In contrast to conventional inactivated influenza vaccines, 4xM2e.HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran

International Nuclear Information System (INIS)

Ebrahimi, Seyyed Mahmoud; Dabaghian, Mehran; Tebianian, Majid; Zabeh Jazi, Mohammad Hossein

2012-01-01

Ideal vaccines against influenza viruses should elicit not only a humoral response, but also a cellular response. Mycobacterium tuberculosis HSP70 (mHSP70) have been found to promote immunogenic APCs function, elicit a strong cytotoxic T lymphocyte (CTL) response, and prevent the induction of tolerance. Moreover, it showed linkage of antigens to the C-terminus of mHSP70 (mHSP70c) can represent them as vaccines resulted in more potent, protective antigen specific responses in the absence of adjuvants or complex formulations. Hence, recombinant fusion protein comprising C-terminus of mHSP70 genetically fused to four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli, purified under denaturing condition, refolding, and then confirmed by SDS–PAGE, respectively. The recombinant fusion protein, 4xM2e.HSP70c, retained its immunogenicity and displayed the protective epitope of M2e by ELISA and FITC assays. A prime-boost administration of 4xM2e.HSP70c formulated in F105 buffer by intramuscular route in mice (Balb/C) provided full protection against lethal dose of mouse-adapted H1N1, H3N2, or H9N2 influenza A isolates from Iran compared to 0–33.34% survival rate of challenged unimmunized and immunized mice with the currently in use conventional vaccines designated as control groups. However, protection induced by immunization with 4xM2e.HSP70c failed to prevent weight loss in challenged mice; they experienced significantly lower weight loss, clinical symptoms and higher lung viral clearance in comparison with protective effects of conventional influenza vaccines in challenged mice. These data demonstrate that C-terminal domain of mHSP70 can be a superior candidate to deliver the adjuvant function in M2e-based influenza A vaccine in order to provide significant protection against multiple influenza A virus strains.

Flunarizine Prevents Hepatitis C Virus Membrane Fusion in a Genotype-dependent Manner by Targeting the Potential Fusion Peptide within E1

Science.gov (United States)

Perin, Paula M.; Haid, Sibylle; Brown, Richard J. P.; Doerrbecker, Juliane; Schulze, Kai; Zeilinger, Carsten; von Schaewen, Markus; Heller, Brigitte; Vercauteren, Koen; Luxenburger, Eva; Baktash, Yasmine M.; Vondran, Florian W. R.; Speerstra, Sietkse; Awadh, Abdullah; Mukhtarov, Furkat; Schang, Luis M; Kirschning, Andreas; Müller, Rolf; Guzman, Carlos A.; Kaderali, Lars; Randall, Glenn; Meuleman, Philip; Ploss, Alexander; Pietschmann, Thomas

2015-01-01

To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies. PMID:26248546

Fluorescent S-layer fusion proteins

International Nuclear Information System (INIS)

Kainz, B.

2010-01-01

This work describes the construction and characterisation of fluorescent S-layer fusion proteins used as building blocks for the fabrication of nanostructured monomolecular biocoatings on silica particles with defined fluorescence properties. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the pH-dependant cyan, green and yellow variant of the green fluorescent protein (GFP) and the red fluorescent protein mRFP1. These fluorescent S-layer fusion proteins, acting as scaffold and optical sensing element simultaneously, were able to reassemble in solution and on silica particles forming 2D nanostructures with p2 lattice symmetry (a=11 ±0.5 nm, b=14 ±0.4 nm, g=80 ±1 o ). The pH-dependant fluorescence behaviour was studied with fluorimetry, confocal microscopy and flow cytometry. These fluorescent S-layer fusion proteins can be used as pH-sensor. 50% of the fluorescence intensity decreases at their calculated pKa values (pH6 - pH5). The fluorescence intensity of the GFP variants vanished completely between pH4 and pH3 whereas the chromophore of the red protein mRFP1 was only slightly affected in acidic conditions. At the isoelectric point of the S-layer coated silica particles (pH4.6 ±0.2) an increase in particle aggregation was detected by flow cytometry. The cyan and yellow fluorescent proteins were chosen to create a bi-fluorescent S-layer tandem fusion protein with the possibility for resonance energy transfer (FRET). A transfer efficiency of 20% and a molecular distance between the donor (ECFP) and acceptor (YFP) chromophores of around 6.2 nm could be shown. This bi-fluorescent ECFP-SgsE-YFP tandem fusion protein was able to reassemble on solid surfaces. The remarkable combination of fluorescence and self-assembly and the design of bi-functional S-layer tandem fusion protein matrices makes them to a promising tool in nanobiotechnology. (author) [de

Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli

Energy Technology Data Exchange (ETDEWEB)

Pasek, Marta [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Boeggeman, Elizabeth; Ramakrishnan, Boopathy [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States); Qasba, Pradman K., E-mail: qasba@helix.nih.gov [Structural Glycobiology Section, SAIC-Frederick, Inc., Center for Cancer Research Nanobiology Program, Center for Cancer Research, NCI-Frederick, Frederick, MD 2170 (United States)

2010-04-09

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.

Identification and quantification of N alpha-acetylated Y. pestis fusion protein F1-V expressed in Escherichia coli using LCMS E.

Science.gov (United States)

Bariola, Pauline A; Russell, Brett A; Monahan, Steven J; Stroop, Steven D

2007-05-31

N-terminal acetylation in E coli is a rare event catalyzed by three known N-acetyl-transferases (NATs), each having a specific ribosomal protein substrate. Multiple, gram-scale lots of recombinant F1-V, a fusion protein constructed from Y. Pestis antigens, were expressed and purified from a single stably transformed E. coli cell bank. A variant form of F1-V with mass increased by 42-43 Da was detected in all purified lots by electrospray orthogonal acceleration time-of-flight mass spectrometry (MS). Peptide mapping LCMS localized the increased mass to an N-terminal Lys-C peptide, residues 1-24, and defined it as +42.0308+/-0.0231 Da using a LockSpray exact mass feature and a leucine enkaphalin mass standard. Sequencing of the variant 1-24 peptide by LCMS and high-energy collision induced dissociation (LCMS(E)) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as N(alpha)-acetylation. The average content of N(alpha)-acetylated F1-V in five lots was 24.7+/-2.6% indicating that a stable acetylation activity for F1-V was established in the E. coli expression system. Alignment of the F1-V N-terminal sequence with those of other known N(alpha)-acetylated ectopic proteins expressed in E. coli reveals a substrate motif analogous to the eukaryote NatA' acetylation pathway and distinct from endogenous E. coli NAT substrates.

Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

Science.gov (United States)

Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

2002-01-01

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

Directory of Open Access Journals (Sweden)

Lijun Chao

Full Text Available The HIV-1 envelope glycoprotein (Env gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521 of the human POB1 (the partner of RalBP1, designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR of gp41, and to the six-helix bundle (6-HB formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

[Construction and prokaryotic expression of recombinant gene EGFRvIII HBcAg and immunogenicity analysis of the fusion protein].

Science.gov (United States)

Duan, Xiao-yi; Wang, Jian-sheng; Guo, You-min; Han, Jun-li; Wang, Quan-ying; Yang, Guang-xiao

2007-01-01

To construct recombinant prokaryotic expression plasmid pET28a(+)/c-PEP-3-c and evaluate the immunogenicity of the fusion protein. cDNA fragment encoding PEP-3 was obtained from pGEM-T Easy/PEP-3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni(2+)-NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA. The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1:16 000 after the fourth immunization and reached 1:2.56x10(5) after the sixth immunization. The titre of anti-PEP-3 antibody reached 1:1.28x10(5) and the titre of anti-HBcAg antibody was less than 1:4x10(3). Fusion gene PEP-3-HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII.

An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

International Nuclear Information System (INIS)

Yin, Guangwen; Qin, Mei; Liu, Xianyong; Suo, Jingxia; Tang, Xinming; Tao, Geru; Han, Qian; Suo, Xun; Wu, Wenxue

2013-01-01

Highlights: •We found a new protective protein – (IMPI) in Eimeria tenella. •EtIMP1-flagellin fusion protein is an effective immunogen against Eimeria infection. •Flagellin can be as an apicomplexan parasite vaccine adjuvant in chickens. -- Abstract: Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens

An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

Energy Technology Data Exchange (ETDEWEB)

Yin, Guangwen; Qin, Mei [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Liu, Xianyong [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Suo, Jingxia; Tang, Xinming; Tao, Geru [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Han, Qian [Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061 (United States); Suo, Xun [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Wu, Wenxue, E-mail: labboard@126.com [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China)

2013-10-25

Highlights: •We found a new protective protein – (IMPI) in Eimeria tenella. •EtIMP1-flagellin fusion protein is an effective immunogen against Eimeria infection. •Flagellin can be as an apicomplexan parasite vaccine adjuvant in chickens. -- Abstract: Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens.

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

Science.gov (United States)

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2015-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

Characterization of HCoV-229E fusion core: Implications for structure basis of coronavirus membrane fusion

International Nuclear Information System (INIS)

Liu Cheng; Feng Youjun; Gao Feng; Zhang Qiangmin; Wang Ming

2006-01-01

Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection

Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

International Nuclear Information System (INIS)

Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

2012-01-01

The crystallization and initial diffraction analysis of human Drp1 GTPase-GED fusion protein are reported. The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity.

Science.gov (United States)

Humenik, Martin; Mohrand, Madeleine; Scheibel, Thomas

2018-04-18

The recombinant spider silk protein eADF4(C16) was genetically fused either with esterase 2 (EST2) or green fluorescent protein (GFP). The fusions EST-eADF4(C16) and GFP-eADF4(C16) were spectroscopically investigated and showed native structures of EST and GFP. The structural integrity was confirmed by the enzymatic activity of EST and the fluorescence of GFP. The spider silk moiety retained its intrinsically unstructured conformation in solution and the self-assembly into either nanofibrils or nanoparticles could be controlled by the concentration of phosphate. Particles, however, showed significantly lower activity of the EST and GFP domains likely caused by a steric hindrance. However, upon self-assembly of EST-eADF4(C16) and GFP-eADF4(C16) into fibrils the protein activities were retained. In general, the fusion of globular enzymes with the spider silk domain allows the generation of fibrous biomaterials with catalytic or light emitting properties.

Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

Science.gov (United States)

Schlatter, Stefan; Senn, Claudia; Fussenegger, Martin

2003-07-20

Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210-225, 2003.

The B isozyme creatine kinase is active as a fusion protein in Escherichia coli

International Nuclear Information System (INIS)

Koretsky, A.P.; Traxler, B.A.

1989-01-01

A cDNA encoding the B isozyme of creatine kinase CK B has been expressed in Escherichia coli from a fusion with lacZ carried by λgtll. Western blots indicate that a stable polypeptide with the appropriate mobility for the Β-galactosidase-creatine kinase Β-gal-CK B ) fusion protein cross-reacts with both Β-gal and CK B antiserum. No significant CK activity is detected in control E. coli; however, extracts from cells containing the λgtll-CK B construct have a CK activity of 1.54j0.07 μmol/min per mg protein. The fusion protein appears to provide this activity bacause immunoprecipitation of protein with Β-gal antiserum leads to a loss of CK activity from extracts. That the enzyme is active in vivo was demonstrated by detection of a phosphocreatine (PCr) peak in the 31 P NMR spectrum from E. coli grown on medium supplemented with creatine. As in mammalian brain and muscle, the PCr peak detected was sensitive to the energy status of the E. coli. (author). 17 refs.; 3 figs.; 1 tab

Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

Directory of Open Access Journals (Sweden)

Somayeh Kadkhodayan

2016-07-01

Full Text Available Abstract Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa could act as a cell penetrating peptide (CPP. In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method. Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3 strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method. Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it. Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.

Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

Energy Technology Data Exchange (ETDEWEB)

Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun

2014-08-26

Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.

Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

Science.gov (United States)

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-12-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

Rational design of highly potent HIV-1 fusion inhibitory proteins: Implication for developing antiviral therapeutics

International Nuclear Information System (INIS)

Ni Ling; Gao, George F.; Tien Po

2005-01-01

Recombinant protein containing one heptad-repeat 1 (HR1) segment and one HR2 segment of the HIV-1 gp41 (HR1-HR2) has been shown to fold into thermally stable six-helix bundle, representing the fusogenic core of gp41. In this study, we have used the fusogenic core as a scaffold to design HIV-1 fusion inhibitory proteins by linking another HR1 to the C terminus of HR1-HR2 (HR121) or additional HR2 to the N terminus of HR1-HR2 (HR212). Both recombinant proteins could be abundantly and solubly expressed and easily purified, exhibiting high stability and potent inhibitory activity on HIV-1 fusion with IC 50 values of 16.2 ± 2.8 and 2.8 ± 0.63 nM, respectively. These suggest that these rationally designed proteins can be further developed as novel anti-HIV-1 therapeutics

Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion

International Nuclear Information System (INIS)

Butrapet, Siritorn; Childers, Thomas; Moss, Kelley J.; Erb, Steven M.; Luy, Betty E.; Calvert, Amanda E.; Blair, Carol D.; Roehrig, John T.; Huang, Claire Y.-H.

2011-01-01

Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, but only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.

Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology.

Science.gov (United States)

Shafiee, Fatemeh; Rabbani, Mohammad; Jahanian-Najafabadi, Ali

2017-01-01

The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli ( E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3), followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM). Finally, the best culture medium was selected. Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml). Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology

Directory of Open Access Journals (Sweden)

Fatemeh Shafiee

2017-01-01

Full Text Available Background: The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Materials and Methods: Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli (E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3, followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM. Finally, the best culture medium was selected. Results: Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml. Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. Conclusion: In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

Energy Technology Data Exchange (ETDEWEB)

Shi, Jian [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Zhang, Huaidong [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Gong, Rui, E-mail: gongr@wh.iov.cn [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China)

2015-05-08

Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL

International Nuclear Information System (INIS)

Douette, Pierre; Navet, Rachel; Gerkens, Pascal; Galleni, Moreno; Levy, Daniel; Sluse, Francis E.

2005-01-01

Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein

Interactions involved in pH protection of the alphavirus fusion protein

Energy Technology Data Exchange (ETDEWEB)

Fields, Whitney; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

2015-12-15

The alphavirus membrane protein E1 mediates low pH-triggered fusion of the viral and endosome membranes during virus entry. During virus biogenesis E1 associates as a heterodimer with the transmembrane protein p62. Late in the secretory pathway, cellular furin cleaves p62 to the mature E2 protein and a peripheral protein E3. E3 remains bound to E2 at low pH, stabilizing the heterodimer and thus protecting E1 from the acidic pH of the secretory pathway. Release of E3 at neutral pH then primes the virus for fusion during entry. Here we used site-directed mutagenesis and revertant analysis to define residues important for the interactions at the E3–E2 interface. Our data identified a key residue, E2 W235, which was required for E1 pH protection and alphavirus production. Our data also suggest additional residues on E3 and E2 that affect their interacting surfaces and thus influence the pH protection of E1 during alphavirus exit.

Fluorescent IgG fusion proteins made in E. coli

Science.gov (United States)

Luria, Yael; Raichlin, Dina; Benhar, Itai

2012-01-01

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals. PMID:22531449

Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway.

Science.gov (United States)

Ko, Su Hyuk; Jeon, Jong Ik; Myung, Hyun Soo; Kim, Young-Jeon; Kim, Jung Mogg

2017-10-01

Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. Copyright © 2017 American Society for Microbiology.

HUWE1 and TRIP12 Collaborate in Degradation of Ubiquitin-Fusion Proteins and Misframed Ubiquitin

DEFF Research Database (Denmark)

Poulsen, Esben G; Steinhauer, Cornelia; Lees, Michael

2012-01-01

In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD) pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized...... in degradation of the UFD substrate Ub(G76V)-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD...... substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1). Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V)-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1...

FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing's sarcoma cells

International Nuclear Information System (INIS)

Yang, Liu; Hu, Hsien-Ming; Zielinska-Kwiatkowska, Anna; Chansky, Howard A.

2010-01-01

Research highlights: → Inducible and reversible siRNA knockdown of an oncogenic fusion protein such as EWS-Fli1 is feasible and more advantageous than other siRNA methods. → The tumor suppressor gene FOXO1 is a new EWS-Fli1 target. → While trans-activators are known for the FOXO1 gene, there has been no report on negative regulators of FOXO1 transcription. → This study provides first evidence that the EWS-Fli1 oncogenic fusion protein can function as a transcriptional repressor of the FOXO1 gene. -- Abstract: Ewing's family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing's cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing's sarcoma cells. Here we show that FOXO1 expression in Ewing's cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing's family tumors.

The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

International Nuclear Information System (INIS)

Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

2010-01-01

The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

Fluorescent sensors based on bacterial fusion proteins

International Nuclear Information System (INIS)

Mateu, Batirtze Prats; Pum, Dietmar; Sleytr, Uwe B; Toca-Herrera, José L; Kainz, Birgit

2014-01-01

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties.

Directory of Open Access Journals (Sweden)

Elodie Beaumont

Full Text Available Various strategies involving the use of hepatitis C virus (HCV E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.

Structural Study of a New HIV-1 Entry Inhibitor and Interaction with the HIV-1 Fusion Peptide in Dodecylphosphocholine Micelles.

Science.gov (United States)

Pérez, Yolanda; Gómara, Maria José; Yuste, Eloísa; Gómez-Gutierrez, Patricia; Pérez, Juan Jesús; Haro, Isabel

2017-08-25

Previous studies support the hypothesis that the envelope GB virus C (GBV-C) E1 protein interferes the HIV-1 entry and that a peptide, derived from the region 139-156 of this protein, has been defined as a novel HIV-1 entry inhibitor. In this work, we firstly focus on the characterization of the structural features of this peptide, which are determinant for its anti-HIV-1 activity and secondly, on the study of its interaction with the proposed viral target (i.e., the HIV-1 fusion peptide). We report the structure of the peptide determined by NMR spectroscopy in dodecylphosphocholine (DPC) micelles solved by using restrained molecular dynamics calculations. The acquisition of different NMR experiments in DPC micelles (i.e., peptide-peptide titration, diffusion NMR spectroscopy, and addition of paramagnetic relaxation agents) allows a proposal of an inhibition mechanism. We conclude that a 18-mer peptide from the non-pathogenic E1 GBV-C protein, with a helix-turn-helix structure inhibits HIV-1 by binding to the HIV-1 fusion peptide at the membrane level, thereby interfering with those domains in the HIV-1, which are critical for stabilizing the six-helix bundle formation in a membranous environment. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression of fusion IL2-B7.1(IgV+C) and effects on T lymphocytes.

Science.gov (United States)

Kong, Linghong; Li, Yaochen; Yang, Ye; Li, Kangsheng

2007-12-01

The search for an effective immunotherapeutic treatment for tumors is an important area of cancer research. To prepare a more effective form of the bifunctional fusion protein IL2-B7.1(IgV+C) and analyze its effect on the stimulation of T lymphocyte proliferation, we used DNAStar 5.03 software to predict the structural diversity and biochemical character of IL2-B7.1(IgV+C). We then prepared fusion protein IL2-B7.1(IgV+C) by establishing its prokaryotic expression system, and tested its effect on the stimulation of T lymphocytes in vitro. The results indicated that IL2-B7.1(IgV+C) correctly formed a secondary structure in which both IL2 and B7.1(IgV+C) maintained their original hydrophilicity and epitopes. Western blot analysis revealed that IL2-B7.1(IgV+C) was efficiently expressed. Our analysis of CTLL-2 and T-cell proliferation showed that recombinant human (rh) IL2-B7.1(IgV+C) exerted the combined stimulating effects of both rhIL2 and rh B7.1(IgV+C) on cell proliferation, and that these effects could be blocked by adding either anti-IL2 or anti-B7.1 monoclonal antibodies. A >2-fold increase in [3H]TdR incorporation compared with that of cells treated with recombinant protein IL2, or B7.1(IgV+C) alone, revealed that rhIL2-B7.1(IgV+C) had dose-dependent synergetic effects on T-cell activation in the presence of anti-CD3 monoclonal antibody. We concluded that the augmented potency of rhIL2-B7.1(IgV+C) resulted in a stronger stimulation of T-cell proliferation than either rhB7.1(IgV+C) or rhIL2 alone.

Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

Science.gov (United States)

Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei

2016-07-08

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection*

Science.gov (United States)

Yun, Bing-Ling; Guan, Xiao-Lu; Liu, Yong-Zhen; Zhang, Yao; Wang, Yong-Qiang; Qi, Xiao-Le; Cui, Hong-Yu; Liu, Chang-Jun; Zhang, Yan-Ping; Gao, Hong-Lei; Gao, Li; Li, Kai; Gao, Yu-Long; Wang, Xiao-Mei

2016-01-01

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. PMID:27226547

The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export*

Science.gov (United States)

Port, Sarah A.; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H.

2016-01-01

Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. PMID:27613868

The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

Science.gov (United States)

Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

2016-10-28

Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A) + RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A) + RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

Science.gov (United States)

Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M.; Iorio, Ronald M.

2012-01-01

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented. PMID:22915815

Localization of a region in the fusion protein of avian metapneumovirus that modulates cell-cell fusion.

Science.gov (United States)

Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M; Iorio, Ronald M; Li, Jianrong

2012-11-01

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.

Biologically active and C-amidated hinnavinII-38-Asn produced from a Trx fusion construct in Escherichia coli.

Science.gov (United States)

Kang, Chang Soo; Son, Seung-Yeol; Bang, In Seok

2008-12-01

The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15-20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.

Low Resolution Structure of RAR1-GST-Tag Fusion Protein in Solution

International Nuclear Information System (INIS)

Taube, M.; Kozak, M.; Jarmolowski, A.

2010-01-01

RAR1 is a protein required for resistance mediated by many R genes and function upstream of signaling pathways leading to H 2 O 2 accumulation. The structure and conformation of RAR1-GST-Tag fusion protein from barley (Hordeum vulgare) in solution was studied by the small angle scattering of synchrotron radiation. It was found that the dimer of RAR1-GST-Tag protein is characterized in solution by radius of gyration R G = 6.19 nm and maximal intramolecular vector D max = 23 nm. On the basis of the small angle scattering of synchrotron radiation SAXS data two bead models obtained by ab initio modeling are proposed. Both models show elongated conformations. We also concluded that molecules of fusion protein form: dimers in solution via interaction of GST domains. (authors)

HUWE1 and TRIP12 collaborate in degradation of ubiquitin-fusion proteins and misframed ubiquitin.

Directory of Open Access Journals (Sweden)

Esben G Poulsen

Full Text Available In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized. Recently the human E3 ubiquitin-protein ligase TRIP12 was connected with the UFD pathway, but little is otherwise known about this system in mammalian cells. In the present work, we utilized high-throughput imaging on cells transfected with a targeted siRNA library to identify components involved in degradation of the UFD substrate Ub(G76V-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1. Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1 and TRIP12 resulted in an additive stabilization of the substrate, suggesting that HUWE1 and TRIP12 function in parallel during UFD. However, even when both HUWE1 and TRIP12 are downregulated, ubiquitylation of the UFD substrate was still apparent, revealing functional redundancy between HUWE1, TRIP12 and yet other ubiquitin-protein ligases.

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

Science.gov (United States)

Brown, W C; Zhao, S; Woods, V M; Tripp, C A; Tetzlaff, C L; Heussler, V T; Dobbelaere, D A; Rice-Ficht, A C

1993-01-01

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1

Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

OpenAIRE

Somayeh Kadkhodayan; Shiva Irani; Seyed Mehdi Sadat; Fatemeh Fotouhi; Azam Bolhassani

2016-01-01

Abstract Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa) could act as a cell penetrating peptide (CPP). In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confi...

Mitochondrial Fusion Proteins and Human Diseases

Directory of Open Access Journals (Sweden)

Michela Ranieri

2013-01-01

Full Text Available Mitochondria are highly dynamic, complex organelles that continuously alter their shape, ranging between two opposite processes, fission and fusion, in response to several stimuli and the metabolic demands of the cell. Alterations in mitochondrial dynamics due to mutations in proteins involved in the fusion-fission machinery represent an important pathogenic mechanism of human diseases. The most relevant proteins involved in the mitochondrial fusion process are three GTPase dynamin-like proteins: mitofusin 1 (MFN1 and 2 (MFN2, located in the outer mitochondrial membrane, and optic atrophy protein 1 (OPA1, in the inner membrane. An expanding number of degenerative disorders are associated with mutations in the genes encoding MFN2 and OPA1, including Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. While these disorders can still be considered rare, defective mitochondrial dynamics seem to play a significant role in the molecular and cellular pathogenesis of more common neurodegenerative diseases, for example, Alzheimer’s and Parkinson’s diseases. This review provides an overview of the basic molecular mechanisms involved in mitochondrial fusion and focuses on the alteration in mitochondrial DNA amount resulting from impairment of mitochondrial dynamics. We also review the literature describing the main disorders associated with the disruption of mitochondrial fusion.

Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

International Nuclear Information System (INIS)

Ou Wu; Silver, Jonathan

2006-01-01

Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion

Immunological evaluation of Escherichia coli-derived hepatitis C virus second envelope protein (E2) variants.

Science.gov (United States)

Dueñas-Carrera, S; Viña, A; Garay, H E; Reyes, O; Alvarez-Lajonchere, L; Guerra, I; González, L J; Morales, J

2001-09-01

Two variants of the hepatitis C virus (HCV) E2 envelope protein, lacking the C-terminal domain and comprising amino acids 458-650 (E2A) and 382-605 (E2C), respectively, were efficiently produced in BL21 (DE3) Escherichia coli cells. E2A and E2C were used to immunize mice. The E2C variant induced the maximal mean antibody titer. Anti-E2C mouse sera reacted mainly with E2 synthetic peptides covering the 70 amino acid N-terminal region of the E2 protein. Moreover, a panel of anti-HCV positive human sera recognized only the E2C protein (28.2%) and the synthetic peptide covering the HVR-1 of the E2 protein (23.1%). These data indicate the existence of an immunologically relevant region in the HVR-1 of the HCV E2 protein.

Preparation of GST Fusion Proteins.

Science.gov (United States)

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-04-01

INTRODUCTIONThis protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

Multimerized CHR-derived peptides as HIV-1 fusion inhibitors.

Science.gov (United States)

Nomura, Wataru; Hashimoto, Chie; Suzuki, Takaharu; Ohashi, Nami; Fujino, Masayuki; Murakami, Tsutomu; Yamamoto, Naoki; Tamamura, Hirokazu

2013-08-01

To date, several HIV-1 fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of an HIV-1 envelope protein gp41 have been discovered. We have shown that a synthetic peptide mimetic of a trimer form of the CHR-derived peptide C34 has potent inhibitory activity against the HIV-1 fusion mechanism, compared to a monomer C34 peptide. The present study revealed that a dimeric form of C34 is evidently structurally critical for fusion inhibitors, and that the activity of multimerized CHR-derived peptides in fusion inhibition is affected by the properties of the unit peptides C34, SC34EK, and T20. The fluorescence-based study suggested that the N36-interactive sites of the C34 trimer, including hydrophobic residues, are exposed outside the trimer and that trimerization of C34 caused a remarkable increase in fusion inhibitory activity. The present results could be useful in the design of fusion inhibitors against viral infections which proceed via membrane fusion with host cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system.

Science.gov (United States)

Rao, Shengqi; Zang, Xiangyu; Yang, Zhenquan; Gao, Lu; Yin, Yongqi; Fang, Weiming

2016-02-01

A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag. Copyright © 2015 Elsevier Inc. All rights reserved.

The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

International Nuclear Information System (INIS)

Liao Maofu; Kielian, Margaret

2005-01-01

The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion

Characterization of the honeybee venom proteins C1q-like protein and PVF1 and their allergenic potential

DEFF Research Database (Denmark)

Russkamp, Dennis; Van Vaerenbergh, Matthias; Etzold, Stefanie

2018-01-01

-like protein (C1q) and PDGF/VEGF-like factor 1 (PVF1). C1q and PVF1 were produced as recombinant proteins in insect cells. Their allergenic properties were examined by determining the level of specific IgE antibodies in the sera of HBV-allergic patients (n = 26) as well as by their capacity to activate...... frugiperda insect cells exhibited specific IgE reactivity with approximately 38.5% of sera of HBV-allergic patients. Interestingly, both proteins were unable to activate basophils of the patients, questioning their role in the context of clinically relevant sensitization. Recombinant C1q and PVF1 can build...

Expression of human mag-1 gene in E. coli and preparation of its antibody

International Nuclear Information System (INIS)

Lin Huiyun; Xu Yuanji; Wang Yan; Chen Huihua; Du Zhiyan; Tan Xiaogang; Lu Yinglin

2006-01-01

Objective: To further investigate the new metastasis associated gene, mag-1 expressed in E. coli and its anti-body was prepared in rabbit. Methods: mag-1 was amplified by PCR from pcDNA3-mag-1 and directly cloned into pET-28a vector. The fusion protein was expressed in BL21 and identified by Western blot using anti-His monoclonal antibody. Rabbit was immunized with partially purified fusion protein subcutaneously. Results: Sequence analysis revealed identity of the sequence obtained to the previous report. The recombinant His-mag-1 could be expressed in E. coli as a fusion protein of 18 x 10 3 . The recombinant protein was mostly expressed in the inclusion bodies on the induction by 0.1 mmol/L IPTG at 37 degree C for 6 hours. Western blot analysis showed that the recombinant protein could be recognized by His monoclonal anti-body. The titer of polyclonal antibody against mag-1 was 1:160000. Conclusion: The mag-1 gene is expressed in E. coli highly and its antibody is prepared successfully. (authors)

IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells.

Directory of Open Access Journals (Sweden)

Luisa Cironi

Full Text Available BACKGROUND: The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT, the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1 for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. METHODOLOGY/PRINCIPAL FINDINGS: To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. CONCLUSION/SIGNIFICANCE: Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity.

Science.gov (United States)

McCoy, Andrea J; Maurelli, Anthony T

2005-07-01

Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D-amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D-alanyl-D-alanine (D-Ala-D-Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics-based approach to demonstrate and characterize the D-Ala-D-Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full-length murC-ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible ara promoter and transformed into a D-Ala-D-Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliDeltaddlADeltaddlB mutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydial murC-ddl thus demonstrating functional ligase activity. Domain mapping of the full-length fusion protein and site-directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala. Chlamydia growth is inhibited by D-cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D-Ala and D-Ala-D-Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

Science.gov (United States)

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome

Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

Directory of Open Access Journals (Sweden)

Sanjukta Chakrabarti

2016-06-01

Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

Science.gov (United States)

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

Directory of Open Access Journals (Sweden)

Aiko Amagai

2012-01-01

Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

Science.gov (United States)

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

DEFF Research Database (Denmark)

Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

2004-01-01

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein...... the expected nanomolar affinities for interaction with SNX1. Truncations of the NK1 receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded...

A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

DEFF Research Database (Denmark)

Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne

2004-01-01

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein...... the expected nanomolar affinities for interaction with SNX1. Truncations of the NK(1) receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded...

Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing's sarcoma in vitro.

Science.gov (United States)

Jully, Babu; Vijayalakshmi, Ramshankar; Gopal, Gopisetty; Sabitha, Kesavan; Rajkumar, Thangarajan

2012-11-12

Ewing's sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251-343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251-280 ectopically in Ewing's sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Our modelling analysis indicated that Junction region (aa 251-343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing's sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing's Sarcoma.

Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

International Nuclear Information System (INIS)

Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis

2006-01-01

The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1 V12 or Cdc42 V12 could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA L63 decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia

Computational and biological characterization of fusion proteins of two insecticidal proteins for control of insect pests.

Science.gov (United States)

Javaid, Shaista; Naz, Sehrish; Amin, Imran; Jander, Georg; Ul-Haq, Zaheer; Mansoor, Shahid

2018-03-19

Sucking pests pose a serious agricultural challenge, as available transgenic technologies such as Bacillus thuringiensis crystal toxins (Bt) are not effective against them. One approach is to produce fusion protein toxins for the control of these pests. Two protein toxins, Hvt (ω-atracotoxin from Hadronyche versuta) and onion leaf lectin, were translationally fused to evaluate the negative effects of fusion proteins on Phenacoccus solenopsis (mealybug), a phloem-feeding insect pest. Hvt was cloned both N-terminally (HL) and then C-terminally (LH) in the fusion protein constructs, which were expressed transiently in Nicotiana tabacum using a Potato Virus X (PVX) vector. The HL fusion protein was found to be more effective against P. solenopsis, with an 83% mortality rate, as compared to the LH protein, which caused 65% mortality. Hvt and lectin alone caused 42% and 45%, respectively, under the same conditions. Computational studies of both fusion proteins showed that the HL protein is more stable than the LH protein. Together, these results demonstrate that translational fusion of two insecticidal proteins improved the insecticidal activity relative to each protein individually and could be expressed in transgenic plants for effective control of sucking pests.

Fusion of the BCL9 HD2 domain to E1A increases the cytopathic effect of an oncolytic adenovirus that targets colon cancer cells

Directory of Open Access Journals (Sweden)

Pittet Anne-Laure

2006-10-01

Full Text Available Abstract Background The Wnt signaling pathway is activated by mutations in the APC and β-catenin genes in many types of human cancer. β-catenin is stabilized by these mutations and activates transcription in part by acting as a bridge between Tcf/LEF proteins and the HD2 domain of the BCL9 coactivator. We have previously described oncolytic adenoviruses with binding sites for Tcf/LEF transcription factors inserted into the early viral promoters. These viruses replicate selectively in cells with activation of the Wnt pathway. To increase the activity of these viruses we have fused the viral transactivator E1A to the BCL9 HD2 domain. Methods Luciferase assays, co-immunoprecipitation and Western blotting, immunofluorescent cell staining and cytopathic effect assays were used to characterize the E1A-HD2 fusion protein and virus in vitro. Growth curves of subcutaneous SW620 colon cancer xenografts were used to characterize the virus in vivo. Results The E1A-HD2 fusion protein binds to β-catenin in vivo and activates a Tcf-regulated luciferase reporter better than wild-type E1A in cells with activated Wnt signaling. Expression of the E1A-HD2 protein promotes nuclear import of β-catenin, mediated by the strong nuclear localization signal in E1A. Tcf-regulated viruses expressing the fusion protein show increased expression of viral proteins and a five-fold increase in cytopathic effect (CPE in colorectal cancer cell lines. There was no change in viral protein expression or CPE in HeLa cells, indicating that E1A-HD2 viruses retain selectivity for cells with activation of the Wnt signaling pathway. Despite increasing the cytopathic effect of the virus in vitro, fusion of the HD2 domain to E1A did not increase the burst size of the virus in vitro or the anti-tumor effect of the virus in an SW620 xenograft model in vivo. Conclusion Despite an increase in the nuclear pool of β-catenin, the effects on viral activity in colon cancer cells were small

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

Science.gov (United States)

Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

2017-04-01

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Fusion protein is the main determinant of metapneumovirus host tropism.

Science.gov (United States)

de Graaf, Miranda; Schrauwen, Eefje J A; Herfst, Sander; van Amerongen, Geert; Osterhaus, Albert D M E; Fouchier, Ron A M

2009-06-01

Human metapneumovirus (HMPV) and avian metapneumovirus subgroup C (AMPV-C) infect humans and birds, respectively. This study confirmed the difference in host range in turkey poults, and analysed the contribution of the individual metapneumovirus genes to host range in an in vitro cell-culture model. Mammalian Vero-118 cells supported replication of both HMPV and AMPV-C in contrast to avian quail fibroblast (QT6) cells in which only AMPV-C replicated to high titres. Inoculation of Vero-118 and QT6 cells with recombinant HMPV in which genes were exchanged with those of AMPV-C revealed that the metapneumovirus fusion (F) protein is the main determinant for host tropism. Chimeric viruses in which polymerase complex proteins were exchanged between HMPV and AMPV-C replicated less efficiently compared with HMPV in QT6 cells. Using mini-genome systems, it was shown that exchanging these polymerase proteins resulted in reduced replication and transcription efficiency in QT6 cells. Examination of infected Vero-118 and QT6 cells revealed that viruses containing the F protein of AMPV-C yielded larger syncytia compared with viruses containing the HMPV F protein. Cell-content mixing assays revealed that the F protein of AMPV-C was more fusogenic compared with the F protein of HMPV, and that the F2 region is responsible for the difference observed between AMPV-C and HMPV F-promoted fusion in QT6 and Vero-118 cells. This study provides insight into the determinants of host tropism and membrane fusion of metapneumoviruses.

[Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

Science.gov (United States)

Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

2011-08-01

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

A Protein Diet Score, Including Plant and Animal Protein, Investigating the Association with HbA1c and eGFR—The PREVIEW Project

Science.gov (United States)

Mikkilä, Vera; Raitakari, Olli T.; Hutri-Kähönen, Nina; Dragsted, Lars O.; Poppitt, Sally D.; Silvestre, Marta P.; Feskens, Edith J.M.

2017-01-01

Higher-protein diets have been advocated for body-weight regulation for the past few decades. However, the potential health risks of these diets are still uncertain. We aimed to develop a protein score based on the quantity and source of protein, and to examine the association of the score with glycated haemoglobin (HbA1c) and estimated glomerular filtration rate (eGFR). Analyses were based on three population studies included in the PREVIEW project (PREVention of diabetes through lifestyle Intervention and population studies in Europe and around the World): NQplus, Lifelines, and the Young Finns Study. Cross-sectional data from food-frequency questionnaires (n = 76,777 subjects) were used to develop a protein score consisting of two components: 1) percentage of energy from total protein, and 2) plant to animal protein ratio. An inverse association between protein score and HbA1c (slope −0.02 ± 0.01 mmol/mol, p < 0.001) was seen in Lifelines. We found a positive association between the protein score and eGFR in Lifelines (slope 0.17 ± 0.02 mL/min/1.73 m2, p < 0.0001). Protein scoring might be a useful tool to assess both the effect of quantity and source of protein on health parameters. Further studies are needed to validate this newly developed protein score. PMID:28714926

The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

Directory of Open Access Journals (Sweden)

Bin Lu

2015-06-01

Full Text Available Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2 on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26 abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9 loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

Structural characterization of Mumps virus fusion protein core

International Nuclear Information System (INIS)

Liu Yueyong; Xu Yanhui; Lou Zhiyong; Zhu Jieqing; Hu Xuebo; Gao, George F.; Qiu Bingsheng; Rao Zihe; Tien, Po

2006-01-01

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins

[Research progress in hirudin fusion protein--review].

Science.gov (United States)

Zhang, Chuan-Ling; Yu, Ai-Ping; Jin, Ji-De; Wu, Chu-Tse

2007-02-01

Natural hirudin extracted from the secretion of medical leech salivary gland is a single-chain peptide containing 65 aminoacid residues with molecular weight of 7000 D, and exists in three isomers of HV1, HV2 and HV3. Hirudin possesses three disulfide bridges forming the structure of core cyclic peptides, which binds to the catalytic site of thrombin so as to inhibit the catalysis of thrombin. Its c-terminus rich in acidic aminoacid residues possesses hydrophilicity, and is free on the molecular surface, and can bind with fibrin recognition site of hirudin. The minimal segment of 12 - 16 C-terminal acidic residues keeps the minimal activity of anti-thrombosis. Thus, hirudin, as a potent and specific inhibitor of thrombin, can be used to protect from and to treat clinically thrombosis. As it has some disadvantages such as short half-life, bleeding side-effect and mono-function, and so on, hirudin has been fused with some other functional proteins in recent years. The obtained fusion proteins can prolong the half life of hirudin, or relieve it bleeding side effect, or bring new functions, such as thrombolysis, inhibiting the platelet aggregation, targeting specifically. The research progress in hirudin fusion protein was summarized in this review.

Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

Science.gov (United States)

Khademi, Farzad; Yousefi-Avarvand, Arshid; Derakhshan, Mohammad; Meshkat, Zahra; Tafaghodi, Mohsen; Ghazvini, Kiarash; Aryan, Ehsan; Sankian, Mojtaba

2017-10-01

The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

Exo-endo cellulase fusion protein

Science.gov (United States)

Bower, Benjamin S [Palo Alto, CA; Larenas, Edmund A [Palo Alto, CA; Mitchinson, Colin [Palo Alto, CA

2012-01-17

The present invention relates to a heterologous exo-endo cellulase fusion construct, which encodes a fusion protein having cellulolytic activity comprising a catalytic domain derived from a fungal exo-cellobiohydrolase and a catalytic domain derived from an endoglucanase. The invention also relates to vectors and fungal host cells comprising the heterologous exo-endo cellulase fusion construct as well as methods for producing a cellulase fusion protein and enzymatic cellulase compositions.

Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing’s Sarcoma in vitro

International Nuclear Information System (INIS)

Jully, Babu; Vijayalakshmi, Ramshankar; Gopal, Gopisetty; Sabitha, Kesavan; Rajkumar, Thangarajan

2012-01-01

Ewing’s sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251–343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251–280 ectopically in Ewing’s sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Our modelling analysis indicated that Junction region (aa 251–343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing’s sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing’s Sarcoma

Analysis of antigenic cross-reactivity between subgroup C avian pneumovirus and human metapneumovirus by using recombinant fusion proteins.

Science.gov (United States)

Luo, L; Sabara, M I; Li, Y

2009-10-01

Avian pneumovirus subgroup C (APV/C) has recently been reported to be more closely related to human metapneumovirus (hMPV) as determined by sequence analysis. To examine the antigenic relationship between APV/C and hMPV, the APV/C fusion (F) gene was cloned and expressed as an uncleaved glycoprotein in a baculovirus system. The reactivity of the APV/C F protein with antibodies against APV subgroups A, B, C, and hMPV was examined by Western blot analysis. The results showed that the expressed APV/C F protein was not only recognized by APV/C-specific antibodies but also by antibodies raised against hMPV. Previously expressed recombinant hMPV F protein also reacted with APV/C-specific antibodies, suggesting that there was significant antigenic cross-reactivity and a potential evolutionary relationship between hMPV and APV/C. Interestingly, the recombinant F proteins from APV/C and hMPV were not recognized by polyclonal antibodies specific to APV subgroups A and B.

Fc-fusion Proteins in Therapy: An Updated View.

Science.gov (United States)

Jafari, Reza; Zolbanin, Naime M; Rafatpanah, Houshang; Majidi, Jafar; Kazemi, Tohid

2017-01-01

Fc-fusion proteins are composed of Fc region of IgG antibody (Hinge-CH2-CH3) and a desired linked protein. Fc region of Fc-fusion proteins can bind to neonatal Fc receptor (FcRn) thereby rescuing it from degradation. The first therapeutic Fc-fusion protein was introduced for the treatment of AIDS. The molecular designing is the first stage in production of Fc-fusion proteins. The amino acid residues in the Fc region and linked protein are very important in the bioactivity and affinity of the fusion proteins. Although, therapeutic monoclonal antibodies are the top selling biologics but the application of therapeutic Fc-fusion proteins in clinic is in progress and among these medications Etanercept is the most effective in therapy. At present, eleven Fc-fusion proteins have been approved by FDA. There are novel Fc-fusion proteins which are in pre-clinical and clinical development. In this article, we review the molecular and biological characteristics of Fc-fusion proteins and then further discuss the features of novel therapeutic Fc-fusion proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

Directory of Open Access Journals (Sweden)

Juan Fontana

2017-08-01

Full Text Available All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV, a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i mutagenesis to insert fluorescent proteins at specific positions, (ii coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.

Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

Science.gov (United States)

Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

2013-08-09

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

Directory of Open Access Journals (Sweden)

Trimpalis Philip

2011-07-01

Full Text Available Abstract Background Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. Results In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. Conclusions This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs.

The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse

DEFF Research Database (Denmark)

Vasyutina, Elena; Martarelli, Benedetta; Brakebusch, Cord

2009-01-01

Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42 mut...... genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts....

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

Science.gov (United States)

Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

2010-01-01

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

Science.gov (United States)

Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

2016-05-01

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

Complete fusion of the 12C+12O, 14N+12C and 15N+12C systems

International Nuclear Information System (INIS)

Conjeaud, M.; Gary, S.; Harar, S.; Wieleczko, J.P.

1978-01-01

Cross sections for evaporation residues following the complete fusion of the 12 C+ 12 C, 14 N+ 12 C and 15 N+ 12 C systems have been measured with a E-ΔE counter telescope in a wide range of incident energies. They are fairly well reproduced by evaporation calculations based on the statistical theory. The total fusion excitation function of the 12 C+ 12 C system shows strong structure, which is compared to the predictions of the reaction cross sections derived from coupled channel calculations and to the integrated inelastic cross sections. Critical angular momenta have been obtained from the fusion cross-section data and these values are discussed in the framework of compound nucleus and entrance channel effects. A striking difference is observed between the fusion cross sections of the 14 N+ 12 C and 15 N+ 12 C systems and shows the importance of the valence nucleons of colliding ions in the fusion process. A possible interpretation might be the influence of the yrast line of the compound nuclei. (Auth.)

“Inclonals”: IgGs and IgG-enzyme fusion proteins produced in an E. coli expression-refolding system

OpenAIRE

Hakim, Rahely; Benhar, Itai

2009-01-01

Full-length antibodies and antibodies that ferry a cargo to target cells are desired biopharmaceuticals. We describe the production of full-length IgGs and IgG-toxin fusion proteins in E. coli. In the presented examples of anti CD30 and anti EGF-receptor antibodies, the antibody heavy and light chains or toxin fusions thereof were expressed in separate bacterial cultures, where they accumulated as insoluble inclusion bodies. Following refolding and purification, high yields (up to 50 mg/L of ...

G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

DEFF Research Database (Denmark)

Dou, Q P; Zhao, S; Levin, A H

1994-01-01

report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes...... complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex...

Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins.

Science.gov (United States)

Takeda, Akiko; Sarma, Nayan J; Abdul-Nabi, Anmaar M; Yaseen, Nabeel R

2010-05-21

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

Chaperone activity of human small heat shock protein-GST fusion proteins.

Science.gov (United States)

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

Science.gov (United States)

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2017-04-01

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

Energy Technology Data Exchange (ETDEWEB)

Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jin, Aishun [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Immunology, College of Basic Medical Sciences, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081 (China); Kishi, Hiroyuki, E-mail: immkishi@med.u-toyama.ac.jp [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Muraguchi, Atsushi [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

2012-06-01

Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion.

Science.gov (United States)

Yen, Li-Chen; Liao, Jia-Teh; Lee, Hwei-Jen; Chou, Wei-Yuan; Chen, Chun-Wei; Lin, Yi-Ling; Liao, Ching-Len

2016-02-01

NS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using a trans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain. The positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained

MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity.

Science.gov (United States)

Lu, Shuo; Zgurskaya, Helen I

2013-11-01

The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC.

Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

Energy Technology Data Exchange (ETDEWEB)

P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

2011-12-31

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

In-silico determination of insecticidal potential of Vip3Aa-Cry1Ac fusion protein against Lepidopteran targets using molecular docking

Directory of Open Access Journals (Sweden)

Aftab eAhmad

2015-12-01

Full Text Available Study and research of Bt (Bacillus thuringiensis transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac insecticidal protein and vegetative insecticidal protein (Vip3Aa have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua and Spodoptera litura revealed that the Ser290, Ser293, Leu337, Thr340 and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.

Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization

International Nuclear Information System (INIS)

Waldhuber, Megan G.; Bateson, Michael; Tan, Judith; Greenway, Alison L.; McPhee, Dale A.

2003-01-01

The human immunodeficiency virus type 1 (HIV-1) Vpr protein is known to arrest the cell cycle in G 2 /M and induce apoptosis following arrest. The functions of Vpr relative to its location in the cell remain unresolved. We now demonstrate that the location and function of Vpr are dependent on the makeup of fusion proteins and that the functions of G 2 /M arrest and apoptosis are separable. Using green fluorescence protein mutants (EGFP or EYFP), we found that fusion at either the N- or C-terminus compromised the ability of Vpr to arrest cell cycling, relative to that of His-Vpr or wild-type protein. Additionally, utilizing the ability to specifically identify cells expressing the fusion proteins, we confirm that Vpr can induce apoptosis, but appears to be independent of cell-cycle arrest in G 2 /M. Both N- and C-terminal Vpr/EYFP fusion proteins induced apoptosis but caused minimal G 2 /M arrest. These studies with Vpr fusion proteins indicate that the functions of Vpr leading to G 2 /M arrest and apoptosis are separable and that fusion of Vpr to EGFP or EYFP affected the localization of the protein. Our findings suggest that nuclear membrane localization and nuclear import and export are strongly governed by modification of the N-terminus of Vpr

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC-eseE Operon.

Science.gov (United States)

Yi, Jia; Xiao, Shui Bing; Zeng, Zhi Xiong; Lu, Jin Fang; Liu, Lu Yi; Laghari, Zubair Ahmed; Nie, Pin; Yu, Hong Bing; Xie, Hai Xia

2016-08-01

Edwardsiella tarda is an important Gram-negative pathogen that employs a type III secretion system (T3SS) to deliver effectors into host cells to facilitate bacterial survival and replication. These effectors are translocated into host cells through a translocon complex composed of three secreted proteins, namely, EseB, EseC, and EseD. The secretion of EseB and EseD requires a chaperone protein called EscC, whereas the secretion of EseC requires the chaperone EscA. In this study, we identified a novel protein (EseE) that also regulates the secretion of EseC. An eseE deletion mutant secreted much less EseC into supernatants, accompanied by increased EseC levels within bacterial cells. We also demonstrated that EseE interacted directly with EseC in a pulldown assay. Interestingly, EseC, EseE, and EscA were able to form a ternary complex, as revealed by pulldown and gel filtration assays. Of particular importance, the deletion of eseE resulted in decreased levels of EseB and EseD proteins in both the bacterial pellet and supernatant fraction. Furthermore, real-time PCR assays showed that EseE positively regulated the transcription of the translocon operon escC-eseE, comprising escC, eseB, escA, eseC, eseD, and eseE These effects of EseE on the translocon components/operon appeared to have a functional consequence, since the ΔeseE strain was outcompeted by wild-type E. tarda in a mixed infection in blue gourami fish. Collectively, our results demonstrate that EseE not only functions as a chaperone for EseC but also acts as a positive regulator controlling the expression of the translocon operon escC-eseE, thus contributing to the pathogenesis of E. tarda in fish. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Semi empirical model for astrophysical nuclear fusion reactions of 1≤Z≤15

International Nuclear Information System (INIS)

Manjunatha, H.C.; Seenappa, L.; Sridhar, K.N.

2017-01-01

The fusion reaction is one of the most important reactions in the stellar evolution. Due to the complicated reaction mechanism of fusion, there is great uncertainty in the reaction rate which limits our understanding of various stellar objects. Low z elements are formed through many fusion reactions such as "4He+"1"2C→"1"6O, "1"2C+"1"2C→"2"0Ne+"4He, "1"2C+"1"2C→"2"3Na, "1"2C+"1"2C→"2"3Mg, "1"6O+"1"6O→"2"8Si+"4He, "1"2C+"1H→"1"3N and "1"3C+"4He→"1"6O. A detail study is required on Coulomb and nuclear interaction in formation of low Z elements in stars through fusion reactions. For astrophysics, the important energy range extends from 1 MeV to 3 MeV in the center of mass frame, which is only partially covered by experiments. In the present work, we have studied the basic fusion parameters such as barrier heights (V_B), positions (R_B), curvature of the inverted parabola (ħω_1) for fusion barrier, cross section and compound nucleus formation probability (P_C_N) and fusion process in the low Z element (1≤Z≤15) formation process. For each isotope, we have studied all possible projectile-target combinations. We have also studied the astrophysical S(E) factor for these reactions. Based on this study, we have formulated the semi empirical relations for barrier heights (V_B), positions (R_B), curvature of the inverted parabola and hence for the fusion cross section and astrophysical S(E) factor. The values produced by the present model compared with the experiments and data available in the literature. (author)

Characterization of EBV gB indicates properties of both class I and class II viral fusion proteins

International Nuclear Information System (INIS)

Backovic, Marija; Leser, George P.; Lamb, Robert A.; Longnecker, Richard; Jardetzky, Theodore S.

2007-01-01

To gain insight into Epstein-Barr virus (EBV) glycoprotein B (gB), recombinant, secreted variants were generated. The role of putative transmembrane regions, the proteolytic processing and the oligomerization state of the gB variants were investigated. Constructs containing 2 of 3 C-terminal hydrophobic regions were secreted, indicating that these do not act as transmembrane anchors. The efficiency of cleavage of the gB furin site was found to depend on the nature of C-terminus. All of the gB constructs formed rosette structures reminiscent of the postfusion aggregates formed by other viral fusion proteins. However, substitution of putative fusion loop residues, WY 112-113 and WLIY 193-196 , with less hydrophobic amino acids from HSV-1 gB, produced trimeric protein and abrogated the ability of the EBV gB ectodomains to form rosettes. These data demonstrate biochemical features of EBV gB that are characteristic of other class I and class II viral fusion proteins, but not of HSV-1 gB

Role of the synaptobrevin C terminus in fusion pore formation

DEFF Research Database (Denmark)

Ngatchou, Annita N; Kisler, Kassandra; Fang, Qinghua

2010-01-01

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore...... stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular...

Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase.

Directory of Open Access Journals (Sweden)

Minh Tan Nguyen

Full Text Available Human growth hormone (hGH is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, protein disulfide bond isomerase (PDI, and the b'a' domain of PDI (PDIb'a', were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

International Nuclear Information System (INIS)

Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

2004-01-01

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d 3 ) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d 3 . In addition, both sCD4-gp120 and sCD4-gp120-mC3d 3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d 3 or sCD4-gp120-mC3d 3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d 3 -DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d 3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d

Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry

DEFF Research Database (Denmark)

Carlsen, Thomas H R; Scheel, Troels K H; Ramirez, Santseharay

2013-01-01

The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a....../release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition...

Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways.

Science.gov (United States)

Rival, Thomas; Macchi, Marc; Arnauné-Pelloquin, Laetitia; Poidevin, Mickael; Maillet, Frédéric; Richard, Fabrice; Fatmi, Ahmed; Belenguer, Pascale; Royet, Julien

2011-03-01

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis.

Science.gov (United States)

Holst, B; Hastrup, H; Raffetseder, U; Martini, L; Schwartz, T W

2001-06-08

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.

HIV-1 gp41 Fusion Intermediate: A Target for HIV Therapeutics

Directory of Open Access Journals (Sweden)

Chungen Pan

2010-02-01

Full Text Available Human immunodeficiency virus (HIV-1 infection is initiated by the binding of gp120 envelope glyco-protein to its cell receptor (CD4 and a coreceptor (CXCR4 or CCR5, followed by a series of conformational changes in the gp41 transmembrane subunit. These changes include insertion of fusion peptide into the target cell membrane and association of C-heptad repeat (CHR peptide with the N-heptad repeat (NHR trimer, a pre-hairpin fusion intermediate. A stable six-helix bundle core is then formed, bringing the viral envelope and target cell membrane into close proximity for fusion. Peptides derived from the CHR region, such as T20 and C34, inhibit HIV-1 fusion by interacting with the gp41 fusion intermediate. A number of anti-HIV-1 peptides and small molecule compounds targeting the gp41 NHR-trimer have been identified. By combining HIV fusion/entry inhibitors targeting different sites in the gp41 fusion intermediate, a potent synergistic effect takes place, resulting in a potential new therapeutic strategy for the HIV infection/AIDS. Here, we present an overview of the current development of anti-HIV drugs, particularly those targeting the gp41 fusion intermediate.

Characterization and immunogenicity of rLipL32/1-LipL21-OmpL1/2 fusion protein as a novel immunogen for a vaccine against Leptospirosis

Directory of Open Access Journals (Sweden)

Zhao Xin

2015-01-01

Full Text Available Vaccination is an effective strategy to prevent leptospirosis, a global zoonotic disease caused by infection with pathogenic Leptospira species. However, the currently used multiple-valence vaccine, which is prepared with whole cells of several Leptospira serovars, has major side effects, while its cross-immunogenicity among different Leptospira serovars is weak. LipL32, LipL21 and 2 OmpL1 have been confirmed as surface-exposed antigens in all pathogenic Leptospira strains, but their immunoprotective efficiency needs to be improved. In the present study, we generated a fusion gene lipL32/1-lipL21-ompL1/2 using primer-linking PCR and an engineered E. coli strain to express the recombinant fusion protein rLipL32/1-LipL21-OmpL1/2 (rLLO. Subsequently, the expression conditions were optimized using a central composite design that increased the fusion protein yield 2.7-fold. Western blot assays confirmed that rLLO was recognized by anti-rLipL32/1, anti-rLipL21, and anti-rOmpL1/2 sera as well as 98.5% of the sera from leptospirosis patients. The microscopic agglutination test (MAT demonstrated that rLLO antiserum had a stronger ability to agglutinate the strains of different Leptospira serovars than the rLipL32/1, rLipL21, and rOmpL1/2 antisera. More importantly, tests in hamsters showed that rLLO provided higher immunoprotective rates (91.7% than rLipL32/1, rLipL21 and rOmpL1/2 (50.0-75.0%. All the data indicate that rLLO, a recombinant fusion protein incorporating three antigens, has increased antigenicity and immunoprotective effects, and so can be used as a novel immunogen to develop a universal genetically engineered vaccine against leptospirosis.

Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

Science.gov (United States)

Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

2015-09-01

The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

Fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75 interact with the CREC proteins, calumenin and reticulocalbin

DEFF Research Database (Denmark)

Hansen, Gry Aune Westergaard; Ludvigsen, Maja; Jacobsen, Christian

2015-01-01

Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify...... the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted...

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

Science.gov (United States)

Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

2018-02-01

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli.

Directory of Open Access Journals (Sweden)

Sang Beum Lee

Full Text Available Myostatin (MSTN is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP as a fusion partner were expressed in E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42-175 (MBP-Pro42-175 exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42-115 (MBP-Pro42-115 or 42-98 (MBP-Pro42-98 also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42-175 is sufficient to maintain the full MSTN-inhibitory capacity.

Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles

DEFF Research Database (Denmark)

Russell, R S; Kawaguchi, K; Meunier, J-C

2009-01-01

Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide. Retrov...

[Eukaryotic expression of Leptospira interrogans lipL32/1-ompL1/1 fusion gene encoding genus-specific protein antigens and the immunoreactivity of expression products].

Science.gov (United States)

Yan, Jie; Zhao, Shou-feng; Mao, Ya-fei; Ruan, Ping; Luo, Yi-hui; Li, Shu-ping; Li, Li-wei

2005-01-01

To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products. PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively. Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1. An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific

The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors

International Nuclear Information System (INIS)

Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna; Onder, Zeynep; Hannah, Erin C.; Mamoor, Shahan; Crosby, Lauren; Moroianu, Junona

2010-01-01

We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7 39-98 localized mostly to the nucleus. The GST-11E7 and GST-11cE7 39-98 were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.

IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

International Nuclear Information System (INIS)

Kwasnicka-Crawford, Dorota A.; Carson, Andrew R.; Scherer, Stephen W.

2006-01-01

The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNA is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions

Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

Science.gov (United States)

Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

2015-05-15

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

The pharmacological efficacy of the anti-IL17 scFv and sTNFR1 bispecific fusion protein in inflammation mouse stimulated by LPS.

Science.gov (United States)

Yang, Yongbi; Zhang, Teng; Cao, Hongxue; Yu, Dan; Zhang, Tong; Zhao, Shaojuan; Jing, Xiaohui; Song, Liying; Liu, Yunye; Che, Ruixiang; Liu, Xin; Li, Deshan; Ren, Guiping

2017-08-01

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our study found that a bispecific fusion protein treatment can ameliorate the lung injury induced by LPS. However, the molecular mechanisms which bispecific fusion protein ameliorates acute lung injury remain unclear. In this study, we found that the bispecific fusion protein treatment inhibited the nuclear transcription of NF-κB in confocal laser scanning fluorescence microscopy, the bispecific fusion protein exert protective effects in the cell model of ALI induced by lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway and mediate inflammation. Moreover, the treatment of the bispecific fusion protein show its efficacy in animal models stimulated by LPS, the results of real-time PCR and ELISA demonstrate that bispecific fusion protein treatment effectively inhibited the over-expression of inflammatory cytokines(tumor necrosis factor α, interleukin 1β and interleukin 17). In addition, LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology, which was meliorated by bispecific fusion protein treatment. Collectively, these results demonstrate that bispecific fusion protein treatment ameliorates LPS-induced ALI through reducing inflammatory cytokines and lung inflammation, which may be associated with the decreased the nuclear transcription of NF-κB. The bispecific fusion protein may be useful as a novel therapy to treat ALI. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE.

Directory of Open Access Journals (Sweden)

Puthayalai Treerat

Full Text Available Many Gram-negative pathogens use a type III secretion system (TTSS for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness.

MEMBRANE-FUSION OF SEMLIKI FOREST VIRUS INVOLVES HOMOTRIMERS OF THE FUSION PROTEIN

NARCIS (Netherlands)

WAHLBERG, JM; WILSCHUT, J; GAROFF, H

1992-01-01

Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion Processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated

Potential allergenicity research of Cry1C protein from genetically modified rice.

Science.gov (United States)

Cao, Sishuo; He, Xiaoyun; Xu, Wentao; Luo, Yunbo; Ran, Wenjun; Liang, Lixing; Dai, Yunqing; Huang, Kunlun

2012-07-01

With the development of genetically modified crops, there has been a growing interest in available approaches to assess the potential allergenicity of novel gene products. We were not sure whether Cry1C could induce allergy. We examined the protein with three other proteins to determine the potential allergenicity of Cry1C protein from genetically modified rice. Female Brown Norway (BN) rats received 0.1 mg peanut agglutinin (PNA), 1mg potato acid phosphatase (PAP), 1mg ovalbumin (OVA) or 5 mg purified Cry1C protein dissolved in 1 mL water by daily gavage for 42 days to test potential allergenicity. Ten days after the last gavage, rats were orally challenged with antigens, and physiologic and immunologic responses were studied. In contrast to sensitization with PNA, PAP and OVA Cry1C protein did not induce antigen-specific IgG2a in BN rats. Cytokine expression, serum IgE and histamine levels and the number of eosinophils and mast cells in the blood of Cry1C group rats were comparable to the control group rats, which were treated with water alone. As Cry1C did not show any allergenicity, we make the following conclusion that the protein could be safety used in rice or other plants. Copyright © 2012 Elsevier Inc. All rights reserved.

Trypsin- and low pH-mediated fusogenicity of avian metapneumovirus fusion proteins is determined by residues at positions 100, 101 and 294.

Science.gov (United States)

Yun, Bingling; Guan, Xiaolu; Liu, Yongzhen; Gao, Yanni; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Li; Li, Kai; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

2015-10-26

Avian metapneumovirus (aMPV) and human metapneumovirus (hMPV) are members of the genus Metapneumovirus in the subfamily Pneumovirinae. Metapneumovirus fusion (F) protein mediates the fusion of host cells with the virus membrane for infection. Trypsin- and/or low pH-induced membrane fusion is a strain-dependent phenomenon for hMPV. Here, we demonstrated that three subtypes of aMPV (aMPV/A, aMPV/B, and aMPV/C) F proteins promoted cell-cell fusion in the absence of trypsin. Indeed, in the presence of trypsin, only aMPV/C F protein fusogenicity was enhanced. Mutagenesis of the amino acids at position 100 and/or 101, located at a putative cleavage region in aMPV F proteins, revealed that the trypsin-mediated fusogenicity of aMPV F proteins is regulated by the residues at positions 100 and 101. Moreover, we demonstrated that aMPV/A and aMPV/B F proteins mediated cell-cell fusion independent of low pH, whereas the aMPV/C F protein did not. Mutagenesis of the residue at position 294 in the aMPV/A, aMPV/B, and aMPV/C F proteins showed that 294G played a critical role in F protein-mediated fusion under low pH conditions. These findings on aMPV F protein-induced cell-cell fusion provide new insights into the molecular mechanisms underlying membrane fusion and pathogenesis of aMPV.

HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

International Nuclear Information System (INIS)

Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique; Villa, Luisa Lina; Rahal, Paula

2016-01-01

Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Calmon, Marilia Freitas [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil); Sichero, Laura [Molecular Biology Laboratory, Centre for Translational Research in Oncology, Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo (Brazil); Boccardo, Enrique [Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo., São Paulo (Brazil); Villa, Luisa Lina [Department of Radiology and Oncology, Faculdade de Medicina, Universidade de São Paulo, São Paulo (Brazil); Rahal, Paula, E-mail: rahalp@yahoo.com.br [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil)

2016-09-15

Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

SymB and SymC, two membrane associated proteins, are required for Epichloë festucae hyphal cell-cell fusion and maintenance of a mutualistic interaction with Lolium perenne.

Science.gov (United States)

Green, Kimberly A; Becker, Yvonne; Tanaka, Aiko; Takemoto, Daigo; Fitzsimons, Helen L; Seiler, Stephan; Lalucque, Hervé; Silar, Philippe; Scott, Barry

2017-02-01

Cell-cell fusion in fungi is required for colony formation, nutrient transfer and signal transduction. Disruption of genes required for hyphal fusion in Epichloë festucae, a mutualistic symbiont of Lolium grasses, severely disrupts the host interaction phenotype. They examined whether symB and symC, the E. festucae homologs of Podospora anserina self-signaling genes IDC2 and IDC3, are required for E. festucae hyphal fusion and host symbiosis. Deletion mutants of these genes were defective in hyphal cell fusion, formed intra-hyphal hyphae, and had enhanced conidiation. SymB-GFP and SymC-mRFP1 localize to plasma membrane, septa and points of hyphal cell fusion. Plants infected with ΔsymB and ΔsymC strains were severely stunted. Hyphae of the mutants colonized vascular bundles, were more abundant than wild type in the intercellular spaces and formed intra-hyphal hyphae. Although these phenotypes are identical to those previously observed for cell wall integrity MAP kinase mutants no difference was observed in the basal level of MpkA phosphorylation or its cellular localization in the mutant backgrounds. Both genes contain binding sites for the transcription factor ProA. Collectively these results show that SymB and SymC are key components of a conserved signaling network for E. festucae to maintain a mutualistic symbiotic interaction within L. perenne. © 2016 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests.

Science.gov (United States)

Boddupally, Dayakar; Tamirisa, Srinath; Gundra, Sivakrishna Rao; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2018-05-31

To evolve rice varieties resistant to different groups of insect pests a fusion gene, comprising DI and DII domains of Bt Cry1Ac and carbohydrate binding domain of garlic lectin (ASAL), was constructed. Transgenic rice lines were generated and evaluated to assess the efficacy of Cry1Ac::ASAL fusion protein against three major pests, viz., yellow stem borer (YSB), leaf folder (LF) and brown planthopper (BPH). Molecular analyses of transgenic plants revealed stable integration and expression of the fusion gene. In planta insect bioassays on transgenics disclosed enhanced levels of resistance compared to the control plants. High insect mortality of YSB, LF and BPH was observed on transgenics compared to that of control plants. Furthermore, honeydew assays revealed significant decreases in the feeding ability of BPH on transgenic plants as compared to the controls. Ligand blot analysis, using BPH insects fed on cry1Ac::asal transgenic rice plants, revealed a modified receptor protein-binding pattern owing to its ability to bind to additional receptors in insects. The overall results authenticate that Cry1Ac::ASAL protein is endowed with remarkable entomotoxic effects against major lepidopteran and hemipteran insects. As such, the fusion gene appears promising and can be introduced into various other crops to control multiple insect pests.

Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

Science.gov (United States)

Komorek, Jessica; Kuppuswamy, Mohan; Subramanian, T; Vijayalingam, S; Lomonosova, Elena; Zhao, Ling-Jun; Mymryk, Joe S; Schmitt, Kimberly; Chinnadurai, G

2010-03-01

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

Modular organization of proteins containing C1q-like globular domain.

Science.gov (United States)

Kishore, U; Reid, K B

1999-05-01

The first step in the activation of the classical pathway of complement cascade by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of immunoglobulin G (IgG) or immunoglobulin M (IgM). The globular heads of C1q are located C-terminal to the six triple-helical stalks present in the molecule, each head is considered to be composed of the C-terminal halves (3 x 135 residues) of one A-, one B- and one C-chain. It is not known if the C-terminal globular regions, present in each of the three types of chains, are independently folded modules (with each chain having distinct binding properties towards immunoglobulins) or whether the different binding functions of C1q are dependent upon a globular structure which relies on contributions from all three chains. Recent reports of recombinant production and characterisation of soluble globular head regions of all the three chains indicate that the globular regions of C1q may adopt a modular organization, i.e., each globular head of C1q may be composed of three, structurally and functionally, independent domains, thus retaining multivalency in the form of a heterotrimer. Modules of the same type as the C1q C-terminal module are also found in a variety of noncomplement proteins that include the C-terminal regions of the human type VIII and type X collagens, precerebellin, the chipmunk hibernation proteins, the human endothelial cell protein, multimerin, the serum protein, Acrp-30 which is secreted from mouse adipocytes, and the sunfish inner-ear specific structural protein. The C1q molecule is the only one of these proteins for which, to date, a function has been ascribed to the module. The existence of a shared structural region between C1q and certain collagens may suggest an evolutionarily common ancestral precursor. Various structural and biochemical data suggest that these modules may be responsible for multimerisation through patches of aromatic residues within them.

Design of Recombinant Stem Cell Factor macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

Science.gov (United States)

Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan

2005-05-01

Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

Eradication of Human Hepatic and Pulmonary Melanoma Metastases in SCID Mice by Antibody--Interleukin 2 Fusion Proteins

Science.gov (United States)

Becker, Jurgen C.; Pancook, James D.; Gillies, Stephen D.; Mendelsohn, John; Reisfeld, Ralph A.

1996-04-01

Antibody--cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody--interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cγ 1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumorspecific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumorspecific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating.

Science.gov (United States)

Rogers, Jason V; Arlow, Tim; Inkellis, Elizabeth R; Koo, Timothy S; Rose, Mark D

2013-12-01

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.

Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

Directory of Open Access Journals (Sweden)

Balart Luis A

2010-10-01

Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

Functional analysis of a novel KRAB/C2H2 zinc finger protein Mipu1

International Nuclear Information System (INIS)

Jiang, Lei; Tang, Daolin; Wang, Kangkai; Zhang, Huali; Yuan, Can; Duan, Dayue; Xiao, Xianzhong

2007-01-01

A novel rat gene, Mipu1, encodes a 608 amino acid protein with an amino-terminal KRAB domain and 14 carboxyl-terminal C 2 H 2 zinc finger motifs. Mipu1 is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region. Using the GST-Mipu1 bound to glutathione-Sepharose beads, a consensus putative DNA binding site (5'-TGTCTTATCGAA-3') was extracted from a random oligonucleotide library. EMSA and target detection assay showed that the probe containing the putative site can bind to purified GST-Mipu1 fusion protein. The oligonucleotide containing the putative site was inserted into the pGL3-promotor vector to produce a reporter construct. The expression of reporter gene was repressed by overexpression of Mipu1 in a dose-dependent manner. Mutation analysis of the consensus sequence indicated that the repression mediated by Mipu1 is sequence-dependent. These results suggest that Mipu1 is a nuclear protein, which functions as a transcriptional repressor

Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication.

Directory of Open Access Journals (Sweden)

Hussin A Rothan

Full Text Available Dengue virus (DENV broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1 and plectasin (PLSN were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro with half-maximal inhibitory concentration (IC50 0.5±0.1 μM. The real-time proliferation assay (RTCA and the end-point proliferation assay (MTT assay showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.

Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

International Nuclear Information System (INIS)

Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

2012-01-01

Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin domain III (R-III) and albumin domain I -RBP-albumin III (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

Science.gov (United States)

Grembecka, Jolanta; He, Shihan; Shi, Aibin; Purohit, Trupta; Muntean, Andrew G; Sorenson, Roderick J; Showalter, Hollis D; Murai, Marcelo J; Belcher, Amalia M; Hartley, Thomas; Hess, Jay L; Cierpicki, Tomasz

2012-01-29

Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

International Nuclear Information System (INIS)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

2009-01-01

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

Bacteriophage SP6 encodes a second tailspike protein that recognizes Salmonella enterica serogroups C2 and C3

International Nuclear Information System (INIS)

Gebhart, Dana; Williams, Steven R.; Scholl, Dean

2017-01-01

SP6 is a salmonella phage closely related to coliphage K1-5. K1-5 is notable in that it encodes two polysaccharide-degrading tailspike proteins, an endosialidase that allows it to infect E. coli K1, and a lyase that enables it to infect K5 strains. SP6 is similar to K1-5 except that it encodes a P22-like endorhamnosidase tailspike, gp46, allowing it to infect group B Salmonella. We show here that SP6 can also infect Salmonella serogroups C 2 and C 3 and that a mutation in a putative second tailspike, gp47, eliminates this specificity. Gene 47 was fused to the coding region of the N-terminal portion of the Pseudomonas aeruginosa R2 pyocin tail fiber and expressed in trans such that the fusion protein becomes incorporated into pyocin particles. These pyocins, termed AvR2-SP47, killed serogroups C 2 and C 3 Salmonella. We conclude that SP6 encodes two tail proteins providing it a broad host range among Salmonella enterica. - Highlights: • SP6 is a “dual specificity” bacteriophage that encodes two different receptor binding proteins giving it a broad host range. • These receptor binding proteins can be used to re-target the spectrum of R-type bacteriocins to Salmonella enterica. • Both SP6 and the engineered R-type bacteriocins can kill the Salmonella serovars most associated with human disease making them attractive for development as antimicrobial agents.

Expression, Purification and Characterization of GMZ2'.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite

NARCIS (Netherlands)

Mistarz, U.H.; Singh, S.K; Nguyen, T.; Roeffen, W.; Lissau, C.; Madsen, S.M.; Vrang, A.; Tiendrebeogo, R.W.; Kana, I.H.; Sauerwein, R.W.; Theisen, M.; Rand, K.D.

2017-01-01

PURPOSE: Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a

Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites

International Nuclear Information System (INIS)

Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

2009-01-01

Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection

FENDL/C-1.0. Charged-particle reaction data library for fusion applications Version 1.0 of November 1991. Summary documentation

International Nuclear Information System (INIS)

Pashchenko, A.B.

1995-01-01

This document describes the FENDL/C-1.0 charged-particle reaction data library which is a sublibrary of FENDL, the evaluated nuclear data library for fusion applications. This file contains evaluated data in ENDF-6 format for the D(d,n), D(d,p), T(d,n), T(t,2n), He-3(d,p) reactions. The processed information, i.e. Maxwellian-averaged reaction rates, and related quantities, calculated from reaction cross-sections, are also included. The data are available from the Nuclear Data Section online via INTERNET by FTP command, or on magnetic tape upon request. (author). 1 tab

Cellulose binding domain fusion proteins

Science.gov (United States)

Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

1998-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin.

Directory of Open Access Journals (Sweden)

Gry Aune Westergaard Hansen

Full Text Available Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted with both proteins with an estimated dissociation constant at 1 μM for reticulocalbin and 150 nM for calumenin. The interaction, at least for calumenin, was dependent upon the presence of Ca2+ with strong interaction at 3.5 mM while no detectable interaction could be found at 0.1 mM. Grp75 binds with an affinity of approximately 3-7 nM with reticulocalbin as well as with calumenin. These interactions suggest functional participation of the CREC proteins in chaperone activity, cell proliferation and transformation, cellular aging, haemostasis and thrombosis as well as modulation of the complement system in fighting bacterial infection.

c-Jun N-terminal kinase mediates AML1-ETO protein-induced connexin-43 expression

International Nuclear Information System (INIS)

Gao Fenghou; Wang Qiong; Wu Yingli; Li Xi; Zhao Kewen; Chen Guoqiang

2007-01-01

AML1-ETO fusion protein, a product of leukemia-related chromosomal translocation t(8;21), was reported to upregulate expression of connexin-43 (Cx43), a member of gap junction-constituted connexin family. However, its mechanism(s) remains unclear. By bioinformatic analysis, here we showed that there are two putative AML1-binding consensus sequences followed by two activated protein (AP)1 sites in the 5'-flanking region upstream to Cx43 gene. AML1-ETO could directly bind to these two AML1-binding sites in electrophoretic mobility shift assay, but luciferase reporter assay revealed that the AML1 binding sites were not indispensable for Cx43 induction by AML1-ETO protein. Conversely, AP1 sites exerted an important role in this event. In agreement, AML1-ETO overexpression in leukemic U937 cells activated c-Jun N-terminal kinase (JNK), while its specific inhibitor SP600125 effectively abrogated AML1-ETO-induced Cx43 expression, indicating that JNK signaling pathway contributes to AML1-ETO induced Cx43 expression. These results would shed new insights for understanding mechanisms of AML1-ETO-associated leukemogenesis

Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

Energy Technology Data Exchange (ETDEWEB)

Choi, Soyoung; Park, Sangeun; Kim, Suhyun [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of); Lim, Chaeseung [Department of Laboratory Medicine, Korea University Guro Hospital, Seoul 152-703 (Korea, Republic of); Kim, Jungho [Department of Life Science, Sogang University, Seoul 121-742 (Korea, Republic of); Cha, Dae Ryong [Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Gyeonggi do 425-020 (Korea, Republic of); Oh, Junseo, E-mail: ohjs@korea.ac.kr [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of)

2012-02-03

Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Directory of Open Access Journals (Sweden)

A.T. Da Poian

2005-06-01

Full Text Available Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Syntaxin binding protein 1 is not required for allergic inflammation via IgE-mediated mast cell activation.

Directory of Open Access Journals (Sweden)

Zhengli Wu

Full Text Available Mast cells play a central role in both innate and acquired immunity. When activated by IgE-dependent FcεRI cross-linking, mast cells rapidly initiate a signaling cascade and undergo an extensive release of their granule contents, including inflammatory mediators. Some SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor proteins and SM (Sec1/Munc18 family proteins are involved in mast cell degranulation. However, the function of syntaxin binding protein 1 (STXBP1, a member of SM family, in mast cell degranulation is currently unknown. In this study, we examined the role of STXBP1 in IgE-dependent mast cell activation. Liver-derived mast cells (LMCs from wild-type and STXBP1-deficient mice were cultured in vitro for the study of mast cell maturation, degranulation, cytokine and chemokine production, as well as MAPK, IκB-NFκB, and NFAT signaling pathways. In addition, in vivo models of passive cutaneous anaphylaxis and late-phase IgE-dependent inflammation were conducted in mast cell deficient W(sh mice that had been reconstituted with wild-type or STXBP1-deficient mast cells. Our findings indicate that STXBP1 is not required for any of these important functional mechanisms in mast cells both in vitro and in vivo. Our results demonstrate that STXBP1 is dispensable during IgE-mediated mast cell activation and in IgE-dependent allergic inflammatory reactions.

TaPP2C1, a Group F2 Protein Phosphatase 2C Gene, Confers Resistance to Salt Stress in Transgenic Tobacco.

Directory of Open Access Journals (Sweden)

Wei Hu

Full Text Available Group A protein phosphatases 2Cs (PP2Cs are essential components of abscisic acid (ABA signaling in Arabidopsis; however, the function of group F2 subfamily PP2Cs is currently less known. In this study, TaPP2C1 which belongs to group F2 was isolated and characterized from wheat. Expression of the TaPP2C1-GFP fusion protein suggested its ubiquitous localization within a cell. TaPP2C1 expression was downregulated by abscisic acid (ABA and NaCl treatments, but upregulated by H2O2 treatment. Overexpression of TaPP2C1 in tobacco resulted in reduced ABA sensitivity and increased salt resistance of transgenic seedlings. Additionally, physiological analyses showed that improved resistance to salt stress conferred by TaPP2C1 is due to the reduced reactive oxygen species (ROS accumulation, the improved antioxidant system, and the increased transcription of genes in the ABA-independent pathway. Finally, transgenic tobacco showed increased resistance to oxidative stress by maintaining a more effective antioxidant system. Taken together, these results demonstrated that TaPP2C1 negatively regulates ABA signaling, but positively regulates salt resistance. TaPP2C1 confers salt resistance through activating the antioxidant system and ABA-independent gene transcription process.

HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation

Energy Technology Data Exchange (ETDEWEB)

Tan, Chye Ling [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Gunaratne, Jayantha [Mass Spectrometry and Systems Biology Laboratory, Institute of Molecular and Cell Biology, A-STAR, Biopolis, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); Lai, Deborah [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Carthagena, Laetitia [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Wang, Qian [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Xue, Yue Zhen; Quek, Ling Shih [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Doorbar, John [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Bachelerie, Francoise [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Thierry, Francoise, E-mail: francoise.thierry@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Bellanger, Sophie, E-mail: sophie.bellanger@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)

2012-07-20

The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

Application of SGT1-Hsp90 chaperone complex for soluble expression of NOD1 LRR domain in E. coli

International Nuclear Information System (INIS)

Hong, Tae-Joon; Hahn, Ji-Sook

2016-01-01

NOD1 is an intracellular sensor of innate immunity which is related to a number of inflammatory diseases. NOD1 is known to be difficult to express and purify for structural and biochemical studies. Based on the fact that Hsp90 and its cochaperone SGT1 are necessary for the stabilization and activation of NOD1 in mammals, SGT1 was chosen as a fusion partner of the leucine-rich repeat (LRR) domain of NOD1 for its soluble expression in Escherichia coli. Fusion of human SGT1 (hSGT1) to NOD1 LRR significantly enhanced the solubility, and the fusion protein was stabilized by coexpression of mouse Hsp90α. The expression level of hSGT1-NOD1 LRR was further enhanced by supplementation of rare codon tRNAs and exchange of antibiotic marker genes. - Highlights: • The NOD1 LRR domain was solubilized by SGT1 fusion in E. coli. • The coexpression of HSP90 stabilized the SGT1-NOD1 LRR fusion protein. • Several optimizations could enhance the expression level of the fusion protein.

Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis

International Nuclear Information System (INIS)

Jiang, Wen-guo; Zhen, Yong-su; Lu, Xin-an; Shang, Bo-yang; Fu, Yan; Zhang, Sheng-hua; Zhou, Daifu; Li, Liang; Li, Yi; Luo, Yongzhang

2013-01-01

Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety. In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer. ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice. The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin

Development of a novel fluorescent imaging probe for tumor hypoxia by use of a fusion protein with oxygen-dependent degradation domain of HIF-1α

Science.gov (United States)

Tanaka, Shotaro; Kizaka-Kondoh, Shinae; Harada, Hiroshi; Hiraoka, Masahiro

2007-02-01

More malignant tumors contain more hypoxic regions. In hypoxic tumor cells, expression of a series of hypoxiaresponsive genes related to malignant phenotype such as angiogenesis and metastasis are induced. Hypoxia-inducible factor-1 (HIF-1) is a master transcriptional activator of such genes, and thus imaging of hypoxic tumor cells where HIF-1 is active, is important in cancer therapy. We have been developing PTD-ODD fusion proteins, which contain protein transduction domain (PTD) and the VHL-mediated protein destruction motif in oxygen-dependent degradation (ODD) domain of HIF-1 alpha subunit (HIF-1α). Thus PTD-ODD fusion proteins can be delivered to any tissue in vivo through PTD function and specifically stabilized in hypoxic cells through ODD function. To investigate if PTD-ODD fusion protein can be applied to construct hypoxia-specific imaging probes, we first constructed a fluorescent probe because optical imaging enable us to evaluate a probe easily, quickly and economically in a small animal. We first construct a model fusion porein PTD-ODD-EGFP-Cy5.5 named POEC, which is PTD-ODD protein fused with EGFP for in vitro imaging and stabilization of fusion protein, and conjugated with a near-infrared dye Cy5.5. This probe is designed to be degraded in normoxic cells through the function of ODD domain and followed by quick clearance of free fluorescent dye. On the other hand, this prove is stabilized in hypoxic tumor cells and thus the dye is stayed in the cells. Between normoxic and hypoxic conditions, the difference in the clearance rate of the dye will reveals suited contrast for tumor-hypoxia imaging. The optical imaging probe has not been optimized yet but the results presented here exhibit a potential of PTD-ODD fusion protein as a hypoxia-specific imaging probe.

Association of papillomavirus E6 proteins with either MAML1 or E6AP clusters E6 proteins by structure, function, and evolutionary relatedness.

Directory of Open Access Journals (Sweden)

Nicole Brimer

2017-12-01

Full Text Available Papillomavirus E6 proteins bind to LXXLL peptide motifs displayed on targeted cellular proteins. Alpha genus HPV E6 proteins associate with the cellular ubiquitin ligase E6AP (UBE3A, by binding to an LXXLL peptide (ELTLQELLGEE displayed by E6AP, thereby stimulating E6AP ubiquitin ligase activity. Beta, Gamma, and Delta genera E6 proteins bind a similar LXXLL peptide (WMSDLDDLLGS on the cellular transcriptional co-activator MAML1 and thereby repress Notch signaling. We expressed 45 different animal and human E6 proteins from diverse papillomavirus genera to ascertain the overall preference of E6 proteins for E6AP or MAML1. E6 proteins from all HPV genera except Alpha preferentially interacted with MAML1 over E6AP. Among animal papillomaviruses, E6 proteins from certain ungulate (SsPV1 from pigs and cetacean (porpoises and dolphins hosts functionally resembled Alpha genus HPV by binding and targeting the degradation of E6AP. Beta genus HPV E6 proteins functionally clustered with Delta, Pi, Tau, Gamma, Chi, Mu, Lambda, Iota, Dyokappa, Rho, and Dyolambda E6 proteins to bind and repress MAML1. None of the tested E6 proteins physically and functionally interacted with both MAML1 and E6AP, indicating an evolutionary split. Further, interaction of an E6 protein was insufficient to activate degradation of E6AP, indicating that E6 proteins that target E6AP co-evolved to separately acquire both binding and triggering of ubiquitin ligase activation. E6 proteins with similar biological function clustered together in phylogenetic trees and shared structural features. This suggests that the divergence of E6 proteins from either MAML1 or E6AP binding preference is a major event in papillomavirus evolution.

Dicty_cDB: Contig-U14112-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available Oryza sativa Japonica Group genom... 77 8e-14 FJ787374_1( FJ787374 |pid:none) Nicotiana repanda protein kin..._1( FJ787369 |pid:none) Nicotiana repanda protein kinase-c... 79 1e-13 AC004260_13( AC004260 |pid:none) Arab...8... 72 2e-12 FJ787371_1( FJ787371 |pid:none) Nicotiana repanda protein kinase-c... 75 2e-12 FB875947_1( FB8

Function of Nup98 subtypes and their fusion proteins, Nup98-TopIIβ and Nup98-SETBP1 in nuclear-cytoplasmic transport.

Science.gov (United States)

Saito, Shoko; Yokokawa, Takafumi; Iizuka, Gemmei; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

2017-05-20

Nup98 is a component of the nuclear pore complex. The nup98-fusion genes derived by chromosome translocations are involved in hematopoietic malignancies. Here, we investigated the functions of Nup98 isoforms and two unexamined Nup98-fusion proteins, Nup98-TopIIβ and Nup98-SETBP1. We first demonstrated that two Nup98 isoforms are expressed in various mouse tissues and similarly localized in the nucleus and the nuclear envelope. We also showed that Nup98-TopIIβ and Nup98-SETBP1 are localized in the nucleus and partially co-localized with full-length Nup98 and a nuclear export receptor XPO1. We demonstrated that Nup98-TopIIβ and Nup98-SETBP1 negatively regulate the XPO1-mediated protein export. Our results will contribute to the understanding of the molecular mechanism by which the Nup98-fusion proteins induce tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

Science.gov (United States)

Abdallah, Cosette; Lejamtel, Charlène; Benzoubir, Nassima; Battaglia, Serena; Sidahmed-Adrar, Nazha; Desterke, Christophe; Lemasson, Matthieu; Rosenberg, Arielle R; Samuel, Didier; Bréchot, Christian; Pflieger, Delphine; Le Naour, François; Bourgeade, Marie-Françoise

2017-08-22

Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

A protein kinase binds the C-terminal domain of the readthrough protein of Turnip yellows virus and regulates virus accumulation

International Nuclear Information System (INIS)

Rodriguez-Medina, Caren; Boissinot, Sylvaine; Chapuis, Sophie; Gereige, Dalya; Rastegar, Maryam; Erdinger, Monique; Revers, Frédéric; Ziegler-Graff, Véronique; Brault, Véronique

2015-01-01

Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT_C_t_e_r) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RT_C_t_e_r. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. - Highlights: • The C-terminal domain of TuYV-RT is required for long-distance movement. • CIPK7 from Arabidopsis interacts with RT_C_t_e_r in yeast and in plants. • CIPK7 overexpression increases virus titer locally but not virus systemic movement. • CIPK7 localizes to plasmodesmata. • CIPK7 could be a defense protein regulating virus export.

Screening effects on 12C+12C fusion reaction

Science.gov (United States)

Koyuncu, F.; Soylu, A.

2018-05-01

One of the important reactions for nucleosynthesis in the carbon burning phase in high-mass stars is the 12C+12C fusion reaction. In this study, we investigate the influences of the nuclear potentials and screening effect on astrophysically interesting 12C+12C fusion reaction observables at sub-barrier energies by using the microscopic α–α double folding cluster (DFC) potential and the proximity potential. In order to model the screening effects on the experimental data, a more general exponential cosine screened Coulomb (MGECSC) potential including Debye and quantum plasma cases has been considered in the calculations for the 12C+12C fusion reaction. In the calculations of the reaction observables, the semi-classical Wentzel-Kramers-Brillouin (WKB) approach and coupled channel (CC) formalism have been used. Moreover, in order to investigate how the potentials between 12C nuclei produce molecular cluster states of 24Mg, the normalized resonant energy states of 24Mg cluster bands have been calculated for the DFC potential. By analyzing the results produced from the fusion of 12C+12C, it is found that taking into account the screening effects in terms of MGECSC is important for explaining the 12C+12C fusion data, and the microscopic DFC potential is better than the proximity potential in explaining the experimental data, also considering that clustering is dominant for the structure of the 24Mg nucleus. Supported by the Turkish Science and Research Council (TÜBİTAK) with (117R015)

Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

Energy Technology Data Exchange (ETDEWEB)

Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

2010-11-19

Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

International Nuclear Information System (INIS)

Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

2010-01-01

Research highlights: → A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. → HCV-3a NS5A increases mature SREBP-1c protein level. → HCV-3a NS5A activates SREBP-1c transcription. → Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. → Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

Impact of fluorescent protein fusions on the bacterial flagellar motor.

Science.gov (United States)

Heo, M; Nord, A L; Chamousset, D; van Rijn, E; Beaumont, H J E; Pedaci, F

2017-10-03

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation- new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.

Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

Science.gov (United States)

Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

2016-07-01

Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824).

Science.gov (United States)

Jochems, Caroline; Tritsch, Sarah R; Pellom, Samuel Troy; Su, Zhen; Soon-Shiong, Patrick; Wong, Hing C; Gulley, James L; Schlom, Jeffrey

2017-09-26

M7824 (MSB0011359C) is a novel first-in-class bifunctional fusion protein consisting of a fully human IgG1 anti-PD-L1 monoclonal antibody (with structural similarities to avelumab) linked to the extracellular domain of two TGFβ receptor 2 (TGFβR2) molecules serving as a TGFβ Trap. Avelumab has demonstrated clinical activity in a range of human cancers and has been approved by the Food and Drug Administration for the therapy of Merkel cell and bladder carcinomas. Preclinical studies have shown this anti-PD-L1 is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). In the studies reported here, it is shown that M7824 is also capable of mediating ADCC of a wide range of human carcinoma cells in vitro , employing natural killer (NK) cells as effectors, albeit not as potent as anti-PD-L1 employing some tumor cells as targets. The addition of the IL-15 superagonist fusion protein complex ALT-803 enhanced the ADCC capacity of both anti-PD-L1 and M7824, and to levels that both agents now demonstrated similar levels of ADCC of tumor cells. TGFβ is a known immunosuppressive entity. Studies reported here show TGFβ1 induced reduction of several NK activation markers as well as reduction of endogenous NK lytic activity and NK-mediated ADCC of tumor cells. These phenomena could be reduced or mitigated, however, by M7824, but not by anti-PD-L1. M7824, but not anti-PD-L1, was also shown to reduce the immunosuppressive activity of regulatory T cells on human CD4 + T-cell proliferation. These studies thus demonstrate the dual functionalities of M7824 and provide the rationale for its further clinical development.

Enhanced SUMOylation of proteins containing a SUMO-interacting motif by SUMO-Ubc9 fusion

International Nuclear Information System (INIS)

Kim, Eui Tae; Kim, Kyeong Kyu; Matunis, Mike J.; Ahn, Jin-Hyun

2009-01-01

Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins.

A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria).

Science.gov (United States)

Formighieri, Cinzia; Melis, Anastasios

2015-11-01

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis

DEFF Research Database (Denmark)

Holst, B; Hastrup, H; Raffetseder, U

2001-01-01

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between ei...

[Prokaryotic expression and immunogenicity analysis of the chimeric HBcAg containing APP beta cleavage site peptide and Aβ(1-15);].

Science.gov (United States)

Feng, Gai-feng; Wang, Jun-yang; Jin, Hui; Wang, Wei-xi; Qian, Yi-hua; Yang, Wei-na; Wang, Quan-ying; Yang, Guang-xiao

2011-11-01

To construct the recombinant prokaryotic expression plasmid pET/c-ABCSP-Aβ(15-c);, and evaluate the immunogenicity of the fusion protein expressed in E.coli. The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The APP beta cleavage site peptide(ABCSP) and Aβ(1-15); gene(ABCSP-Aβ(15);) was amplified by PCR and inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-17-ABCSP-Aβ(15); were connected with HBc88-144, yielding the recombinant gene c-ABCSP-Aβ(15-c);. c-ABCSP-Aβ(15-c); gene was subcloned into pET-28a(+).The fusion protein expressed in transformed E.coli BL21 was induced with IPTG and analyzed by SDS-PAGE. The virus-like particles (VLP) formed by fusion protein was observed with Transmission Electron Microscope (TEM). 4 Kunming (KM) mice received intraperitoneal injection (i.p) of fusion protein VLP. The antibody was detected by indirect ELISA. The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, fusion protein was expressed and mainly existed in the sediment of the bacterial lysate. The expression level was 40% of all the proteins in the sediment. The fusion protein could form VLP. After 5 times of immunization, the titer of anti-ABCSP and anti-Aβantibody in sera of KM mice reached up to 1:5 000 and 1:10 000 respectively, while the anti-HBc antibody was undetectable. Recombinant c-ABCSP-Aβ(15-c); gene can be expressed in E.coli. The expressed protein could form VLP and has a strong immunogenicity. This study lays the foundation for the study of AD genetic engineering vaccine.

Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

International Nuclear Information System (INIS)

Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

1987-01-01

C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of [ 32 P]C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of 32 [P]. Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

Science.gov (United States)

Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

2015-01-01

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

Directory of Open Access Journals (Sweden)

Anna Grazia Recchia

Full Text Available Chronic Myeloid Leukemia (CML is characterized by a balanced translocation juxtaposing the Abelson (ABL and breakpoint cluster region (BCR genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i CML can be properly diagnosed at onset, (ii follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1 when BCR-ABL1IS transcripts are between 1-10%, and (iii rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

International Nuclear Information System (INIS)

Mukai, Atsushi; Hashimoto, Naohiro

2008-01-01

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells

Direct regulation of E-cadherin by targeted histone methylation of TALE-SET fusion protein in cancer cells.

Science.gov (United States)

Cho, Hyun-Soo; Kang, Jeong Gu; Lee, Jae-Hye; Lee, Jeong-Ju; Jeon, Seong Kook; Ko, Jeong-Heon; Kim, Dae-Soo; Park, Kun-Hyang; Kim, Yong-Sam; Kim, Nam-Soon

2015-09-15

TALE-nuclease chimeras (TALENs) can bind to and cleave specific genomic loci and, are used to engineer gene knockouts and additions. Recently, instead of using the FokI domain, epigenetically active domains, such as TET1 and LSD1, have been combined with TAL effector domains to regulate targeted gene expression via DNA and histone demethylation. However, studies of histone methylation in the TALE system have not been performed. Therefore, in this study, we established a novel targeted regulation system with a TAL effector domain and a histone methylation domain. To construct a TALE-methylation fusion protein, we combined a TAL effector domain containing an E-Box region to act as a Snail binding site and the SET domain of EHMT 2 to allow for histone methylation. The constructed TALE-SET module (TSET) repressed the expression of E-cadherin via by increasing H3K9 dimethylation. Moreover, the cells that overexpressed TSET showed increased cell migration and invasion. This is the first phenotype-based study of targeted histone methylation by the TALE module, and this new system can be applied in new cancer therapies to reduce side effects.

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

Directory of Open Access Journals (Sweden)

Hideki Kusunoki

2017-03-01

Full Text Available Hepatitis B virus X protein (HBx is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.

Overproduction, purification and characterization of human interferon alpha2a-human serum albumin fusion protein produced in methilotropic yeast Pichia pastoris

Science.gov (United States)

Ningrum, R. A.; Santoso, A.; Herawati, N.

2017-05-01

Human interferon alpha2a (hIFNα2a) is a therapeutic protein that used in cancer and hepatitis B/C therapy. The main problem of using hIFNα-2a is its short elimination half life due to its low molecular weight. Development of higher molecular weight protein by albumin fusion technology is a rational strategy to solve the problem. In our previous research we constructed an open reading frame (ORF) encoding hIFNα2a-human serum albumin (HSA) fusion protein that expressed in Pichia pastoris (P. pastoris) protease deficient strain SMD1168. This research was performed to overproduce, purify and characterize the fusion protein. To overproduce the protein, cultivation was performed in buffered complex medium containing glyserol (BMGY) for 24 h and protein overproduction was applied in buffered complex medium containing methanol (BMMY) for 48 hours at 30°C. The fusion protein was purified by blue sepharose affinity chromatography. Molecular weight characterization by SDS PAGE corresponds with its theoretical size, 85 kDa. Western blot analysis demonstrated that the fusion protein was recognized by anti hIFNα2 and anti HSA monoclonal antibody as well. Amino acid sequence of the fusion protein was determined by LC MS/MS2 mass spectrometry with trypsin as proteolitic enzyme. There were three fragments that identified as hIFNα2a and seven fragments that identified as HSA. Total identified amino acids were 150 residues with 20% coverage from total residues. To conclude, hIFNα2a-HSA fusion protein was overproduced, purified and characterized. Characterization based on molecular weight, antibody recognition and amino acid sequence confirmed that the fusion protein has correct identity as theoretically thought.

Overproduction, purification and characterization of human interferon alpha2a-human serum albumin fusion protein produced in methilotropic yeast Pichia pastoris

International Nuclear Information System (INIS)

Ningrum, R A; Santoso, A; Herawati, N

2017-01-01

Human interferon alpha2a (hIFNα2a) is a therapeutic protein that used in cancer and hepatitis B/C therapy. The main problem of using hIFNα-2a is its short elimination half life due to its low molecular weight. Development of higher molecular weight protein by albumin fusion technology is a rational strategy to solve the problem. In our previous research we constructed an open reading frame (ORF) encoding hIFNα2a-human serum albumin (HSA) fusion protein that expressed in Pichia pastoris (P. pastoris) protease deficient strain SMD1168. This research was performed to overproduce, purify and characterize the fusion protein. To overproduce the protein, cultivation was performed in buffered complex medium containing glyserol (BMGY) for 24 h and protein overproduction was applied in buffered complex medium containing methanol (BMMY) for 48 hours at 30°C. The fusion protein was purified by blue sepharose affinity chromatography. Molecular weight characterization by SDS PAGE corresponds with its theoretical size, 85 kDa. Western blot analysis demonstrated that the fusion protein was recognized by anti hIFNα2 and anti HSA monoclonal antibody as well. Amino acid sequence of the fusion protein was determined by LC MS/MS2 mass spectrometry with trypsin as proteolitic enzyme. There were three fragments that identified as hIFNα2a and seven fragments that identified as HSA. Total identified amino acids were 150 residues with 20% coverage from total residues. To conclude, hIFNα2a-HSA fusion protein was overproduced, purified and characterized. Characterization based on molecular weight, antibody recognition and amino acid sequence confirmed that the fusion protein has correct identity as theoretically thought. (paper)

cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane Fusion Sites.

Science.gov (United States)

Xu, Shuting; Ducroux, Aurélie; Ponnurangam, Aparna; Vieyres, Gabrielle; Franz, Sergej; Müsken, Mathias; Zillinger, Thomas; Malassa, Angelina; Ewald, Ellen; Hornung, Veit; Barchet, Winfried; Häussler, Susanne; Pietschmann, Thomas; Goffinet, Christine

2016-10-12

Upon sensing cytoplasmic retroviral DNA in infected cells, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide cGAMP, which activates STING to trigger a type I interferon (IFN) response. We find that membrane fusion-inducing contact between donor cells expressing the HIV envelope (Env) and primary macrophages endogenously expressing the HIV receptor CD4 and coreceptor enable intercellular transfer of cGAMP. This cGAMP exchange results in STING-dependent antiviral IFN responses in target macrophages and protection from HIV infection. Furthermore, under conditions allowing cell-to-cell transmission of HIV-1, infected primary T cells, but not cell-free virions, deliver cGAMP to autologous macrophages through HIV-1 Env and CD4/coreceptor-mediated membrane fusion sites and induce a STING-dependent, but cGAS-independent, IFN response in target cells. Collectively, these findings identify an infection-specific mode of horizontal transfer of cGAMP between primary immune cells that may boost antiviral responses, particularly in infected tissues in which cell-to-cell transmission of virions exceeds cell-free infection. Copyright © 2016 Elsevier Inc. All rights reserved.

The interaction of mammalian Class C Vps with nSec-1/Munc18-a and syntaxin 1A regulates pre-synaptic release

International Nuclear Information System (INIS)

Kim, Bong Yoon; Sahara, Yoshinori; Yamamoto, Akitsugu; Kominami, Eiki; Kohsaka, Shinichi; Akazawa, Chihiro

2006-01-01

Membrane docking and fusion in neurons is a highly regulated process requiring the participation of a large number of SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and SNARE-interacting proteins. We found that mammalian Class C Vps protein complex associated specifically with nSec-1/Munc18-a, and syntaxin 1A both in vivo and in vitro. In contrast, VAMP2 and SNAP-25, other neuronal core complex proteins, did not interact. When co-transfected with the human growth hormone (hGH) reporter gene, mammalian Class C Vps proteins enhanced Ca 2+ -dependent exocytosis, which was abolished by the Ca 2+ -channel blocker nifedipine. In hippocampal primary cultures, the lentivirus-mediated overexpression of hVps18 increased asynchronous spontaneous synaptic release without changing mEPSCs. These results indicate that mammalian Class C Vps proteins are involved in the regulation of membrane docking and fusion through an interaction with neuronal specific SNARE molecules, nSec-1/Munc18-a and syntaxin 1A

Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Science.gov (United States)

Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

2016-01-01

Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

Directory of Open Access Journals (Sweden)

Perera Rajika L

2004-12-01

Full Text Available Abstract Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44, kinases (EGFR-cytoplasmic domain, CDK2 and 4, proteases (MMP1, CASP2, signal transduction proteins (GRB2, RAF1, HRAS and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX. Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY, or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx and maltose binding protein (MBP were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases

Impact of fluorescent protein fusions on the bacterial flagellar motor

NARCIS (Netherlands)

Heo, M.; Nord, A. L.; Chamousset, D.; van Rijn, E.; Beaumont, H.J.E.; Pedaci, F.

2017-01-01

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side

Dicty_cDB: Contig-U14908-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available id:none) Oryza sativa (japonica cultivar... 86 2e-15 FJ787361_1( FJ787361 |pid:none) Nicotiana repanda prote...|pid:none) Oryza sativa (japonica cultivar-gr... 84 8e-15 FJ787374_1( FJ787374 |pid:none) Nicotiana repanda ...15 FJ787369_1( FJ787369 |pid:none) Nicotiana repanda protein kinase-c... 84 8e-15 EU722820_1( EU722820 |pid:... 1e-14 AB016885_14( AB016885 |pid:none) Arabidopsis thaliana genomic DNA,... 83 1e-14 FJ787371_1( FJ787371 |pid:none) Nicotiana repan...da protein kinase-c... 83 1e-14 A84518( A84518 ) probable receptor-like protein kin

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

International Nuclear Information System (INIS)

Waldron, Richard T.; Whitelegge, Julian P.; Faull, Kym F.; Rozengurt, Enrique

2007-01-01

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic 32 P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

International Nuclear Information System (INIS)

Noisakran, Sansanee; Sengsai, Suchada; Thongboonkerd, Visith; Kanlaya, Rattiyaporn; Sinchaikul, Supachok; Chen, Shui-Tein; Puttikhunt, Chunya

2008-01-01

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells

Viral membrane fusion

International Nuclear Information System (INIS)

Harrison, Stephen C.

2015-01-01

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism

Viral membrane fusion

Energy Technology Data Exchange (ETDEWEB)

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

Bacteriophage SP6 encodes a second tailspike protein that recognizes Salmonella enterica serogroups C{sub 2} and C{sub 3}

Energy Technology Data Exchange (ETDEWEB)

Gebhart, Dana; Williams, Steven R.; Scholl, Dean, E-mail: dean@avidbiotics.com

2017-07-15

SP6 is a salmonella phage closely related to coliphage K1-5. K1-5 is notable in that it encodes two polysaccharide-degrading tailspike proteins, an endosialidase that allows it to infect E. coli K1, and a lyase that enables it to infect K5 strains. SP6 is similar to K1-5 except that it encodes a P22-like endorhamnosidase tailspike, gp46, allowing it to infect group B Salmonella. We show here that SP6 can also infect Salmonella serogroups C{sub 2} and C{sub 3} and that a mutation in a putative second tailspike, gp47, eliminates this specificity. Gene 47 was fused to the coding region of the N-terminal portion of the Pseudomonas aeruginosa R2 pyocin tail fiber and expressed in trans such that the fusion protein becomes incorporated into pyocin particles. These pyocins, termed AvR2-SP47, killed serogroups C{sub 2} and C{sub 3}Salmonella. We conclude that SP6 encodes two tail proteins providing it a broad host range among Salmonella enterica. - Highlights: • SP6 is a “dual specificity” bacteriophage that encodes two different receptor binding proteins giving it a broad host range. • These receptor binding proteins can be used to re-target the spectrum of R-type bacteriocins to Salmonella enterica. • Both SP6 and the engineered R-type bacteriocins can kill the Salmonella serovars most associated with human disease making them attractive for development as antimicrobial agents.

Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae

Directory of Open Access Journals (Sweden)

André L.F. Souza

2008-01-01

Full Text Available In prokaryotes molybdenum is taken up by a high-affinity ABC-type transporter system encoded by the modABC genes. The endophyte β-Proteobacterium Herbaspirillum seropedicae has two modABC gene clusters and two genes encoding putative Mo-dependent regulator proteins (ModE1 and ModE2. Analysis of the amino acid sequence of the ModE1 protein of H. seropedicae revealed the presence of an N-terminal domain containing a DNA-binding helix-turn-helix motif (HTH and a C-terminal domain with a molybdate-binding motif. The second putative regulator protein, ModE2, contains only the helix-turn-helix motif, similar to that observed in some sequenced genomes. We cloned the modE1 (810 bp and modE2 (372 bp genes and expressed them in Escherichia coli as His-tagged fusion proteins, which we subsequently purified. The over-expressed recombinant His-ModE1 was insoluble and was purified after solubilization with urea and then on-column refolded during affinity chromatography. The His-ModE2 was expressed as a soluble protein and purified by affinity chromatography. These purified proteins were analyzed by DNA band-shift assays using the modA2 promoter region as probe. Our results indicate that His-ModE1 and His-ModE2 are able to bind to the modA2 promoter region, suggesting that both proteins may play a role in the regulation of molybdenum uptake and metabolism in H. seropedicae.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of hepatitis B virus core fusion protein corresponding to octahedral particles

International Nuclear Information System (INIS)

Kikuchi, Masaki; Iwabuchi, Shinichiro; Kikkou, Tatsuhiko; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi; Kawata, Masaaki; Sato, Chikara; Matsumoto, Osamu

2013-01-01

Novel hepatitis B virus-like particles of recombinant dimeric core–GFP fusion protein were expressed, purified and crystallized. The crystals diffracted to 2.15 Å resolution and belonged to space group F432, with unit-cell parameters a = b = c = 219.7 Å. Recombinant hepatitis B virus core proteins dimerize to form building blocks that are capable of self-assembly into a capsid. A core capsid protein dimer (CPD) linked to a green fluorescent protein variant, EGFP, at the C-terminus has been designed. The recombinant fusion CPD was expressed in Escherichia coli, assembled into virus-like particles (VLPs), purified and crystallized. The single crystal diffracted to 2.15 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 219.7 Å. The fusion proteins assembled into icosahedral VLPs in aqueous solution, but were rearranged into octahedral symmetry through the crystal-packing process under the crystallization conditions

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

Science.gov (United States)

Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

1995-04-04

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

Influence of rhTPO/GM-CSF fusion protein on hemopoiesis in mice irradiated with 60Co γ-rays

International Nuclear Information System (INIS)

Cao Hua; Ge Zhongliang; Zhang Qunwei; Liu Xiuzhen

1999-01-01

Objective: To find a new biological therapy for secondary hematopoietic failure including anemia, infection and hemorrhage after administration of chemotherapeutic drugs etc. Methods: hGM-CSF gene was ligated with hTPO gene isolated from human fetal liver mRNA and a new fusion protein rh TPO/GM-CSF obtained. Results: The new fusion protein could promote recovery of peripheral WBC and PLT of 5.0 Gy irradiated mice. BFU-E, CFU-Meg and CFU-GM in bone marrow of mice after irradiation recovered significantly by treatment with rhTPO/GM-CSF fusion protein for 10 days. Conclusion: These results suggest that the new fusion protein has the biological activity of both hTPO and hGM-CSF simultaneously and can stimulate the proliferation of megakaryocytes and granulocyte progenitors

Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

Science.gov (United States)

Salehi, Nasrin; Peng, Ching-An

2016-07-08

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

Fusion Technology 1996. Proceedings. Volume 1 and 2

International Nuclear Information System (INIS)

Varandas, C.; Serra, F.

1997-01-01

The objective of these proceedings was to provide a platform for the exchange of information on the design, construction and operation of fusion experiments. The technology which is being developed for the next step devices and fusion reactors was also covered. Sections in volume 1 concern (A) first wall, divertors and vacuum systems; (B) plasma heating and control; (C) plasma engineering and control; and (D) experimental systems. The sections in volume 2 deal with (E) magnet and related power supplies; (F) fuel cycle and tritium processing systems; (G) blanket technology/materials; (H) assembly, remote handling and waste management and storage; and (I) safety and environment, and reactor studies

A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

Science.gov (United States)

Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

2018-01-01

In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

Science.gov (United States)

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Feature Fusion Based Road Extraction for HJ-1-C SAR Image

Directory of Open Access Journals (Sweden)

Lu Ping-ping

2014-06-01

Full Text Available Road network extraction in SAR images is one of the key tasks of military and civilian technologies. To solve the issues of road extraction of HJ-1-C SAR images, a road extraction algorithm is proposed based on the integration of ratio and directional information. Due to the characteristic narrow dynamic range and low signal to noise ratio of HJ-1-C SAR images, a nonlinear quantization and an image filtering method based on a multi-scale autoregressive model are proposed here. A road extraction algorithm based on information fusion, which considers ratio and direction information, is also proposed. By processing Radon transformation, main road directions can be extracted. Cross interferences can be suppressed, and the road continuity can then be improved by the main direction alignment and secondary road extraction. The HJ-1-C SAR image acquired in Wuhan, China was used to evaluate the proposed method. The experimental results show good performance with correctness (80.5% and quality (70.1% when applied to a SAR image with complex content.

An Alphavirus E2 Membrane-Proximal Domain Promotes Envelope Protein Lateral Interactions and Virus Budding

Directory of Open Access Journals (Sweden)

Emily A. Byrd

2017-11-01

Full Text Available Alphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses. The virus membrane is covered by a lattice composed of 80 spikes, each a trimer of heterodimers of the E2 and E1 transmembrane proteins. During virus endocytic entry, the E1 glycoprotein mediates the low-pH-dependent fusion of the virus membrane with the endosome membrane, thus initiating virus infection. While much is known about E1 structural rearrangements during membrane fusion, it is unclear how the E1/E2 dimer dissociates, a step required for the fusion reaction. A recent Alphavirus cryo-electron microscopy reconstruction revealed a previously unidentified D subdomain in the E2 ectodomain, close to the virus membrane. A loop within this region, here referred to as the D-loop, contains two highly conserved histidines, H348 and H352, which were hypothesized to play a role in dimer dissociation. We generated Semliki Forest virus mutants containing the single and double alanine substitutions H348A, H352A, and H348/352A. The three D-loop mutations caused a reduction in virus growth ranging from 1.6 to 2 log but did not significantly affect structural protein biosynthesis or transport, dimer stability, virus fusion, or specific infectivity. Instead, growth reduction was due to inhibition of a late stage of virus assembly at the plasma membrane. The virus particles that are produced show reduced thermostability compared to the wild type. We propose the E2 D-loop as a key region in establishing the E1-E2 contacts that drive glycoprotein lattice formation and promote Alphavirus budding from the plasma membrane.

Automatic Registration Method for Fusion of ZY-1-02C Satellite Images

Directory of Open Access Journals (Sweden)

Qi Chen

2013-12-01

Full Text Available Automatic image registration (AIR has been widely studied in the fields of medical imaging, computer vision, and remote sensing. In various cases, such as image fusion, high registration accuracy should be achieved to meet application requirements. For satellite images, the large image size and unstable positioning accuracy resulting from the limited manufacturing technology of charge-coupled device, focal plane distortion, and unrecorded spacecraft jitter lead to difficulty in obtaining agreeable corresponding points for registration using only area-based matching or feature-based matching. In this situation, a coarse-to-fine matching strategy integrating two types of algorithms is proven feasible and effective. In this paper, an AIR method for application to the fusion of ZY-1-02C satellite imagery is proposed. First, the images are geometrically corrected. Coarse matching, based on scale invariant feature transform, is performed for the subsampled corrected images, and a rough global estimation is made with the matching results. Harris feature points are then extracted, and the coordinates of the corresponding points are calculated according to the global estimation results. Precise matching is conducted, based on normalized cross correlation and least squares matching. As complex image distortion cannot be precisely estimated, a local estimation using the structure of triangulated irregular network is applied to eliminate the false matches. Finally, image resampling is conducted, based on local affine transformation, to achieve high-precision registration. Experiments with ZY-1-02C datasets demonstrate that the accuracy of the proposed method meets the requirements of fusion application, and its efficiency is also suitable for the commercial operation of the automatic satellite data process system.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

International Nuclear Information System (INIS)

Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

2012-01-01

Highlights: ► AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. ► AtPP2CG1 up-regulates the expression of marker genes in different pathways. ► AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2–3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter–GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

Energy Technology Data Exchange (ETDEWEB)

Liu, Xin, E-mail: fangfei6073@126.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhai, Hong, E-mail: Zhai.h@neigaehrb.ac.cn [Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150040 (China); Cai, Hua, E-mail: small-big@sohu.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Ji, Wei, E-mail: iwei_j@hotmail.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Luo, Xiao, E-mail: luoxiao2010@yahoo.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Li, Jing, E-mail: lijing@neau.edu.cn [Plant Secondary Metabolism Laboratory, Northeast Agricultural University, Harbin 150030 (China); Bai, Xi, E-mail: baixi@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China)

2012-06-15

Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

Solution structure of a Plasmodium falciparum AMA-1/MSP 1 chimeric protein vaccine candidate (PfCP-2.9 for malaria

Directory of Open Access Journals (Sweden)

Jin Changwen

2010-03-01

Full Text Available Abstract Background The Plasmodium falciparum chimeric protein PfCP-2.9 is a promising asexual-stage malaria vaccine evaluated in clinical trials. This chimeric protein consists of two cysteine-rich domains: domain III of the apical membrane antigen 1 (AMA-1 [III] and the C-terminal region of the merozoite surface protein 1 (MSP1-19. It has been reported that the fusion of these two antigens enhanced their immunogenicity and antibody-mediated inhibition of parasite growth in vitro. Methods The 15N-labeled and 13C/15N-labeled PfCP-2.9 was produced in Pichia pastoris for nuclear magnetic resonance (NMR structure analysis. The chemical shift assignments of PfCP-2.9 were compared with those previously reported for the individual domains (i.e., PfAMA-1(III or PfMSP 1-19. The two-dimensional spectra and transverse relaxation rates (R2 of the PfMSP1-19 alone were compared with that of the PfCP-2.9. Results Confident backbone assignments were obtained for 122 out of 241 residues of PfCP-2.9. The assigned residues in PfCP-2.9 were very similar to those previously reported for the individual domains. The conformation of the PfMSP1-19 in different constructs is essentially the same. Comparison of transverse relaxation rates (R2 strongly suggests no weak interaction between the domains. Conclusions These data indicate that the fusion of AMA-1(III and MSP1-19 as chimeric protein did not change their structures, supporting the use of the chimeric protein as a potential malaria vaccine.

C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

Science.gov (United States)

Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

2015-09-11

In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells

Directory of Open Access Journals (Sweden)

Liu Chen

2011-09-01

Full Text Available Abstract Interferon Regulatory Factor-3 (IRF-3 plays a central role in the induction of interferon (IFN production and succeeding interferon-stimulated genes (ISG expression en route for restraining hepatitis C virus (HCV infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT in Huh7.5-IRF3ER cells. Expression of IFN-α, IFN-β, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR. Peak expression of IFN-α and IFN-β was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M inhibited HCV internal ribosomal entry site (IRES-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRES-dependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.

Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

Science.gov (United States)

Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

2018-02-01

Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

Localization of poly(3-hydroxybutyrate) (PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16.

Science.gov (United States)

Pfeiffer, Daniel; Jendrossek, Dieter

2012-11-01

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins

Comparative immunoblot analysis with 10 different, partially overlapping recombinant fusion proteins derived from 5 different cytomegalovirus proteins

NARCIS (Netherlands)

van Zanten, J.; LAZZAROTTO, T; CAMPISI, B; VORNHAGEN, R; JAHN, G; LANDINI, MP; The, T. Hauw

Ten fusion proteins derived from five various CMV encoded proteins were used for the detection of specific antibody response by immunoblot technique in sera from renal transplant recipients. The fusion proteins were derived from the following CMV specific proteins: the assembly protein ppUL80a with

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1.

Directory of Open Access Journals (Sweden)

Matthew C Clifton

Full Text Available Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors.

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

International Nuclear Information System (INIS)

Groppo, Rachel; Brown, Bradley A.; Palmenberg, Ann C.

2011-01-01

Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3C pro . Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.

Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

Science.gov (United States)

Desai, Tanay M; Marin, Mariana; Sood, Chetan; Shi, Jiong; Nawaz, Fatima; Aiken, Christopher; Melikyan, Gregory B

2015-10-29

HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

Extensive overproduction of the AdhE protein by rng mutations depends on mutations in the cra gene or in the Cra-box of the adhE promoter.

Science.gov (United States)

Kaga, Naoko; Umitsuki, Genryou; Clark, David P; Nagai, Kazuo; Wachi, Masaaki

2002-07-05

Escherichia coli RNase G encoded by the rng gene is involved in degradation of adhE mRNA. Overproduction of the AdhE protein by rng mutants was found to depend on the genetic background of strains derived from DC272 (adhC81) or MC1061. We found that DC272 carried a point mutation in the Cra-binding site of the adhE promoter. The Cra protein encoded by the cra gene is known to act as a repressor of adhE. P1-phage-mediated transduction and lacZ fusion analysis with the mutant adhE promoter confirmed that this mutation is responsible for overproduction. On the other hand, Southern hybridization revealed that MC1061 had a 0.85-kb deletion of the cra gene. Overproduction of AdhE in the MC1061 background was reversed to the wild-type levels by introduction of a plasmid carrying the cra(+) gene. These results indicated that expression of the adhE gene was regulated transcriptionally by Cra and posttranscriptionally by RNase G. (c) 2002 Elsevier Science (USA).

Dicty_cDB: Contig-U14348-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 71_1( FJ787371 |pid:none) Nicotiana repanda protein kinase-c... 94 3e-18 AC124968_9( AC124968 |pid:none) Med... 5e-17 FJ787374_1( FJ787374 |pid:none) Nicotiana repanda protein kinase-c... 90 5e-17 AE014298_1862( AE01429...08048_1( AY708048 |pid:none) Zea mays salt-inducible putative p... 90 5e-17 FJ787369_1( FJ787369 |pid:none) Nicotiana repanda

In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins

International Nuclear Information System (INIS)

Mack, John; Vermeiren, Christie; Heinrichs, David E.; Stillman, Martin J.

2004-01-01

We report the first characterization of the in vivo porphyrin scavenging abilities of two components of a newly discovered heme scavenging system involving iron-regulated surface determinant (Isd) proteins. These proteins are present within the cell envelope of the Gram-positive human pathogen Staphylococcus aureus. IsdC and IsdE, when expressed heterologously in Escherichia coli, efficiently scavenged intracellular heme and resulted in de novo heme synthesis in excess of 100-fold above background. Magnetic circular dichroism analyses showed that the heme-binding properties of the two proteins differ significantly from one another. IsdC bound almost exclusively free-base protoporphyrin IX, whereas the IsdE protein was associated with low spin Fe(III) and Fe(II) heme. These properties provide important insight into the possible mechanisms of iron scavenging from bound heme by Isd proteins

Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1).

Science.gov (United States)

Ahmad, Muhammad Khairi; Tabana, Yasser M; Ahmed, Mowaffaq Adam; Sandai, Doblin Anak; Mohamed, Rafeezul; Ismail, Ida Shazrina; Zulkiflie, Nurulisa; Yunus, Muhammad Amir

2017-12-01

A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.

Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like-protein polymer.

Science.gov (United States)

Huang, Yan-Shan; Wen, Xiao-Fang; Wu, Yi-Liang; Wang, Ye-Fei; Fan, Min; Yang, Zhi-Yu; Liu, Wei; Zhou, Lin-Fu

2010-03-01

The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications. (c) 2009 Elsevier B.V. All rights reserved.

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

International Nuclear Information System (INIS)

Nordlund, Henri R.; Laitinen, Olli H.; Uotila, Sanna T.H.; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S.

2005-01-01

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.

Science.gov (United States)

Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S

2005-10-14

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

NARCIS (Netherlands)

Pecheur, EI; Martin, [No Value; Bienvenue, A; Ruysschaert, JM; Hoekstra, D

2000-01-01

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or

Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins.

Directory of Open Access Journals (Sweden)

Michelle P Christie

Full Text Available The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.

Site-specific modification of genome with cell-permeable Cre fusion protein in preimplantation mouse embryo

International Nuclear Information System (INIS)

Kim, Kyoungmi; Kim, Hwain; Lee, Daekee

2009-01-01

Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26 tm1Sor , revealed that treatment of 1-cell or 2-cell embryos with 3 μM of Cre fusion protein for 2 h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.

In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF.

Science.gov (United States)

Moldes, Cristina; García, Pedro; García, José L; Prieto, María A

2004-06-01

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.

Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

Science.gov (United States)

Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

2014-07-15

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

Science.gov (United States)

Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

2001-01-01

The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

TcA, the putative transposase of the C. elegans Tc1 transposon, has an N-terminal DNA binding domain.

OpenAIRE

Schukkink, R F; Plasterk, R H

1990-01-01

Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains. In those strains Tc1 insertion is the main cause of spontaneous mutations. The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of Tc1. We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded). A fusion pr...

Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes.

Science.gov (United States)

Zipeto, Donato; Matucci, Andrea; Ripamonti, Chiara; Scarlatti, Gabriella; Rossolillo, Paola; Turci, Marco; Sartoris, Silvia; Tridente, Giuseppe; Bertazzoni, Umberto

2006-05-01

Human immunodeficiency virus-1 (HIV-1) infects cells by membrane fusion that is mediated by the envelope proteins gp120/gp41 and the cellular receptors CD4 and CCR5. During this process, some conserved viral epitopes are temporarily exposed and may induce a neutralizing antibody response when fixed in the fusogenic conformation. These transient structures are conserved and may be effective antigens for use in an anti-HIV-1 vaccine. In this study we tested different conditions of preparation of fusion complexes inducing neutralizing antibodies against both R5 and X4 tropic HIV-1 strains. Cell lines expressing HIV-1 gp120/gp41 and CD4-CCR5 were prepared and conditions for producing fusion complexes were tested. Complexes produced at different temperature and fixative combinations were used to immunize mice. Results indicated that (a) fusion complexes prepared at either 21 degrees C, 30 degrees C or 37 degrees C were immunogenic and induced neutralizing antibodies against both R5 and X4 HIV-1 heterologous isolates; (b) after extensive purification of antibodies there was no cytotoxic effect; (c) complexes prepared at 37 degrees C were more immunogenic and induced higher titers of neutralizing antibodies than complexes prepared at either 21 degrees C or 30 degrees C; (d) the fixative used did not affect the titer of neutralizing antibodies except for glutaraldehyde which was ineffective; (e) the neutralizing activity was retained after CD4-CCR5 antibody removal. The production of higher titers of neutralizing antibody with fusion complexes prepared at 37 degrees C, as compared to lower temperatures, may be related to the induction of antibodies against many different conformation intermediates that subsequently act synergistically at different steps in the fusion process.

Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein

DEFF Research Database (Denmark)

Björnberg, Olof; Østergaard, Henrik; Winther, Jakob R

2006-01-01

Redox-sensitive yellow fluorescent protein (rxYFP) contains a dithiol disulfide pair that is thermodynamically suitable for monitoring intracellular glutathione redox potential. Glutaredoxin 1 (Grx1p) from yeast is known to catalyze the redox equilibrium between rxYFP and glutathione, and here, we...... have generated a fusion of the two proteins, rxYFP-Grx1p. In comparison to isolated subunits, intramolecular transfer of reducing equivalents made the fusion protein kinetically superior in reactions with glutathione. The rate of GSSG oxidation was thus improved by a factor of 3300. The reaction...... separately and in the fusion. This could not be ascribed to the lack of an unproductive side reaction to glutaredoxin disulfide. Instead, slower alkylation kinetics with iodoacetamide indicates a better leaving-group capability of the remaining cysteine residue, which can explain the increased activity....

Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA.

Science.gov (United States)

Wang, Shunfang; Liu, Shuhui

2015-12-19

An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one.

Expression and purification of PprI protein from D.radiodurans R1 in escherichia coli

International Nuclear Information System (INIS)

Zhang Yongqin; Zhou Hui; Chen Jie; Yang Zhanshan

2011-01-01

In order to express and purify PprI protein from D.radiodurans R1 in E. coli, the full length of pprI gene was gained by PCR amplification using pCMV-HA-pprI as a template. The gene segment was inserted into vector pET-28a after digested by two restriction endonucleases Nco I and EcoR I. Then the recombinant vector pET-28a-His-pprI was transfected into E. coli BL21(DE3) RP. The PprI protein expression was induced by IPTG and the fusion protein was confirmed by SDS-PAGE and Western blotting. The expressive conditions of the protein such as E. coli' A 600 , concentration of IPTG, time and temperature of culture, were optimized. Finally the fusion protein was purified by Ni-NTA His Bind Resins and molecule boult. The experimental results show the fusion protein confirmed by Western blotting is 6 x His-PprI and its molecular weight is 37 kDa. The ladders of PprI protein at molecular weight 37 kDa were different due to difference of the PprI protein expression conditions if E. coli. The PprI protein exists both in supernatant and precipitation. The concentration of purified protein is about 0.15 mg/mL which was measured by BCA method. It is concluded that the recombinant plasmid pET-28a-His-pprI is constructed and the PprI fusion protein is expressed and purified. The results lay a solid foundation for studying the radio-resistance and immunity of PprI protein. (authors)

Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.

Science.gov (United States)

Twerdochlib, Adriana L; Chubatsu, Leda S; Souza, Emanuel M; Pedrosa, Fábio O; Steffens, M Berenice R; Yates, M Geoffrey; Rigo, Liu U

2003-07-01

NtrC is a bacterial enhancer-binding protein (EBP) that activates transcription by the sigma54 RNA polymerase holoenzyme. NtrC has a three domain structure typical of EBP family. In Herbaspirillum seropedicae, an endophytic diazotroph, NtrC regulates several operons involved in nitrogen assimilation, including glnAntrBC. In order to over-express and purify the NtrC protein, DNA fragments containing the complete structural gene for the whole protein, and for the N-terminal+Central and Central+C-terminal domains were cloned into expression vectors. The NtrC and NtrC(N-terminal+Central) proteins were over-expressed as His-tag fusion proteins upon IPTG addition, solubilized using N-lauryl-sarcosyl and purified by metal affinity chromatography. The over-expressed His-tag-NtrC(Central+C-terminal) fusion protein was partially soluble and was also purified by affinity chromatography. DNA band-shift assays showed that the NtrC protein and the Central+C-terminal domains bound specifically to the H. seropedicae glnA promoter region. The C-terminal domain is presumably necessary for DNA-protein interaction and DNA-binding does not require a phosphorylated protein.

The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

Directory of Open Access Journals (Sweden)

Chengliang Zhang

Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor.

Science.gov (United States)

Cook, Jonathan D; Soto-Montoya, Hazel; Korpela, Markus K; Lee, Jeffrey E

2015-07-24

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Alcohol binding in the C1 (C1A + C1B) domain of protein kinase C epsilon

Science.gov (United States)

Pany, Satyabrata; Das, Joydip

2015-01-01

Background Alcohol regulates the expression and function of protein kinase C epsilon (PKCε). In a previous study we identified an alcohol binding site in the C1B, one of the twin C1 subdomains of PKCε. Methods In this study, we investigated alcohol binding in the entire C1 domain (combined C1A and C1B) of PKCε. Fluorescent phorbol ester, SAPD and fluorescent diacylglycerol (DAG) analog, dansyl-DAG were used to study the effect of ethanol, butanol, and octanol on the ligand binding using fluorescence resonance energy transfer (FRET). To identify alcohol binding site(s), PKCεC1 was photolabeled with 3-azibutanol and 3-azioctanol, and analyzed by mass spectrometry. The effects of alcohols and the azialcohols on PKCε were studied in NG108-15 cells. Results In the presence of alcohol, SAPD and dansyl-DAG showed different extent of FRET, indicating differential effects of alcohol on the C1A and C1B subdomains. Effects of alcohols and azialcohols on PKCε in NG108-15 cells were comparable. Azialcohols labeled Tyr-176 of C1A and Tyr-250 of C1B. Inspection of the model structure of PKCεC1 reveals that these residues are 40 Å apart from each other indicating that these residues form two different alcohol binding sites. Conclusions The present results provide evidence for the presence of multiple alcohol-binding sites on PKCε and underscore the importance of targeting this PKC isoform in developing alcohol antagonists. PMID:26210390

Aberrant chimeric RNA GOLM1-MAK10 encoding a secreted fusion protein as a molecular signature for human esophageal squamous cell carcinoma

Science.gov (United States)

Zhang, Hao; Lin, Wan; Kannan, Kalpana; Luo, Liming; Li, Jing; Chao, Pei-Wen; Wang, Yan; Chen, Yu-Ping; Gu, Jiang; Yen, Laising