pathway during thorax development. [source] Functional analysis of the cis -acting elements responsible for the induction of the Cyp6a8 and Cyp6g1 genes of Drosophila melanogaster by DDT, phenobarbital and caffeineINSECT MOLECULAR BIOLOGY, Issue 1 2010R. Morra Abstract Many Drosophila cytochrome P450 or Cyp genes are induced by caffeine and phenobarbital (PB). To understand the induction mechanism, we created Drosophila S2 cell lines stably transformed with different luciferase reporter plasmids carrying upstream DNAs of Cyp6a8 allele of the resistant 91-R strain, and the 1.1-kb upstream DNAs of Cyp6g1 of the 91-R and the susceptible 91-C strains. Following 24 h treatment with dichlorodiphenyltrichloroethane (DDT), caffeine or PB, luciferase activity of all cell lines was determined. Results showed that the 0.1-kb DNA of Cyp6a8 and the upstream DNAs of Cyp6g1 from both strains are not induced by these chemicals in S2 cells. However, the 0.2-, 0.5- and 0.8-kb DNAs of Cyp6a8 showed 13,24-, 4,5- and 2.2,2.7-fold induction with caffeine, PB and DDT, respectively. These DNAs also showed a 2,3-fold synergistic effect of caffeine and PB but not of caffeine and DDT. The results suggest that the cis -regulatory elements for all three chemicals are located within the -11/-199 DNA of Cyp6a8. Furthermore, caffeine and PB inductions appear to be mediated via different cis -elements, whereas caffeine and DDT induction may involve common regulatory elements. These stably transformed cell lines should help understand the mechanism of resistance-associated Cyp gene overexpression in Drosophila. [source] Cactus-independent nuclear translocation of Drosophila RELISHJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001William D. Cornwell Abstract Insects can effectively and rapidly clear microbial infections by a variety of innate immune responses including the production of antimicrobial peptides. Induction of these antimicrobial peptides in Drosophila has been well established to involve NF-,B elements. We present evidence here for a molecular mechanism of Lipopolysaccharide (LPS)-induced signaling involving Drosophila NF-,B, RELISH, in Drosophila S2 cells. We demonstrate that LPS induces a rapid processing event within the RELISH protein releasing the C-terminal ankyrin-repeats from the N-terminal Rel homology domain (RHD). Examination of the cellular localization of RELISH reveals that the timing of this processing coincides with the nuclear translocation of the RHD and the retention of the ankyrin-repeats within the cytoplasm. Both the processing and the nuclear translocation immediately precede the expression of antibacterial peptide genes cecropin A1, attacin, and diptericin. Over-expression of the RHD but not full-length RELISH results in an increase in the promoter activity of the cecropin A1 gene in the absence of LPS. Furthermore, the LPS-induced expression of these antibacterial peptides is greatly reduced when RELISH expression is depleted via RNA-mediated interference. In addition, loss of cactus expression via RNAi revealed that RELISH activation and nuclear translocation is not dependent on the presence of cactus. Taken together, these results suggest that this signaling mechanism involving the processing of RELISH followed by nuclear translocation of the RHD is central to the induction of at least part of the antimicrobial response in Drosophila, and is largely independent of cactus regulation. J. Cell. Biochem. 82: 22,37, 2001. © 2001 Wiley-Liss, Inc. [source] Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptorsJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Zewen Liu Abstract Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the , subunits and another three loops (D, E, and F) of the non-, subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-, subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens, a target-site mutation (Y151S) was found within two , subunits (Nl,1 and Nl,3). In Drosophila S2 cells and Xenopus oocytes, Nl,1 can form functional receptors with rat ,2 subunit. However, the same thing was not observed in Nl,3. In the present paper, by exchanging the corresponding regions between Nl,1 and Nl,3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding. [source] 1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cellsACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Kausik Chattopadhyay Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure, murine GITRL demonstrated a unique `strand-exchanged' dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8,Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli -expressed proteins. The S2 cell-expressed murine GITRL adopts an identical `strand-exchanged' dimeric structure to that observed for the E. coli -expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL. [source] Purification, crystallization and preliminary crystallographic studies of the complex of interferon-,1 with its receptorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Eugenia Magracheva Human interferon-,1 (IFN-,1Ins) and the extracellular domain of interferon-,1 receptor (IFN-,1R1) were expressed in Drosophila S2 cells and purified to homogeneity. Both IFN-,1Ins and interferon-,1 produced from Escherichia coli (IFN-,1Bac) were coupled with IFN-,1R1 at room temperature and the complexes were purified by gel filtration. Both complexes were crystallized; the crystals were flash-frozen at 100,K and diffraction data were collected to 2.16 and 2.1,Å, respectively. Although the IFN-,1Bac,IFN-,1R1 and IFN-,1Ins,IFN-,1R1 complexes differed only in the nature of the expression system used for the ligand, their crystallization conditions and crystal forms were quite different. A search for heavy-atom derivatives as well as molecular-replacement trials are in progress. [source] Expression, purification, crystallization and preliminary X-ray analysis of the N-terminal domain of GNBP3 from Drosophila melanogasterACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Yumiko Mishima Gram-negative bacteria-binding protein 3 (GNBP3) is a pattern-recognition receptor which contributes to the defensive response against fungal infection in Drosophila. The protein consists of an N-terminal domain, which is considered to recognize ,-glucans from the fungal cell wall, and a C-terminal domain, which is homologous to bacterial glucanases but devoid of activity. The N-terminal domain of GNBP3 (GNBP3-Nter) was successfully purified after expression in Drosophila S2 cells. Diffraction-quality crystals were produced by the hanging-drop vapour-diffusion method using PEG 2000 and PEG 8000 as precipitants. Preliminary X-ray diffraction analysis revealed that the GNBP3-Nter crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 134.79, b = 30.55, c = 51.73,Å, , = 107.4°, and diffracted to 1.7,Å using synchrotron radiation. The asymmetric unit is expected to contain two copies of GNBP3-Nter. Heavy-atom derivative data were collected and a samarium derivative showed one high-occupancy site per molecule. [source] Rabies virus glycoprotein expression in Drosophila S2 cells: Influence of re-selection on protein expressionBIOTECHNOLOGY JOURNAL, Issue 11 2009Alexandra Souza dos Santos Abstract The aim of this study was to achieve expression of recombinant rabies virus glycoprotein (rRVGP) in Drosophila S2 cells. For this, a cDNA coding for the selection hygromycin antibiotic and the cDNA encoding the RVGP protein under the control of the constitutive actin promoter (Ac) were cloned in an expression plasmid, which was transfected into S2 cells. S2 cell populations (S2AcRVGPHy) showed rRVGP expression in cell lysates, attaining concentrations up to 1.5 ,g/107 cells (705 ,g/L). Of the transfected cells, 20% were shown to express the rRVGP. Cell subpopulations selected by limiting dilution expressed higher rRVGP yields and 90% of the cells were shown to express the rRVGP. Cell populations re-selected by addition of hygromycin were shown to express 10 times higher rRVGP yields. The data presented here show that Drosophila S2 cells can be efficiently transfected with an expression/selection plasmid for rRVGP expression, allowing its synthesis with a high degree of physical and biological integrity. The importance of subpopulation selection was indicated by the increasing rRVGP yields during these procedures. [source] | |||||||||||||