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Guo, Xin-Yu, Xiao-Dong Gao y Morihisa Fujita. "Sulfation of a FLAG tag mediated by SLC35B2 and TPST2 affects antibody recognition". PLOS ONE 16, n.º 5 (5 de mayo de 2021): e0250805. http://dx.doi.org/10.1371/journal.pone.0250805.
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A FLAG tag consisting of DYKDDDDK is an epitope tag that is frequently and widely used to detect recombinant proteins of interest. In this study, we performed a CRISPR-based genetic screening to identify factors involved in the detection of a FLAG-tagged misfolded model protein at the cell surface. In the screening, SLC35B2, which encodes 3’-phosphoadenosine-5’-phosphosulfate transporter 1, was identified as the candidate gene. The detection of FLAG-tagged misfolded proteins at the cell surface was significantly increased in SLC35B2-knockout cells. Furthermore, protein tyrosine sulfation mediated by tyrosyl-protein sulfotransferase 2 (TPST2) suppressed FLAG-tagged protein detection. Localization analysis of the FLAG-tagged misfolded proteins confirmed that defects in tyrosine sulfation are only responsible for enhancing anti-FLAG staining on the plasma membrane but not inducing the localization change of misfolded proteins on the plasma membrane. These results suggest that a FLAG tag on the misfolded protein would be sulfated, causing a reduced detection by the M2 anti-FLAG antibody. Attention should be required when quantifying the FLAG-tagged proteins in the secretory pathway.
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Zhao, Xinyu, Guoshun Li y Shufang Liang. "Several Affinity Tags Commonly Used in Chromatographic Purification". Journal of Analytical Methods in Chemistry 2013 (2013): 1–8. http://dx.doi.org/10.1155/2013/581093.
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Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags.
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Seago, Julian, Terry Jackson, Claudia Doel, Elizabeth Fry, David Stuart, Michiel M. Harmsen, Bryan Charleston y Nicholas Juleff. "Characterization of epitope-tagged foot-and-mouth disease virus". Journal of General Virology 93, n.º 11 (1 de noviembre de 2012): 2371–81. http://dx.doi.org/10.1099/vir.0.043521-0.
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Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals with an almost-worldwide distribution. Conventional FMD vaccines consisting of chemically inactivated viruses have aided in the eradication of FMD from Europe and remain the main tool for control in endemic countries. Although significant steps have been made to improve the quality of vaccines, such as improved methods of antigen concentration and purification, manufacturing processes are technically demanding and expensive. Consequently, there is large variation in the quality of vaccines distributed in FMD-endemic countries compared with those manufactured for emergency use in FMD-free countries. Here, we have used reverse genetics to introduce haemagglutinin (HA) and FLAG tags into the foot-and-mouth disease virus (FMDV) capsid. HA- and FLAG-tagged FMDVs were infectious, with a plaque morphology similar to the non-tagged parental infectious copy virus and the field virus. The tagged viruses utilized integrin-mediated cell entry and retained the tag epitopes over serial passages. In addition, infectious HA- and FLAG-tagged FMDVs were readily purified from small-scale cultures using commercial antibodies. Tagged FMDV offers a feasible alternative to the current methods of vaccine concentration and purification, a potential to develop FMD vaccine conjugates and a unique tool for FMDV research.
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Laird, Melissa E. y Ronald C. Desrosiers. "Infectivity and Neutralization of Simian Immunodeficiency Virus with FLAG Epitope Insertion in gp120 Variable Loops". Journal of Virology 81, n.º 20 (8 de agosto de 2007): 10838–48. http://dx.doi.org/10.1128/jvi.00831-07.
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ABSTRACT A FLAG epitope tag was substituted within variable loop 1 (V1), 2 (V2), or 4 (V4) of the gp120 envelope glycoprotein of simian immunodeficiency virus strain 239 (SIV239) to evaluate the extent to which each variable loop may serve as a target for antibody-mediated neutralization. Two sites within each variable loop of SIV239 were chosen for individual epitope tag insertions. FLAG epitope substitutions were also made in the V1, V2, and V4 loops of a neutralization-sensitive derivative of SIV239, SIV316. Of the 10 FLAG-tagged recombinant viruses analyzed, three (SIV239FV1b, SIV239FV2b, and SIV239FV4a) replicated with kinetics similar to those of the parental strain, SIV239, in both CEMx174 cells and the immortalized rhesus monkey T-cell line 221. The SIV316FV1b and SIV316FV4a FLAG variants replicated with a substantial lag, and the five remaining recombinants did not replicate detectably. Both gp160 and gp120 from replication-competent FLAG variants could be immunoprecipitated from transfected 293T cells by the anti-gp120 rhesus monoclonal antibody (RhMAb) 3.11H, the anti-FLAG MAb M2, and CD4-immunoglobulin, whereas only unprocessed gp160 was detected in 293T cells transfected with replication-defective variants. Furthermore, gp120 was detectably incorporated only into virions that were infectious. SIV239FV1b was sensitive to neutralization by MAb M2, with a 50% inhibitory concentration of 1 μg/ml. Neither SIV239FV2b nor SIV239FV4a was sensitive to M2 neutralization. The ability of the M2 antibody to neutralize SIV239FV1b infectivity was associated with an increased ability of the M2 antibody to detect native, oligomeric SIV239FV1b envelope protein on the surfaces of cells relative to that for the other SIV FLAG variants. Furthermore, SIV239FV1b was globally more sensitive to antibody-mediated neutralization than was parental SIV239 when these strains were screened with a panel of anti-SIV MAbs of various specificities. These results indicate that the V1 loop can serve as an effective target for neutralization on SIV239FV1b. However, antibody-mediated neutralization of this variant, similar to that of other SIV239 variants that have been studied previously, was associated with a global increase in neutralization sensitivity. These results suggest that the variable loops on the neutralization-resistant SIV239 strain are difficult for antibodies to access effectively and that mutations that allow neutralization have global effects on the trimeric envelope glycoprotein structure and accessibility.
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Plemper, Richard K., Anthea L. Hammond, Denis Gerlier, Adele K. Fielding y Roberto Cattaneo. "Strength of Envelope Protein Interaction Modulates Cytopathicity of Measles Virus". Journal of Virology 76, n.º 10 (15 de mayo de 2002): 5051–61. http://dx.doi.org/10.1128/jvi.76.10.5051-5061.2002.
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ABSTRACT To understand the molecular determinants of measles virus (MV) cytopathicity, we have characterized mutant viruses exhibiting a more-extensive cell-to-cell fusion while maintaining efficient replication to high titers. A virus which is modified by the addition of an 8-amino-acid Flag epitope tag at the cytoplasmic tail of its H (for MV hemagglutinin) envelope glycoprotein replicates efficiently, has an increased cytopathicity, possesses a greater infectivity per particle, and has an altered protein composition compared with that of unmodified MV. The mutant phenotype is not specifically linked to the epitope sequence, since an alternatively added HA (for influenza virus-derived hemagglutinin) epitope tag caused similar effects. We demonstrate that both epitope tags weaken the interaction between the H and fusion (F) glycoproteins in virus-infected cells. This reduction in strength of H/F interaction is independent of the presence of the viral matrix (M) protein. Viruses with this less stable complex are more sensitive to neutralization by a soluble octameric form of the CD46 receptor, consistent with their increased fusogenicity. Similar analyses of glycoproteins derived from MV strains with reduced cytopathicities confirm that the strength of H and F glycoprotein interaction is a modulator of viral fusogenicity.
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BLONG, M. Renee, Elliott BEDOWS y Oksana LOCKRIDGE. "Tetramerization domain of human butyrylcholinesterase is at the C-terminus". Biochemical Journal 327, n.º 3 (1 de noviembre de 1997): 747–57. http://dx.doi.org/10.1042/bj3270747.
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Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type Km and kcat values (8 μM and 24000 min-1 for butyrylthiocholine) and showed substrate activation. The Cys-571→Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.
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Joyce, Charles W., Gregory S. Shelness, Matthew A. Davis, Richard G. Lee, Kelly Skinner, Richard A. Anderson y Lawrence L. Rudel. "ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane". Molecular Biology of the Cell 11, n.º 11 (noviembre de 2000): 3675–87. http://dx.doi.org/10.1091/mbc.11.11.3675.
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A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser269) in the cytosol and the analogous residue in ACAT2 (Ser249) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.
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Mazurov, Dmitriy, Alexandra Maslennikova, Dmitriy Komkov y Anastasia Zotova. "Application of SORTS, a Novel Gene-Edited Cell Selection Method for HIV Study and Therapy". Proceedings 50, n.º 1 (4 de junio de 2020): 13. http://dx.doi.org/10.3390/proceedings2020050013.
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We have recently developed surface oligopeptide knock-in for rapid target selection (SORTS), a novel method to isolate mammalian cells with gene modifications using FACS-sorting. It relies on CRISPR/Cas9-mediated targeted knock-in of a very short promoterless expression construct (250 bp) comprising a Flag or HA epitope embedded into the smallest GPI-protein CD52, and a polyA signal from the beta-globin. CD52 efficiently delivers the epitope to the cell surface, where it serves as a marker for selection, while polyA terminates transcription and silences target gene expression. Primarily, SORTS was developed to knock out genes encoding intracellular or secreted proteins, which cannot be used as markers for selection of live cells. Using in-frame modification of SORTS, we demonstrated the possibility of HIV-1 provirus inactivation via sorting of GPI-tag positive cells. In order to make the “cured” cells resistant to a subsequent HIV-1 infection, the epitope tag in the CD52 molecule was substituted by one of the fusion inhibitor peptides from the CHR-domain of gp41. We selected a series of cell-surface-expressed, GPI-anchored, C34-based peptides that confer a strong cellular resistance to HIV-1 infection mediated by NL4-3, JRFL, or ZM153 Env. These findings together with a monoclonal antibody raised against the C34 peptide provide an opportunity to generate and select HIV-resistant lymphocytes for a therapeutic goal. SORTS was also adapted to engineer transgenic HIV-1 effector Т cells and to study cell-to-cell transmission. To facilitate transgenesis, we developed a knock-in strategy to express GPI-tag from the intronic region of the human PPP1R12C gene (AAVS1 locus) and delivered FRT sites of recombination into both alleles. In summary, SORTS is a novel instrument to isolate rare cells with precise genomic modifications with broad applications, including HIV biology. This work was supported by the Russian Science Foundation (grant 18-14-00333) and the Russian Foundation for Basic Research (grants 18-29-07052, 18-04-01016).
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Elvira, Bernat, Jamshed Warsi, Myriam Fezai, Carlos Munoz y Florian Lang. "SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels". Cellular Physiology and Biochemistry 37, n.º 5 (2015): 2032–42. http://dx.doi.org/10.1159/000438563.
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Background/Aims: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. Methods: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1). Results: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. Conclusions: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity.
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Kaltwasser, Marcus, Thomas Wiegert y Wolfgang Schumann. "Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis". Applied and Environmental Microbiology 68, n.º 5 (mayo de 2002): 2624–28. http://dx.doi.org/10.1128/aem.68.5.2624-2628.2002.
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ABSTRACT Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3′ terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein+, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.
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Wegelt, Anne, Ilona Reimann, Harald Granzow y Martin Beer. "Characterization and purification of recombinant bovine viral diarrhea virus particles with epitope-tagged envelope proteins". Journal of General Virology 92, n.º 6 (1 de junio de 2011): 1352–57. http://dx.doi.org/10.1099/vir.0.029330-0.
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Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. The lipid membrane of the virions is supposed to contain the three glycosylated envelope proteins Erns, E1 and E2, but detailed studies of virus assembly are complicated because no efficient purification method for pestiviruses has been described so far. In this study, we generated infectious BVDV with N-terminally FLAG-tagged Erns or E2 proteins, respectively. The expression of the epitope-tagged Erns and E2 proteins could be shown by immunofluorescence and Western blot experiments. Furthermore, an affinity tag purification protocol for the isolation and concentration of infectious BVDV was established. In the preparation with a titre of 108.75 TCID50 ml−1, spherical particles with a diameter of 43–58 nm (mean diameter: 48 nm) could be detected by negative staining electron microscopy, and immunogold labelling located both Erns and E2 proteins at the virus membrane.
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Jerlström-Hultqvist, Jon, Elin Einarsson y Staffan G. Svärd. "Stable Transfection of the Diplomonad Parasite Spironucleus salmonicida". Eukaryotic Cell 11, n.º 11 (14 de septiembre de 2012): 1353–61. http://dx.doi.org/10.1128/ec.00179-12.
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ABSTRACT Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis , an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida , a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3×HA [where HA is hemagglutinin], 2× Escherichia coli OmpF linker and mouse langerin fusion sequence [2×OLLAS], 3×MYC) and purification of proteins (2× Strep-Tag II–FLAG tandem-affinity purification tag or streptavidin binding peptide–glutathione S -transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase ( pac ), blasticidin S -deaminase ( bsr ), and neomycin phosphotransferase ( nptII ), were successfully applied for the generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an S. salmonicida double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both Giardia and Spironucleu s.
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Nita, Akihiro, Akinobu Matsumoto, Ronghao Tang, Chisa Shiraishi, Kazuya Ichihara, Daisuke Saito, Mikita Suyama et al. "A ubiquitin-like protein encoded by the “noncoding” RNA TINCR promotes keratinocyte proliferation and wound healing". PLOS Genetics 17, n.º 8 (5 de agosto de 2021): e1009686. http://dx.doi.org/10.1371/journal.pgen.1009686.
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Although long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins by definition, some lncRNAs actually contain small open reading frames that are translated. TINCR (terminal differentiation–induced ncRNA) has been recognized as a lncRNA that contributes to keratinocyte differentiation. However, we here show that TINCR encodes a ubiquitin-like protein that is well conserved among species and whose expression was confirmed by the generation of mice harboring a FLAG epitope tag sequence in the endogenous open reading frame as well as by targeted proteomics. Forced expression of this protein promoted cell cycle progression in normal human epidermal keratinocytes, and mice lacking this protein manifested a delay in skin wound healing associated with attenuated cell cycle progression in keratinocytes. We termed this protein TINCR-encoded ubiquitin-like protein (TUBL), and our results reveal a role for TINCR in the regulation of keratinocyte proliferation and skin regeneration that is dependent on TUBL.
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Jiang, Xiaofeng, Tracee S. Panetti y Michael E. Bromberg. "The Cytoplasmic Domain of Tissue Factor Inhibits Migration and Enhances Adhesion of Human Breast Cancer Cells." Blood 106, n.º 11 (16 de noviembre de 2005): 1939. http://dx.doi.org/10.1182/blood.v106.11.1939.1939.
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Abstract Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that when complexed with its cofactor, factor VIIa (FVIIa), initiates blood coagulation. Apart from hemostasis, TF has been shown to have roles in cellular signaling, development, inflammation, metastasis and angiogenesis. We showed previously that both the cytoplasmic and extracellular domains of TF are required for the full metastatic effect of TF. Recently, we showed that TF-FVIIa-FXa complex induces cellular signaling in human breast cancer cells and is associated with enhanced cell migration and prevention of apoptosis. However, the role of the cytoplasmic domain of TF in tumor cell function is not fully known. In the present study, the role of the cytoplasmic domain of TF in cell migration and adhesion was investigated using the Adr-MCF-7 cell line, a multidrug resistant subline of the human breast cancer cell line, MCF-7. The Adr-MCF-7 cell line has high endogenous expression of TF and expression of PAR1 and PAR2. Adr-MCF-7 cells were retrovirally transfected with either a cDNA construct encoding a FLAG epitope tag fused to the transmembrane and cytoplasmic domains of TF (known as FLAG-TFCD) or vector (LXSN) alone as a control, and stable, polyclonal cell lines selected using G418. Expression of the FLAG-TFCD construct was verified by RT-PCR, Western blot analysis and flow cytometry. To test the effect of overexpression of the FLAG-TFCD construct on cell motility a modified Boyden chamber chemotaxis assay was used. The control LXSN cell line had a nearly 9 fold increase in cell migration [33.5± 3.2 cells/hpf (mean± SEM)]using the combination of rFVIIa (10 nM) and FX (150 nM) as the chemoattractant compared with 0.1% bovine serum albumin (BSA) [3.9± 1.2 cells/hpf]. In contrast, the FLAG-TFCD cell line had no increase in migration of using the combination of rFVIIa and FX [6.6± 0.57 cells/hpf] compared with BSA [5.4± 0.67 cells/hpf]. We then examined the ability of the transfected cell lines to adhere to type IV collagen. The number of adherent cells for the transfected cell line, FLAG-TFCD, was nearly 3 fold higher than that for the LXSN line using a colorimetric MTS assay (0.295± 0.041 vs 0.119± 0.011). Moreover, treatment of the FLAG-TFCD cells with the combination of rFVIIa and FX increased the adhesion by nearly 2 fold compared with untreated FLAG-TFCD cells (0.561±0.055 vs 0.296±0.044). In summary, overexpression of TF cytoplasmic domain leads to inhibition of tumor cell migration and enhancement of cell adhesion and potentially acts as a dominant negative in these cellular processes. These data suggest that a function of the cytoplasmic domain of TF in metastasis is to regulate tumor cell migration and adhesion.
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Walker, Michelle Portlance y W. Ian Lipkin. "Characterization of the Nuclear Localization Signal of the Borna Disease Virus Polymerase". Journal of Virology 76, n.º 16 (15 de agosto de 2002): 8460–67. http://dx.doi.org/10.1128/jvi.76.16.8460-8467.2002.
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ABSTRACT Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. BDV proteins involved in replication and transcription must pass through the nuclear envelope to associate with the genomic viral RNA. The RNA-dependent RNA polymerase (L) of BDV is postulated to be the catalytic enzyme of replication and transcription. We demonstrated previously that BDV L localizes to the nucleus of BDV-infected cells and L-transfected cells. Nuclear localization of the protein presupposes the presence of a nuclear localization signal (NLS) within its primary amino acid sequence or cotransport to the nucleus with another karyophilic protein. Because L localized to the nucleus in the absence of other viral proteins, we investigated the possibility that L contains an NLS. The minimal sequence required for nuclear localization of L was identified by analyzing the subcellular distribution of deletion mutants of L fused to a flag epitope tag or β-galactosidase. Although the majority of the L fusion proteins localized to the cytoplasm of transfected BSR-T7 cells, a strong NLS (844RVVKLRIAP852) with basic and proline residues was identified. Mutation of this sequence resulted in cytoplasmic distribution of L, confirming that this sequence was necessary and sufficient to drive the nuclear localization of L.
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Maslennikova, Aleksandra, Dmitriy Komkov, Anastasia Zotova y Dmitriy Mazurov. "Cell Surface-Expressed GPI-Anchored Peptides from the CHR Domain of gp41 Are Potent Inhibitors of HIV-1 Fusion". Proceedings 50, n.º 1 (16 de junio de 2020): 70. http://dx.doi.org/10.3390/proceedings2020050070.
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Current antiretroviral therapy efficiently suppresses viral replication but cannot eliminate latent HIV reservoirs. Moreover, the associated high costs, side effects, and drug resistance have stimulated a need for the development of alternative methods of HIV-1/AIDS treatment, such as peptide inhibitors or gene editing. Recently, we have developed Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a method for the rapid selection of CRISPR/Cas9 gene-edited cells via knock-in of the Flag and HA epitope tags embedded into the shortest GPI-protein, CD52. By targeting the capsid region of the HIV-1 genome, we demonstrate that SORTS can be applied in provirus eradication. However, the cells with inactivated provirus will be susceptible to HIV re-infection. We hypothesized that knocking in one of the peptides from the CHR-domain of gp41, which are known potent inhibitors of HIV-1 fusion, instead of the epitope tag, will provide “post-curable” HIV-1 resistance. While these peptides were extensively studied as soluble substances, their inhibitory effects on HIV after expression on cell surfaces via GPI-anchor are largely unknown. In this study, we established HEK293T/CD4/R5 and Raji/CD4/R5 HIV-1 permissive cell lines that stably expressed one of the gp41 peptides C34, MT-C34, MT-C34-R, and MT34-15D, or alfa-helix mimetics HP23L, p52, and MT-WQ-IDL. For cell surface delivery, the indicated peptides were embedded into the CD52 molecule, and upstream GFP was used to select transformed cells. Using a single-cycle replication assay with the inLuc reporter vector and different Envs, we demonstrated that C34-based GPI-anchored peptides inhibited both cell-free and cell-to-cell HIV-1 infection by at least two orders of magnitude. With the exception of HP23L, the alfa-helix mimetics were less potent inhibitors. Thus, peptides from gp41 associated with lipid rafts and exerted a strong inhibitory activity which can far exceed that determined for soluble peptides, but this should be tested further.
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Cristea, Ileana M., Heather Rozjabek, Kelly R. Molloy, Sophiya Karki, Laura L. White, Charles M. Rice, Michael P. Rout, Brian T. Chait y Margaret R. MacDonald. "Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication". Journal of Virology 84, n.º 13 (14 de abril de 2010): 6720–32. http://dx.doi.org/10.1128/jvi.01983-09.
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ABSTRACT Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3×Flag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
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Peters, Kathryn W., Juanjuan Qi, Simon C. Watkins y Raymond A. Frizzell. "Syntaxin 1A inhibits regulated CFTR trafficking inXenopus oocytes". American Journal of Physiology-Cell Physiology 277, n.º 1 (1 de julio de 1999): C174—C180. http://dx.doi.org/10.1152/ajpcell.1999.277.1.c174.
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The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302–305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopusoocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxin’s effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.
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Bewarder, Moritz, Lorenz Thurner, Frank Neumann, Natalie Fadle, Evi Regitz, Maria Kemele, Helene Will et al. "BAR-Bodies: B-Cell Receptor Antigens As the Targeting Moiety of Antibodies in Substitution for the Variable Region of Heavy and Light Chains". Blood 132, Supplement 1 (29 de noviembre de 2018): 2940. http://dx.doi.org/10.1182/blood-2018-99-115348.
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Abstract Background Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type and 45% of mantle cell lymphomas (MCLs), respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). The optimal therapeutic format BARs can be integrated in has yet to be found. Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed and tested an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. Material and methods To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector for fusion with the FLAG tag and transfected into HEK293 cells for production. Purification of the BAR-body was performed via anti-FLAG antibody affinity chromatography. The BAR-body was detected by western blot analysis and binding capacity to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and the not ARS2-reactive control DLBCL cell line TMD8 was assessed by flow cytometry. ARS2 BAR-body induced cytotoxicity of lymphoma cells with an ARS2 reactive BCR was measured by LDH release assays with human PBMCs as effector cells at an E:T ratio of 10:1. Results We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis, which showed the construct at approximately 150 kD as was to be expected. The BAR-body bound specifically to the ARS2-reactive lymphoma cell lines U2932 and OCI-Ly3 and did not bind to the DLBCL cell line TMD8, which has a B-cell receptor of different specificity or to lymphoma cell lines of different entities. In LDH release assays with 5 x 104 PBMCs and 5 x 103 lymphoma cells (E:T ratio of 10:1) the ARS2 BAR-body induced PBMC mediated specific lysis of the ARS2 reactive lymphoma cell lines U2932 and OCI-Ly3 but not the control DLBCL cell line TMD8 starting at a concentration of 0,1µg/ml. Cytotoxic effects were dose dependent, reached a maximum of 50% specific lysis at a concentration of 1µg/ml and did not increase at concentrations of 10µg/ml. Conclusion Here, we show that BARs can substitute for the variable domains as binding moiety in antibody like constructs to target the BCR of B-cell lymphomas. Because approaches using their specific cognate antigen for targeting the malignant B cells have an exclusive specificity for the BCR of the malignant clone, they can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells, because they leave normal B-lymphocytes unaffected. By incorporating BARs into the well-known format of an antibody we hope to capitalize on years of experience with this therapeutic format from conducting and interpreting in vivo experiments to the translation of the BAR approach into the clinic. Disclosures Stilgenbauer: Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Bewarder, Moritz, Christina Körbel, Matthias W. Laschke, Michael D. Menger, Maximilian Kiefer, Dominic Kaddu-Mulindwa, Helene Will et al. "The B-Cell Receptor Antigen ARS2 Can be Integrated into a BAR-Body Format to Treat Diffuse Large B-Cell Lymphomas in Xenograft Mouse Models". Blood 134, Supplement_1 (13 de noviembre de 2019): 2860. http://dx.doi.org/10.1182/blood-2019-131434.
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Chronic antigenic stimulation of the B-cell receptor (BCR) seems to play a critical role in the pathogenesis of B-cell lymphomas. We recently identified ARS2, LRPAP1 and Neurabin-I as the autoantigenic targets of the B-cell receptors of approximately 25% of diffuse large B cell lymphomas (DLBCLs) of the ABC type, 45% of mantle cell lymphomas (MCLs) and 66% of primary CNS lymphomas, respectively. These BCR antigens can be used to target lymphoma cells in an approach we designated as BAR (B-cell receptor antigens for reverse targeting). Since the most established approach to deliver therapeutic payloads to specific targets are antibodies which have well-defined pharmacokinetics, we constructed an antibody like construct (BAR-body) incorporating the DLBCL-BAR ARS2 in substitution for the variable domains of the heavy and light chains. This ARS2 containing BAR-body showed promising efficacy in in-vitro experiments. Here, we report the results of the initial in-vivo experiments using lymphoma xenograft mouse models. To create the ARS2 BAR-body, we exchanged the heavy and light chain variable region sequences of an IgG1 antibody with a sequence of similar length (approximately 120 amino acids) of the ARS2 protein (aa 343 - 466) containing the DLBCL reactive epitope (aa 343 - 375). The construct was assembled in a pCR2.1 vector, then transferred to a pSfi FLAG Tag vector and transfected into HEK293 cells for production. Flow cytometry was used for binding experiments of the ARS2 BAR-body to lymphoma cell lines. For mouse experiments we inoculated 1x107 cells of the human DLBCL cell line U2932 (expresses a BCR with reactivity for ARS2) subcutaneously into the left flank of 12 NOD SCID mice. Tumor volume was calculated from day 14 measuring the long and short diameters of the tumor mass in millimeters. 6 mice were treated with 60 mg/kg ARS2 BAR-body intraperitoneally on days 23, 25, 30, 32, 37, 39, 44 and 46 after tumor inoculation. 6 control mice were mock-treated with PBS following the same time schedule. Tumor growth was evaluated by calculating the change in tumor volume from the first measurement at day 14. We cloned, expressed and characterized an ARS2 containing BAR-body incorporating 4 molecules of the lymphoma-reactive epitope of ARS2 resulting in an antibody like construct using a BAR (ARS2) as binding moiety instead of normal variable regions. The ARS2 BAR-body could successfully be cloned and expressed as confirmed by western blot analysis. Flow cytometric binding assays confirmed specific binding to the DLBCL cell line U2932 which expresses a BCR receptor with reactivity for ARS2 while control cell lines could not be stained by the ARS2 BAR-body. U2932 cells could successfully be transplanted subcutaneously into the flank of NOD SCID mice to generate a xenograft mouse model. Tumors in the treatment group increased their mean volume 14.1 times while tumors in the control group grew by a factor of 18.3 as compared to the initial mean tumor volume analyzed at day 14. Approaches using the cognate antigen of B-cell receptors to target malignant B cells have an exclusive specificity for the BCR of the malignant clone and can be expected to be less toxic than the currently available antibody derived therapies targeting B-cells. The ARS2 BAR-body shows promising activity against ARS2 reactive B-cell lymphoma cells in in-vivo mouse experiments. Toxic effects using the ARS2 BAR-body for the first time in-vivo were not observed. Further studies are necessary to reproduce and optimize the current experiments to form the basis for the translational development of BAR-bodies. Disclosures Stilgenbauer: AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.
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Stalder, Elizabeth S., Lauren H. Nagy, Pilar Batalla, Terrance M. Arthur, Nancy E. Thompson y Richard R. Burgess. "The epitope for the polyol-responsive monoclonal antibody 8RB13 is in the flap-domain of the beta-subunit of bacterial RNA polymerase and can be used as an epitope tag for immunoaffinity chromatography". Protein Expression and Purification 77, n.º 1 (mayo de 2011): 26–33. http://dx.doi.org/10.1016/j.pep.2010.12.011.
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Dooher, Julia E., Ido Paz-Priel, Simone Houng, Albert S. Baldwin y Alan D. Friedman. "C/EBPα, C/EBPα Oncoproteins, or C/EBPβ Preferentially Bind NF-κB p50 Compared with p65 Via Conserved Residues In the C/EBP Basic Region". Blood 116, n.º 21 (19 de noviembre de 2010): 708. http://dx.doi.org/10.1182/blood.v116.21.708.708.
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Abstract Abstract 708 Nuclear Factor-κB (NF-κB) is a key mediator of the inflammatory response and also inhibits apoptosis. Activation of NF-κB is prevalent in AML, Hodgkin's lymphoma and subsets of non-Hodgkin's lymphoma, as well as in multiple solid tumors. NF-κB p50 (NF-κB1) and p65 (Rel A) are the most prevalent NF-κB subunits. NF-κB p65 has 30–50 fold higher affinity than p50 for the cytoplasmic protein IκB, and so under basal conditions p50:p65 heterodimers are held in the cytoplasm whereas p50:p50 homodimers are found in the nucleus bound to chromatin where they repress NF-κB target genes via association with HDACs. Canonical NF-κB activation signals lead to IκB proteosome-mediated degradation and translocation of p50:p65 heterodimers into the nucleus to replace inhibitory p50:p50 dimers with activating complexes on chromatin. We postulate an alternative means to activate NF-κB target genes wherein induction or activation of C/EBP proteins allows them to bind chromatin-bound p50 and displace HDACs in cells lacking canonical NF-κB activation. We found that C/EBPα induces bcl-2 and FLIP RNA expression via promoter interaction dependent upon the presence of NF-κB p50, that C/EBPα present in two myeloid leukemia cell lines preferentially binds p50 compared with p65, and that mutation of the C/EBPα basic region prevents bcl-2 induction and weakens interaction with p50 but not p65 (Paz-Priel et al 2005; 2009; Wang et al 2009). Using p50 and p65 variants containing the FLAG epitope at either their N- or C-termini we now demonstrate using reciprocal co-immunoprecipitation that p50 has much higher affinity than p65 for C/EBPα, for N-terminal or leucine zipper mutants of C/EBPα expressed in a subset of AML cases, and for the longer LAP or shorter LIP isoforms of C/EBPβ. The FLAG-tagged p65 proteins retained the ability to bind DNA in a gel shift assay and to activate a reporter containing two κB sites as effectively as untagged p65. Deletion of the p65 trans-activation domain (TAD) did not increase p65 affinity for C/EBPα, indicating that an inhibitory effect of the TAD does not account for specificity of C/EBPα:p50 interaction. Moreover, the increased affinity of the p50 rel homolog domain (RHD) for C/EBPα compared with the p65 RHD suggests that residues that differ between these RHDs rather than those in common make key contacts wih the C/EBP basic region. Germline deletion of the gene encoding p65, by injecting p65(flox/flox); Mx1-CRE mice with pIpC, did not reduce C/EBPα expression, prevent interaction of C/EBPα with the bcl-2 promoter in a ChIP assay, or reduce bcl-2 RNA expression, in contrast to our prior findings with p50 gene deletion. A C/EBPα variant harboring a single point mutation in the basic region, C/EBPα(R300A), has minimal affinity for p50, and further saturating mutagenesis of the C/EBPα basic region identifies an additional block of six amino acids, RERNNI, identical in C/EBPβ and critical for interaction with NF-κB p50. In conclusion, increased affinity of C/EBP proteins for p50 compared with p65 lends support to the hypothesis that C/EBP interaction with p50:p50 complexes provides an alternative means for normal cells or cancer cells to activate NF-κB target genes, for example in response to C/EBP induction or activation. C/EBP interaction with p50:p65 in activated cells may also occur, via p50, to further activate genes regulated by NF-κB. In addition, these findings suggest that targeting the C/EBP:p50 interface rather than C/EBP:p65 interaction will prove more effective in the therapy of inflammatory or malignant conditions, and our mapping of relevant basic region residues and the finding that the p50 RHD retains increased affinity for C/EBPα compared wih the p65 RHD provides a first step towards rational design of such inhibitors. Disclosures: No relevant conflicts of interest to declare.
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Götzke, Hansjörg, Markus Kilisch, Markel Martínez-Carranza, Shama Sograte-Idrissi, Abirami Rajavel, Thomas Schlichthaerle, Niklas Engels et al. "The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications". Nature Communications 10, n.º 1 (27 de septiembre de 2019). http://dx.doi.org/10.1038/s41467-019-12301-7.
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Abstract Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG®- or myc-tag. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.
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Imagawa, Toshifumi, Masahiko Ito, Mami Matsuda, Kenji Nakashima, Yuhei Tokunaga, Isao Ohta, Tian-Cheng Li, Ryosuke Suzuki y Tetsuro Suzuki. "Virus-like particles with FLAG-tagged envelope protein as a tetravalent dengue vaccine candidate". Scientific Reports 11, n.º 1 (2 de septiembre de 2021). http://dx.doi.org/10.1038/s41598-021-97038-4.
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AbstractThe global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future.
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Yang, Hui, Yu Wang y Chengjin Ye. "Rapid Generation of Attenuated Infectious Bursal Disease Virus from Dual-Promoter Plasmids by Reduction of Viral Ribonucleoprotein Activity". Journal of Virology 94, n.º 7 (8 de enero de 2020). http://dx.doi.org/10.1128/jvi.01569-19.
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ABSTRACT Infectious bursal disease virus (IBDV) of the Birnaviridae family leads to immunosuppression of young chickens by destroying B cells in the bursa of Fabricius (BFs). Given the increasing number of variant IBDV strains, we urgently require a method to produce attenuated virus for vaccine development. To accomplish this goal, the dual-promoter plasmids in which the RNA polymerase II and RNA polymerase I (Pol I) promoters were placed upstream of the IBDV genomic sequence, which was followed by mouse Pol I terminator and a synthetic polyadenylation signal, were developed for rapid generation of IBDV. This approach did not require trans-supplementation of plasmids for the expression of VP1 and VP3, the main components of IBDV ribonucleoprotein (RNP). Based on the finding in this study that the IBDV RNP activity was partially retained by VP1-FLAG, we successfully rescued the replication-competent IBDV/1FLAG expressing VP1-FLAG. Compared with its parental counterpart, IBDV/1FLAG formed smaller size plaques in cultured cells and induced the same 100% immune protection in vivo. However, neither retarded development nor severe BFs lesion was observed in the IBDV/1FLAG-inoculated chickens. Collectively, this is the first report that viral RNP activity was affected by the addition of an epitope tag on the componential viral proteins. Furthermore, this work demonstrates the rapid generation of attenuated IBDV from dual-promoter plasmids via reducing viral RNP activity by a fused FLAG tag on the C terminus of VP1. This would be a convenient strategy to attenuate epidemic variant IBDV strains for rapid and efficient vaccine development. IMPORTANCE Immunosuppression in chickens as a result of infectious bursal disease virus (IBDV) infection leads to significant economic losses in the poultry industry worldwide every year. Currently, vaccination is still the best way to prevent the prevalence of IBDV. However, with the occurrence of increasing numbers of variant IBDV strains, it is challenging to develop antigen-matched live attenuated vaccine. Here, we first developed a dual-promoter reverse-genetic system for the rapid generation of IBDV. Using this system, the attenuated IBDV/1FLAG expressing VP1-FLAG, which displays the decreased viral RNP activity, was rescued. Moreover, IBDV/1FLAG inoculation induced a similar level of neutralizing antibodies to that of its parental counterpart, protecting chickens against lethal challenge. Our study, for the first time, describes a dual-promoter reverse-genetic approach for the rapid generation of attenuated IBDV while maintaining entire parental antigenicity, suggesting a potential new method to attenuate epidemic variant IBDV strains for vaccine development.
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Carakushansky, M., AJ Whatmore, PE Clayton, SM Shalet, HK Gleeson, DA Price, MA Levine y R. Salvatori. "A new missense mutation in the growth hormone-releasing hormone receptor gene in familial isolated GH deficiency". European Journal of Endocrinology, 1 de enero de 2003, 25–30. http://dx.doi.org/10.1530/eje.0.1480025.
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OBJECTIVE: Mutations in the GH-releasing hormone (GHRH) receptor (GHRHR) gene (GHRHR) cause autosomal recessive familial isolated GH deficiency (IGHD). We searched for GHRHR mutations in two siblings with IGHD type IB and a history of parental consanguinity. DESIGN: We analyzed peripheral genomic DNA of an index patient. After identifying a novel mutation in the GHRHR, we performed functional studies in order to confirm that the mutation causes receptor malfunction. METHODS: The entire GHRHR was analyzed in the index case by denaturing gradient gel electrophoresis. Abnormally migrating bands were isolated and sequenced. The mutated area was then sequenced in all family members whose DNA was available. The newly found mutation was inserted into a GHRHR cDNA. Wild-type and mutant cDNAs were expressed into CHO cells and the cyclic AMP (cAMP) response to GHRH was measured. In order to determine whether the mutant receptor was properly expressed on the cell membrane surface, CHO cells were transfected with wild-type or mutant GHRHR cDNA containing a FLAG epitope tag in the extracellular N-terminus. RESULTS: Both patients were homozygous for a new missense mutation in codon 176, corresponding to the second transmembrane domain of the receptor protein that replaces alanine with valine (A176V). The mother and three unaffected siblings were heterozygous for the mutation; DNA from the father was not available. Cells expressing the A176V receptor had a significantly reduced cAMP response to GHRH, despite appropriate expression on the cell surface. CONCLUSIONS: We describe two siblings with IGHD due to a new mutation in the GHRHR that disrupts GHRH signaling and leads to GHRH resistance.