A new fusion protein platform for quantitatively measuring activity of multiple proteases

PubMed Central

2014-01-01

Background Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases. Results We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract. Conclusion The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with

A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation▿

PubMed Central

Gardner, Amanda E.; Dutch, Rebecca E.

2007-01-01

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion. PMID:17507474

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

PubMed

Porotto, Matteo; Devito, Ilaria; Palmer, Samantha G; Jurgens, Eric M; Yee, Jia L; Yokoyama, Christine C; Pessi, Antonello; Moscona, Anne

2011-12-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.

Purification and Crystallization Reveal Two Types of Interactions of the Fusion Protein Homotrimer of Semliki Forest Virus

PubMed Central

Gibbons, Don L.; Reilly, Brigid; Ahn, Anna; Vaney, Marie-Christine; Vigouroux, Armelle; Rey, Felix A.; Kielian, Margaret

2004-01-01

The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein. PMID:15016874

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Liu, E-mail: lyang@u.washington.edu; Medical Research Service, VA Puget Sound Health Care System, Seattle, WA 98108; Hu, Hsien-Ming

2010-11-05

Research highlights: {yields} Inducible and reversible siRNA knockdown of an oncogenic fusion protein such as EWS-Fli1 is feasible and more advantageous than other siRNA methods. {yields} The tumor suppressor gene FOXO1 is a new EWS-Fli1 target. {yields} While trans-activators are known for the FOXO1 gene, there has been no report on negative regulators of FOXO1 transcription. {yields} 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 fusionmore » 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.« less

IgG-Paraoxonase-1 Fusion Protein for Targeted Drug Delivery Across the Human Blood-Brain Barrier

PubMed Central

Boado, Ruben J.; Zhang, Yun; Zhang, Yufeng; Wang, Yuntao; Pardridge, William M.

2009-01-01

Paraoxonase (PON)-1 is the most potent human protein with organophosphatase activity against organophosphate (OP) toxins. OP compounds readily cross the blood-brain barrier (BBB), and have lethal mechanisms of action within the brain. The production of a brain penetrating form of human PON1, which crosses the BBB, is possible with the re-engineering of the enzyme as a fusion protein with a monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry the PON1 into brain. The human PON1 was fused to the carboxyl terminus of the heavy chain of the chimeric HIRMAb. COS cells were dual transfected with the heavy chain gene and the light chain gene, and the HIRMAb-PON1 fusion protein was affinity purified with protein A chromatography. Western blotting with antibodies to human IgG or human PON1 showed the heavy chain of the HIRMAb-PON1 fusion protein was 40 kDa larger than the heavy chain of the chimeric HIRMAb. The ED50 of binding to the HIR extracellular domain was 0.55 ± 0.07 nM and 1.1 ±0.1 nM, respectively, for the chimeric HIRMAb and the HIRMAb-PON1 fusion protein. The PON1 enzyme activity of the fusion protein was approximately 25% of the enzyme activity in human plasma, based on a fluorometric enzymatic assay. In conclusion, human PON1 has been re-engineered as an IgG-organophosphatase fusion protein that penetrates the human BBB. PMID:19434854

Fusion Protein Vaccines Targeting Two Tumor Antigens Generate Synergistic Anti-Tumor Effects

PubMed Central

Cheng, Wen-Fang; Chang, Ming-Cheng; Sun, Wei-Zen; Jen, Yu-Wei; Liao, Chao-Wei; Chen, Yun-Yuan; Chen, Chi-An

2013-01-01

Introduction Human papillomavirus (HPV) has been consistently implicated in causing several kinds of malignancies, and two HPV oncogenes, E6 and E7, represent two potential target antigens for cancer vaccines. We developed two fusion protein vaccines, PE(ΔIII)/E6 and PE(ΔIII)/E7 by targeting these two tumor antigens to test whether a combination of two fusion proteins can generate more potent anti-tumor effects than a single fusion protein. Materials and Methods In vivo antitumor effects including preventive, therapeutic, and antibody depletion experiments were performed. In vitro assays including intracellular cytokine staining and ELISA for Ab responses were also performed. Results PE(ΔIII)/E6+PE(ΔIII)/E7 generated both stronger E6 and E7-specific immunity. Only 60% of the tumor protective effect was observed in the PE(ΔIII)/E6 group compared to 100% in the PE(ΔIII)/E7 and PE(ΔIII)/E6+PE(ΔIII)/E7 groups. Mice vaccinated with the PE(ΔIII)/E6+PE(ΔIII)/E7 fusion proteins had a smaller subcutaneous tumor size than those vaccinated with PE(ΔIII)/E6 or PE(ΔIII)/E7 fusion proteins alone. Conclusion Fusion protein vaccines targeting both E6 and E7 tumor antigens generated more potent immunotherapeutic effects than E6 or E7 tumor antigens alone. This novel strategy of targeting two tumor antigens together can promote the development of cancer vaccines and immunotherapy in HPV-related malignancies. PMID:24058440

Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

PubMed

Steinmetz, Eric J; Auldridge, Michele E

2017-11-01

The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

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

PubMed

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.

Dynamin-related protein-1 controls fusion pore dynamics during platelet granule exocytosis.

PubMed

Koseoglu, Secil; Dilks, James R; Peters, Christian G; Fitch-Tewfik, Jennifer L; Fadel, Nathalie A; Jasuja, Reema; Italiano, Joseph E; Haynes, Christy L; Flaumenhaft, Robert

2013-03-01

Platelet granule exocytosis serves a central role in hemostasis and thrombosis. Recently, single-cell amperometry has shown that platelet membrane fusion during granule exocytosis results in the formation of a fusion pore that subsequently expands to enable the extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore expansion and collapse are not known. We identified dynamin-related protein-1 (Drp1) in platelets and found that an inhibitor of Drp1, mdivi-1, blocked exocytosis of both platelet dense and α-granules. We used single-cell amperometry to monitor serotonin release from individual dense granules and, thereby, measured the effect of Drp1 inhibition on fusion pore dynamics. Inhibition of Drp1 increased spike width and decreased prespike foot events, indicating that Drp1 influences fusion pore formation and expansion. Platelet-mediated thrombus formation in vivo after laser-induced injury of mouse cremaster arterioles was impaired after infusion of mdivi-1. These results demonstrate that inhibition of Drp1 disrupts platelet fusion pore dynamics and indicate that Drp1 can be targeted to control thrombus formation in vivo.

Preparation of GST Fusion Proteins.

PubMed

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.

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

PubMed

Srivastava, Preeti; Deb, J K

2002-07-02

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism

PubMed Central

Douam, Florian; Mancip, Jimmy; Mailly, Laurent; Montserret, Roland; Ding, Qiang; Verhoeyen, Els; Baumert, Thomas F.; Ploss, Alexander; Carbone, Alessandra

2018-01-01

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao Maofu; Kielian, Margaret

2005-02-05

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 residuesmore » 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.« less

In vivo characterization of fusion protein comprising of A1 subunit of Shiga toxin and human GM-CSF: Assessment of its immunogenicity and toxicity.

PubMed

Oloomi, Mana; Bouzari, Saeid; Shariati, Elaheh

2010-10-01

Most cancer cells become resistant to anti-cancer agents. In the last few years, a new approach for targeted therapy of human cancer has been developed using immunotoxins which comprise both the cell targeting and the cell killing moieties. In the present study, the recombinant Shiga toxin A1 subunit fused to human granulocyte-macrophage colony stimulating factor (A1-GM-CSF), previously produced in E. coli, was further characterized. The recombinant protein could cause 50% cytotoxicity and induced apoptosis in cells bearing GM-CSF receptors. The non-specific toxicity of the fusion protein was assessed in C57BL/6 and BALB/c mice. No mortality was observed in either group of mice, with different concentration of fusion protein. The lymphocyte proliferation assay, induction of specific IgG response and a mixed (Th1/Th2) response were observed only in BALB/c mice. The mixed response in BALB/c mice (Th1/Th2) could be explained on the basis of the two components of the fusion protein i.e. A1 and GM-CSF.

The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

PubMed

Rizo, Josep; Südhof, Thomas C

2012-01-01

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

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

PubMed

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.

A recombinant fusion protein and DNA vaccines against foot-and-mouth disease virus type Asia 1 infection in guinea pigs.

PubMed

Zhang, Q; Zhu, M W; Yang, Y Q; Shao, M; Zhang, Z Y; Lan, H Y; Yan, W Y; Wu, J J; Zheng, Z X

2003-01-01

On the basis of amino acid (aa) sequence of the tandem repeat 133-158-20-34-133-158 which consisted of aa 133-158 of VP1 and aa 20-34 of VP4 of Foot-and-mouth disease virus (FMDV) type Asia 1 a recombinant prokaryotic expression vector pAS1-P encoding a fusion protein and eukaryotic expression vectors pAS1-E and pAS1-EdeltaCpG-ODN representing DNA vaccines were constructed. Guinea pigs immunized with these vaccines showed both neutralizing antibody and T cell proliferation responses. FMDV challenge tests for the first time showed that the recombinant fusion protein and pAS1-E and pAS1-EdeltaCpG-ODN vaccines protected 86%, 60% and 43% of guinea pigs from FMDV type Asia1 challenge, respectively. The results also indicated that the immune response of animals treated with the vector pAS1-E containing an oligodeoxynucleotide (ODN), which consisted of immunostimulatory cytosine-phosphate-guanosine (CpG) motifs, was augmented by CpG ODN.

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kar, Rekha; Department of Biochemistry, UT Health Science Center at San Antonio, San Antonio, TX 78229; Mishra, Nandita

2010-09-03

Research highlights: {yields} Chemical inhibition of fission protein Drp1 leads to mitochondrial fusion. {yields} Increased fusion stimulates molecular changes in mitochondrial fusion protein OPA1. {yields} Proteolysis of larger isoforms, new synthesis and ubiquitination of OPA1 occur. {yields} Loss of mitochondrial tubular rigidity and disorganization of cristae. {yields} Generation of large swollen dysfunctional mitochondria. -- Abstract: We showed earlier that 15 deoxy {Delta}{sup 12,14} prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion . However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria bymore » 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.« less

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

PubMed

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.

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

PubMed

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.

Transduced PEP-1-Heme Oxygenase-1 Fusion Protein Attenuates Lung Injury in Septic Shock Rats

PubMed Central

Yan, Xue-Tao; Wang, Yan-Lin; Zhang, Zong-Ze; Tang, Jun-Jiao

2018-01-01

Oxidative stress and inflammation have been identified to play a vital role in the pathogenesis of lung injury induced by septic shock. Heme oxygenase-1 (HO-1), an effective antioxidant and anti-inflammatory and antiapoptotic substance, has been used for the treatment of heart, lung, and liver diseases. Thus, we postulated that administration of exogenous HO-1 protein transduced by cell-penetrating peptide PEP-1 has a protective role against septic shock-induced lung injury. Septic shock produced by cecal ligation and puncture caused severe lung damage, manifested in the increase in the lung wet/dry ratio, oxidative stress, inflammation, and apoptosis. However, these changes were reversed by treatment with the PEP-1-HO-1 fusion protein, whereas lung injury in septic shock rats was alleviated. Furthermore, the septic shock upregulated the expression of Toll-like receptor 4 (TLR4) and transcription factor NF-κB, accompanied by the increase of lung injury. Administration of PEP-1-HO-1 fusion protein reversed septic shock-induced lung injury by downregulating the expression of TLR4 and NF-κB. Our study indicates that treatment with HO-1 protein transduced by PEP-1 confers protection against septic shock-induced lung injury by its antioxidant, anti-inflammatory, and antiapoptotic effects. PMID:29682161

Beyond ALK-RET, ROS1 and other oncogene fusions in lung cancer

PubMed Central

Nakaoku, Takashi; Tsuta, Koji; Tsuchihara, Katsuya; Matsumoto, Shingo; Yoh, Kiyotaka; Goto, Koichi

2015-01-01

Fusions of the RET and ROS1 protein tyrosine kinase oncogenes with several partner genes were recently identified as new targetable genetic aberrations in cases of non-small cell lung cancer (NSCLC) lacking activating EGFR, KRAS, ALK, BRAF, or HER2 oncogene aberrations. RET and ROS1 fusion-positive tumors are mainly observed in young, female, and/or never smoking patients. Studies based on in vitro and in vivo (i.e., mouse) models and studies of several fusion-positive patients indicate that inhibiting the kinase activity of the RET and ROS1 fusion proteins is a promising therapeutic strategy. Accordingly, there are several ongoing clinical trials aimed at examining the efficacy of tyrosine kinase inhibitors (TKIs) against RET and ROS1 proteins in patients with fusion-positive lung cancer. Other gene fusions (NTRK1, NRG1, and FGFR1/2/3) that are targetable by existing TKIs have also been identified in NSCLCs. Options for personalized lung cancer therapy will be increased with the help of multiplex diagnosis systems able to detect multiple druggable gene fusions. PMID:25870798

Microneedle delivery of an M2e-TLR5 ligand fusion protein to skin confers broadly cross-protective influenza immunity.

PubMed

Wang, Bao-Zhong; Gill, Harvinder S; He, Cheng; Ou, Changbo; Wang, Li; Wang, Ying-Chun; Feng, Hao; Zhang, Han; Prausnitz, Mark R; Compans, Richard W

2014-03-28

Influenza vaccines with broad cross-protection are urgently needed to prevent an emerging influenza pandemic. A fusion protein of the Toll-like receptor (TLR) 5-agonist domains from flagellin and multiple repeats of the conserved extracellular domain of the influenza matrix protein 2 (M2e) was constructed, purified and evaluated as such a vaccine. A painless vaccination method suitable for possible self-administration using coated microneedle arrays was investigated for skin-targeted delivery of the fusion protein in a mouse model. The results demonstrate that microneedle immunization induced strong humoral as well as mucosal antibody responses and conferred complete protection against homo- and heterosubtypic lethal virus challenges. Protective efficacy with microneedles was found to be significantly better than that seen with conventional intramuscular injection, and comparable to that observed with intranasal immunization. Because of its advantages for administration, safety and storage, microneedle delivery of M2e-flagellin fusion protein is a promising approach for an easy-to-administer universal influenza vaccine. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural characterization of Mumps virus fusion protein core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Yueyong; Xu Yanhui; Lou Zhiyong

2006-09-29

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,more » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.

2006-07-05

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{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra ormore » SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup 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.« less

Exo-endo cellulase fusion protein

DOEpatents

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.

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

PubMed

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.

Fc-fusion Proteins in Therapy: An Updated View.

PubMed

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.

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

PubMed Central

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

2014-01-01

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 Ixodesricinus. 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 1H–15N 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 13C/15N 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. PMID:25628987

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

PubMed

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.

Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion.

PubMed Central

Morrison, T; McQuain, C; McGinnes, L

1991-01-01

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion. Images PMID:1987376

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

PubMed

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.

Spring-Loaded Model Revisited: Paramyxovirus Fusion Requires Engagement of a Receptor Binding Protein beyond Initial Triggering of the Fusion Protein▿

PubMed Central

Porotto, Matteo; DeVito, Ilaria; Palmer, Samantha G.; Jurgens, Eric M.; Yee, Jia L.; Yokoyama, Christine C.; Pessi, Antonello; Moscona, Anne

2011-01-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry. PMID:21976650

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

PubMed

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.

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

PubMed Central

Costa, Sofia; Almeida, André; Castro, António; Domingues, Lucília

2014-01-01

Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system. PMID:24600443

Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits.

PubMed

Gajadeera, Chathurada S; Weber, Joachim

2013-09-13

The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.

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.

PubMed

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.

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*

PubMed Central

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

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits.

PubMed

Suga, Hinako; Haga, Tatsuya

2007-01-01

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.

Human antibodies and fusion proteins as HIV-1 therapeutic | NCI Technology Transfer Center | TTC

Cancer.gov

Available for licensing from the NCI are novel human anti-HIV-1 domain antibodies and their fusion proteins for anti-HIV-1 antibodies and anti-retroviral as therapeutics and/or preventatives for infection by different HIV-1 strains.

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

PubMed

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.

PubMed

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

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

PubMed Central

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

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes.

PubMed

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian; Kielian, Margaret

2016-07-15

The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

PubMed Central

Dubé, Mathieu; Etienne, Loïc; Fels, Maximilian

2016-01-01

ABSTRACT The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. IMPORTANCE Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium

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

PubMed

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 cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

PubMed

Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J

2015-03-01

Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. © 2015. Published by The Company of Biologists Ltd.

Surface Density of the Hendra G Protein Modulates Hendra F Protein-Promoted Membrane Fusion: Role for Hendra G Protein Trafficking and Degradation

PubMed Central

Whitman, Shannon D.; Dutch, Rebecca Ellis

2007-01-01

Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F. PMID:17328935

T-cell immunotherapy for human MK-1-expressing tumors using a fusion protein of the superantigen SEA and anti-MK-1 scFv antibody.

PubMed

Ueno, Aruto; Arakawa, Fumiko; Abe, Hironori; Matsumoto, Hisanobu; Kudo, Toshio; Asano, Ryutaro; Tsumoto, Kohei; Kumagai, Izumi; Kuroki, Motomu; Kuroki, Masahide

2002-01-01

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we constructed a recombinant fusion protein of SEA and the single-chain variable fragment (scFv) of the FU-MK-1 antibody, which recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas. We employed recombinant DNA techniques to fuse recombinant mutant SEA to an scFv antibody derived from FU-MK-1 and the resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system, purified with a metal-affinity column, and characterized for its MK-1-binding specificity and its antitumor activity. The SEA/FUscFv fusion protein retained the reactivity with MK-1-expressing tumor cells, introduced a specific cytotoxicity of lymphokine-activated killer T-cells to the tumor cells, and consequently suppressed the tumor growth in a SCID mouse xenograft model. This genetically engineered SEA/FUscFv fusion protein may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyuhas, Ronit; Noy, Hava; Fishman, Sigal

2009-08-21

HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited {approx}1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect onmore » 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.« less

Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nayak, Vinod; Dessau, Moshe; Kucera, Kaury

Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flavivirusesmore » and are part of the 'pH sensor' that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.« less

Hexahistidine (6xHis) fusion-based assays for protein-protein interactions.

PubMed

Puckett, Mary C

2015-01-01

Fusion-protein tags provide a useful method to study protein-protein interactions. One widely used fusion tag is hexahistidine (6xHis). This tag has unique advantages over others due to its small size and the relatively low abundance of naturally occurring consecutive histidine repeats. 6xHis tags can interact with immobilized metal cations to provide for the capture of proteins and protein complexes of interest. In this chapter, a description of the benefits and uses of 6xHis-fusion proteins as well as a detailed method for performing a 6xHis-pulldown assay are described.

ChiPPI: a novel method for mapping chimeric protein-protein interactions uncovers selection principles of protein fusion events in cancer.

PubMed

Frenkel-Morgenstern, Milana; Gorohovski, Alessandro; Tagore, Somnath; Sekar, Vaishnovi; Vazquez, Miguel; Valencia, Alfonso

2017-07-07

Fusion proteins, comprising peptides deriving from the translation of two parental genes, are produced in cancer by chromosomal aberrations. The expressed fusion protein incorporates domains of both parental proteins. Using a methodology that treats discrete protein domains as binding sites for specific domains of interacting proteins, we have cataloged the protein interaction networks for 11 528 cancer fusions (ChiTaRS-3.1). Here, we present our novel method, chimeric protein-protein interactions (ChiPPI) that uses the domain-domain co-occurrence scores in order to identify preserved interactors of chimeric proteins. Mapping the influence of fusion proteins on cell metabolism and pathways reveals that ChiPPI networks often lose tumor suppressor proteins and gain oncoproteins. Furthermore, fusions often induce novel connections between non-interactors skewing interaction networks and signaling pathways. We compared fusion protein PPI networks in leukemia/lymphoma, sarcoma and solid tumors finding distinct enrichment patterns for each disease type. While certain pathways are enriched in all three diseases (Wnt, Notch and TGF β), there are distinct patterns for leukemia (EGFR signaling, DNA replication and CCKR signaling), for sarcoma (p53 pathway and CCKR signaling) and solid tumors (FGFR and EGFR signaling). Thus, the ChiPPI method represents a comprehensive tool for studying the anomaly of skewed cellular networks produced by fusion proteins in cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy in Chlamydomonas.

PubMed

Liu, Yanjie; Misamore, Michael J; Snell, William J

2010-05-01

The plasma membranes of gametes are specialized for fusion, yet, once fusion occurs, in many organisms the new zygote becomes incapable of further membrane fusion reactions. The molecular mechanisms that underlie this loss of fusion capacity (block to polygamy) remain unknown. During fertilization in the green alga Chlamydomonas, the plus gamete-specific membrane protein FUS1 is required for adhesion between the apically localized sites on the plasma membranes of plus and minus gametes that are specialized for fusion, and the minus-specific membrane protein HAP2 is essential for completion of the membrane fusion reaction. HAP2 (GCS1) family members are also required for fertilization in Arabidopsis, and for the membrane fusion reaction in the malaria organism Plasmodium berghei. Here, we tested whether Chlamydomonas gamete fusion triggers alterations in FUS1 and HAP2 and renders the plasma membranes of the cells incapable of subsequent fusion. We find that, even though the fusogenic sites support multi-cell adhesions, triploid zygotes are rare, indicating a fusion-triggered block to the membrane fusion reaction. Consistent with the extinction of fusogenic capacity, both FUS1 and HAP2 are degraded upon fusion. The rapid, fusion-triggered cleavage of HAP2 in zygotes is distinct from degradation occurring during constitutive turnover in gametes. Thus, gamete fusion triggers specific degradation of fusion-essential proteins and renders the zygote incapable of fusion. Our results provide the first molecular explanation for a membrane block to polygamy in any organism.

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

PubMed

Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

2010-10-01

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

Immunogenicity of a recombinant fusion protein of tandem repeat epitopes of foot-and-mouth disease virus type Asia 1 for guinea pigs.

PubMed

Zhang, Q; Yang, Y Q; Zhang, Z Y; Li, L; Yan, W Y; Jiang, W J; Xin, A G; Lei, C X; Zheng, Z X

2002-01-01

In this study, the sequences of capsid protein VPI regions of YNAs1.1 and YNAs1.2 isolates of foot-and-mouth disease virus (FMDV) were analyzed and a peptide containing amino acids (aa) 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia I was assumed to contain B and T cell epitopes, because it is hypervariable and includes a cell attachment site RGD located in the G-H loop. The DNA fragments encoding aa 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia 1 were chemically synthesized and ligated into a tandem repeat of aa 133-158-20 approximately 34-133-158. In order to enhance its immunogenicity, the tandem repeat was inserted downstream of the beta-galactosidase gene in the expression vector pWR590. This insertion yielded a recombinant expression vector pAS1 encoding the fusion protein. The latter reacted with sera from FMDV type Asia 1-infected animals in vitro and elicited high levels of neutralizing antibodies in guinea pigs. The T cell proliferation in immunized animals increased following stimulation with the fusion protein. It is reported for the first time that a recombinant fusion protein vaccine was produced using B and T cell epitopes of FMDV type Asia 1 and that this fusion protein was immunogenic. The fusion protein reported here can serve as a candidate of fusion epitopes for design of a vaccine against FMDV type Asia 1.

Design and testing for a nontagged F1-V fusion protein as vaccine antigen against bubonic and pneumonic plague.

PubMed

Powell, Bradford S; Andrews, Gerard P; Enama, Jeffrey T; Jendrek, Scott; Bolt, Chris; Worsham, Patricia; Pullen, Jeffrey K; Ribot, Wilson; Hines, Harry; Smith, Leonard; Heath, David G; Adamovicz, Jeffrey J

2005-01-01

A two-component recombinant fusion protein antigen was re-engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine-tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea-denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N-terminal methionine. Purity, quality, and higher-order structure were compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy, and multi-angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V protein also protected mice from wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1-V as the active pharmaceutical ingredient of the next plague vaccine.

Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Jian; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071; Zhang, Huaidong

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 Imore » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Claire Y.-H., E-mail: CHuang1@cdc.go; Butrapet, Siritorn; Moss, Kelly J.

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 notmore » 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.« less

A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion Proteins is Critical for Proper F Protein Folding†

PubMed Central

Gardner, Amanda E.; Martin, Kimberly L.; Dutch, Rebecca E.

2008-01-01

Paramyxoviruses are a diverse family which utilizes a fusion (F) protein to enter cells via fusion of the viral lipid bilayer with a target cell membrane. Although certain regions of F are known to play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequence alignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region of conserved amino acid sequence in a large domain between the heptad repeats of F1, designated CBF1. We employed site-directed mutagenesis to analyze the function of completely conserved residues of CBF1 in both the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF1 point mutants were deficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For some SV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30°C, and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 F V402A displayed a hyperfusogenic phenotype at both 30°C and 37°C, indicating this mutation allows for efficient fusion with only an extremely small amount of cleaved, active protein. The recently published prefusogenic structure of PIV5/SV5 F [Yin, H.S., et al. (2006) Nature 439, 38–44] indicates that residues within and flanking CBF1 interact with the fusion peptide domain. Together, these data suggest that CBF1-fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from across this important viral family, and can also modulate subsequent membrane fusion promotion. PMID:17417875

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton

2010-08-15

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmicmore » tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger.« less

The actin cytoskeleton inhibits pore expansion during PIV5 fusion protein-promoted cell-cell fusion

PubMed Central

Wurth, Mark A.; Schowalter, Rachel M.; Smith, Everett Clinton; Moncman, Carole L.; Dutch, Rebecca Ellis; McCann, Richard O.

2010-01-01

Paramyxovirus fusion (F) proteins promote both virus-cell fusion, required for viral entry, and cell-cell fusion, resulting in syncytia formation. We used the F-actin stabilizing drug, jasplakinolide, and the G-actin sequestrant, latrunculin A, to examine the role of actin dynamics in cell-cell fusion mediated by the parainfluenza virus 5 (PIV5) F protein. Jasplakinolide treatment caused a dose-dependent increase in cell-cell fusion as measured by both syncytia and reporter gene assays, and latrunculin A treatment also resulted in fusion stimulation. Treatment with jasplakinolide or latrunculin A partially rescued a fusion pore opening defect caused by deletion of the PIV5 F protein cytoplasmic tail, but these drugs had no effect on fusion inhibited at earlier stages by either temperature arrest or by a PIV5 heptad repeat peptide. These data suggest that the cortical actin cytoskeleton is an important regulator of fusion pore enlargement, an energetically costly stage of viral fusion protein-mediated membrane merger. PMID:20537366

Membrane Fusion Proteins as Nanomachines

NASA Astrophysics Data System (ADS)

Tamm, Lukas

2009-03-01

Membrane fusion is key to fertilization, virus infection, and neurotransmission. Specific proteins work like nanomachines to stitch together fluid, yet highly ordered lipid bilayers. The energy gained from large exothermic conformational changes of these proteins is utilized to fuse lipid bilayers that do not fuse spontaneously. Structural studies using x-ray crystallography and NMR spectroscopy have yielded detailed information about architecture and inner workings of these molecular machines. The question now is: how is mechanical energy gained from such protein transformations harnessed to transform membrane topology? To answer this question, we have determined that a boomerang-shaped structure of the influenza fusion peptide is critical to generate a high-energy binding intermediate in the target membrane and to return the ``boomerang'' to its place of release near the viral membrane for completion of the fusion cycle. In presynaptic exocytosis, receptor and acceptor SNAREs are zippered to form a helical bundle that is arrested shortly before the membrane. Ca binding to interlocked synaptotagmin releases the fusion block. Structural NMR and single molecule fluorescence data are combined to arrive at and further refine this picture.

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

PubMed

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.

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

PubMed

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.

C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein

PubMed Central

Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

2011-01-01

The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the viral-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of HA tag addition varied with other fusion proteins, as parainfluenza virus 5 F-HA showed decreased surface expression and no stimulation in fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in modulation of the membrane fusion reaction promoted by these viral glycoproteins. PMID:21175223

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots.

PubMed

Wang, Yuh-Shuh; Motes, Christy M; Mohamalawari, Deepti R; Blancaflor, Elison B

2004-10-01

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins. Copyright 2004 Wiley-Liss, Inc.

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

PubMed

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

SAFE Software and FED Database to Uncover Protein-Protein Interactions using Gene Fusion Analysis.

PubMed

Tsagrasoulis, Dimosthenis; Danos, Vasilis; Kissa, Maria; Trimpalis, Philip; Koumandou, V Lila; Karagouni, Amalia D; Tsakalidis, Athanasios; Kossida, Sophia

2012-01-01

Domain Fusion Analysis takes advantage of the fact that certain proteins in a given proteome A, are found to have statistically significant similarity with two separate proteins in another proteome B. In other words, the result of a fusion event between two separate proteins in proteome B is a specific full-length protein in proteome A. In such a case, it can be safely concluded that the protein pair has a common biological function or even interacts physically. In this paper, we present the Fusion Events Database (FED), a database for the maintenance and retrieval of fusion data both in prokaryotic and eukaryotic organisms and the Software for the Analysis of Fusion Events (SAFE), a computational platform implemented for the automated detection, filtering and visualization of fusion events (both available at: http://www.bioacademy.gr/bioinformatics/projects/ProteinFusion/index.htm). Finally, we analyze the proteomes of three microorganisms using these tools in order to demonstrate their functionality.

SAFE Software and FED Database to Uncover Protein-Protein Interactions using Gene Fusion Analysis

PubMed Central

Tsagrasoulis, Dimosthenis; Danos, Vasilis; Kissa, Maria; Trimpalis, Philip; Koumandou, V. Lila; Karagouni, Amalia D.; Tsakalidis, Athanasios; Kossida, Sophia

2012-01-01

Domain Fusion Analysis takes advantage of the fact that certain proteins in a given proteome A, are found to have statistically significant similarity with two separate proteins in another proteome B. In other words, the result of a fusion event between two separate proteins in proteome B is a specific full-length protein in proteome A. In such a case, it can be safely concluded that the protein pair has a common biological function or even interacts physically. In this paper, we present the Fusion Events Database (FED), a database for the maintenance and retrieval of fusion data both in prokaryotic and eukaryotic organisms and the Software for the Analysis of Fusion Events (SAFE), a computational platform implemented for the automated detection, filtering and visualization of fusion events (both available at: http://www.bioacademy.gr/bioinformatics/projects/ProteinFusion/index.htm). Finally, we analyze the proteomes of three microorganisms using these tools in order to demonstrate their functionality. PMID:22267904

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

PubMed

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.

[Construction and expression of HSV-2gD-Hsp70 fusion protein gene].

PubMed

Fan, Jian-Yong; Yang, Hui-Lan; Wang, Ying; Guan, Lei

2006-11-01

To construct and express Hsp70-HSV2gD fusion protein. Genes of Hsp70 and HSV-2gD were subcloned into vectors pGEX-4T-1 respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pGEX-4T-HSP-gD was transformed into E. coli DH5alpha and induced to express with IPTG. The expressed protein was characterized by SDS-PAGE and Western blot after purified. BALB/c mice were immunized with fusion proteins respectively via intra-m uscular injection. The proliferation of spleen lymphocytes, the level of y-IFN in culture and anti-HSV-2gD IgG antibody in serum was detected was detected. The expressed protein was analyzed by SDS-PAGE after induced with IPTG, which showed a new band with an apparent molecular mass corresponding to the predicted size (118 kD). Western Blotting analysis demonstrates that the purified Hsp70-HSV2gD fusion protein had specific binding activity. The stimulation indexes of spleen lymphocytes, the level of gamma-IFN in culture and anti-HSV-2gD IgG antibody in serum of GST-Hsp70-gD group was obviously higher than that of other groups (P < 0.05 respectively). The successful expression of the Hsp70-HSV2gD fusion protein, which can induce immune responses, laid a solid foundation for its further research.

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

PubMed

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.

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

PubMed Central

Jin, Chunsheng; Liu, Jining; Karlsson, Niclas G.; Holgersson, Jan

2016-01-01

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galβ1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive β-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galβ1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases. PMID:27456831

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

PubMed

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.

Fluorescence fluctuation analysis of BACE1-GFP fusion protein in cultured HEK293 cells

NASA Astrophysics Data System (ADS)

Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

2016-10-01

Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion.

PubMed

Zeng, Xiancheng; Mukhopadhyay, Suchetana; Brooks, Charles L

2015-02-17

Alphavirus envelope proteins, organized as trimers of E2-E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections.

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

PubMed

Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

2016-03-18

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli.

PubMed

Hwang, Peter M; Pan, Jonathan S; Sykes, Brian D

2014-01-21

Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by site-specific protease cleavage. In this review, we present an alternative approach to protein production using fusion partners specifically designed to accumulate in insoluble inclusion bodies. The strategy is appropriate for the mass production of short peptides, intrinsically disordered proteins, and proteins that can be efficiently refolded in vitro. There are many fusion protein systems now available for insoluble expression: TrpLE, ketosteroid isomerase, PurF, and PagP, for example. The ideal fusion partner is effective at directing a wide variety of target proteins into inclusion bodies, accumulates in large quantities in a highly pure form, and is readily solubilized and purified in commonly used denaturants. Fusion partner removal under denaturing conditions is biochemically challenging, requiring harsh conditions (e.g., cyanogen bromide in 70% formic acid) that can result in unwanted protein modifications. Recent advances in metal ion-catalyzed peptide bond cleavage allow for more mild conditions, and some methods involving nickel or palladium will likely soon appear in more biological applications. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Butrapet, Siritorn; Childers, Thomas; Moss, Kelley J.

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, butmore » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 mRNAmore » 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.« less

Tandem SUMO fusion vectors for improving soluble protein expression and purification.

PubMed

Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

2015-12-01

Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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.

A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

PubMed

Sharapova, Olga A; Yurkova, Maria S; Fedorov, Alexey N

2016-02-01

We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

PubMed Central

West, Dava S.; Sheehan, Michael S.; Segeleon, Patrick K.; Dutch, Rebecca Ellis

2005-01-01

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. PMID:15650180

Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jufeng; Wang, Zhanli; Wei, Fang

2007-08-17

Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. Themore » toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.« less

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

PubMed

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.

Insertion of inter-domain linkers improves expression and bioactivity of Zygote arrest (Zar) fusion proteins.

PubMed

Cook, Jonathan M; Charlesworth, Amanda

2017-04-01

Developmentally important proteins that are crucial for fertilization and embryogenesis are synthesized through highly regulated translation of maternal mRNA. The Zygote arrest proteins, Zar1 and Zar2, are crucial for embryogenesis and have been implicated in binding mRNA and repressing mRNA translation. To investigate Zar1 and Zar2, the full-length proteins had been fused to glutathione-S-transferase (GST) or MS2 protein tags with minimal inter-domain linkers derived from multiple cloning sites; however, these fusion proteins expressed poorly and/or lacked robust function. Here, we tested the effect of inserting additional linkers between the fusion domains. Three linkers were tested, each 17 amino acids long with different physical and chemical properties: flexible hydrophilic, rigid extended or rigid helical. In the presence of any of the three linkers, GST-Zar1 and GST-Zar2 had fewer breakdown products. Moreover, in the presence of any of the linkers, MS2-Zar1 was expressed to higher levels, and in dual luciferase tethered assays, both MS2-Zar1 and MS2-Zar2 repressed luciferase translation to a greater extent. These data suggest that for Zar fusion proteins, increasing the length of linkers, regardless of their physical or chemical properties, improves stability, expression and bioactivity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Fusion of Protegrin-1 and Plectasin to MAP30 Shows Significant Inhibition Activity against Dengue Virus Replication

PubMed Central

Rothan, Hussin A.; Bahrani, Hirbod; Mohamed, Zulqarnain; Abd Rahman, Noorsaadah; Yusof, Rohana

2014-01-01

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. PMID:24722532

The fate of fusion Cry1Ab/1Ac proteins from Bt-transgenic rice in soil and water.

PubMed

Liu, Yongbo; Li, Junsheng; Luo, Zunlan; Wang, Huaru; Liu, Fang

2016-02-01

Toxin proteins form transgenic crops entering into the environment are likely affect non-target organisms. To investigate the entry route and fate of fusion Cry1Ab/1Ac proteins from transgenic rice expressing insecticide toxins from Bacillus thuringiensis (Bt) in soil and water, we conducted greenhouse and field experiments in 2013 and 2014. Cry1Ab/1Ac proteins from Bt-transgenic rice in soil was found within a horizontal range of 25cm, where most of plant roots distributed. Concentration of Cry1Ab/1Ac proteins was lower in water than in soil in the greenhouse experiment, and no Cry1Ab/1Ac protein was detected in field water. Cry1Ab/1Ac concentration from rice straws was higher in ditch water than in distilled water due to the existence of aquatic organisms in ditch water. Bt proteins from transgenic crops enter into soil ecosystems mainly through root exudates and into aquatic ecosystems through plant residues, which determines Bt fate in the environment. Copyright © 2015 Elsevier Inc. All rights reserved.

Complications, revision fusions, readmissions, and utilization over a 1-year period after bone morphogenetic protein use during primary cervical spine fusions.

PubMed

Goode, Adam P; Richardson, William J; Schectman, Robin M; Carey, Timothy S

2014-09-01

Nationwide estimates examining bone morphogenetic protein (BMP) use with cervical spine fusions have been limited to perioperative outcomes. To determine the 1-year risk of complications, cervical revision fusions, hospital readmissions, and health care services utilization. A retrospective cohort study from 2002 to 2009 using a nationwide claims database. There were 61,937 primary cervical spine fusions of which 1,677 received BMP. Complications, revision fusions, 30-day hospital readmission, and health care utilization. Data for these analyses come from the Thomson Reuters MarketScan Commercial Claims and Encounters Database 2010. Patients were aged 18 to 64 years, receiving and not receiving BMP with a primary (C2-C7) cervical spine fusion. All outcomes were defined by International Classification of Diseases, 9th edition Clinical Modification and Current Procedural and Terminology, 4th edition codes. Complications were analyzed as any complication and stratified by nervous system, wound, and dysphagia or hoarseness. Cervical revision fusions were determined in the 1-year follow-up. Hospital readmission discharge records defined 30-day hospital readmission and reason for the readmission. The utilization of at least one health care service of cervical spine imaging, epidural usage or rehabilitation service was examined. Poisson regression models were used to estimate the relative risk and 95% confidence interval (CI). Linear regression was used to determine the time to hospital readmission. Results were stratified by anterior or posterior and circumferential approaches. Patients receiving BMP were 29% more likely to have a complication (adjusted relative risk [aRR]=1.29 [95% CI, 1.14-1.46]) and a nervous system complication (aRR=1.42 [95% CI, 1.10-1.83]). Cervical revision fusions were more likely among patients receiving BMP (aRR=1.69 [95% CI, 1.35-2.13]). The risk of 30-day readmission was greater with BMP use (aRR=1.37 [95% CI, 1.07-1.73]) and readmission

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

PubMed

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.

Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy

PubMed Central

2011-01-01

Background Genetic fusion of the major birch pollen allergen (Bet v1) to bacterial surface-(S)-layer proteins resulted in recombinant proteins exhibiting reduced allergenicity as well as immunomodulatory capacity. Thus, S-layer/allergen fusion proteins were considered as suitable carriers for new immunotherapeutical vaccines for treatment of Type I hypersensitivity. Up to now, endotoxin contamination of the fusion protein which occurred after isolation from the gram-negative expression host E. coli had to be removed by an expensive and time consuming procedure. In the present study, in order to achieve expression of pyrogen-free, recombinant S-layer/allergen fusion protein and to study the secretion of a protein capable to self-assemble, the S-layer/allergen fusion protein rSbpA/Bet v1 was produced in the gram-positive organism Bacillus subtilis 1012. Results The chimaeric gene encoding the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 as well as Bet v1 was cloned and expressed in B. subtilis 1012. For that purpose, the E. coli-B. subtilis shuttle vectors pHT01 for expression in the B. subtilis cytoplasm and pHT43 for secretion of the recombinant fusion protein into the culture medium were used. As shown by western blot analysis, immediately after induction of expression, B. subtilis 1012 was able to secret rSbpA/Bet v1 mediated by the signal peptide amyQ of Bacillus amyloliquefaciens. Electron microscopical investigation of the culture medium revealed that the secreted fusion protein was able to form self-assembly products in suspension but did not recrystallize on the surface of the B. subtilis cells. The specific binding mechanism between the N-terminus of the S-layer protein and a secondary cell wall polymer (SCWP), located in the peptidoglycan-containing sacculi of Ly. sphaericus CCM 2177, could be used for isolation and purification of the secreted fusion protein from the culture medium. Immune reactivity of rSbpA/Bet v1 could be demonstrated in

Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies

PubMed Central

Simpson, Sean; Collins, Bruce; Sommer, Jeff; Petters, Robert M.; Caballero, Ignacio; Platt, Jeff L.

2017-01-01

Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3–5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model. PMID:28081156

Overview of Fusion Tags for Recombinant Proteins.

PubMed

Kosobokova, E N; Skrypnik, K A; Kosorukov, V S

2016-03-01

Virtually all recombinant proteins are now prepared using fusion domains also known as "tags". The use of tags helps to solve some serious problems: to simplify procedures of protein isolation, to increase expression and solubility of the desired protein, to simplify protein refolding and increase its efficiency, and to prevent proteolysis. In this review, advantages and disadvantages of such fusion tags are analyzed and data on both well-known and new tags are generalized. The authors own data are also presented.

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

PubMed Central

Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

2013-01-01

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

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

PubMed Central

Chakrabarti, Sanjukta; Barrow, Colin J.; Kanwar, Rupinder K.; Ramana, Venkata; Kanwar, Jagat R.

2016-01-01

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. PMID:27294920

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

PubMed Central

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

2013-01-01

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. PMID:24152736

Green fluorescence protein-based content-mixing assay of SNARE-driven membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heo, Paul; Kong, Byoungjae; Jung, Young-Hun

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regardingmore » the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening. - Highlights: • SNARE proteins drive membrane fusion and open a pore for cargo release. • Biotinylated GFP and DyStrp was used as the reporter pair of fusion pore opening. • Procedure for efficient SNARE reconstitution and reporter encapsulation was established. • The FRET pair reported opening of a large fusion pore bigger than 5�

Cellulose binding domain fusion proteins

DOEpatents

Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

1998-02-17

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. 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. 16 figs.

Cellulose binding domain fusion proteins

DOEpatents

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.

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Yeongseon; Choi, Won Tae; Heller, William T.

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermalmore » driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. Lastly, these results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.« less

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins

DOE PAGES

Jang, Yeongseon; Choi, Won Tae; Heller, William T.; ...

2017-07-27

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermalmore » driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. Lastly, these results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.« less

Protein accumulation and rumen stability of wheat γ-gliadin fusion proteins in tobacco and alfalfa.

PubMed

Sun, Xiaodong; Chi-Ham, Cecilia L; Cohen-Davidyan, Tamar; DeBen, Christopher; Getachew, Girma; DePeters, Edward; Putnam, Daniel; Bennett, Alan

2015-09-01

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine-rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine-rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ-gliadin-δ-zein and γ-δ-zein, as well as δ-zein co-expressed with β-zein, all formed protein bodies. However, the γ-gliadin-δ-zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ-gliadin-δ-zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ-gliadin-δ-zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ-gliadin-GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ-gliadin-δ-zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ-gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Mutations in the Parainfluenza Virus 5 Fusion Protein Reveal Domains Important for Fusion Triggering and Metastability

PubMed Central

Bose, Sayantan; Heath, Carissa M.; Shah, Priya A.; Alayyoubi, Maher; Jardetzky, Theodore S.

2013-01-01

Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger. PMID:24089572

HIV-1 Fusion Is Blocked through Binding of GB Virus C E2D Peptides to the HIV-1 gp41 Disulfide Loop

PubMed Central

Eissmann, Kristin; Mueller, Sebastian; Sticht, Heinrich; Jung, Susan; Zou, Peng; Jiang, Shibo; Gross, Andrea; Eichler, Jutta; Fleckenstein, Bernhard; Reil, Heide

2013-01-01

A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors. PMID:23349893

Molecular characterization of epithelioid haemangioendotheliomas identifies novel WWTR1-CAMTA1 fusion variants.

PubMed

Patel, Nimesh R; Salim, Alaa A; Sayeed, Hadi; Sarabia, Stephen F; Hollingsworth, Faith; Warren, Mikako; Jakacky, Jared; Tanas, Munir; Oliveira, Andre M; Rubin, Brian P; Lazar, Alexander J; López-Terrada, Dolores; Wang, Wei-Lien

2015-11-01

Epithelioid haemangioendothelioma (EHE) is a malignant vascular neoplasm. Subsets have been characterized previously by translocations resulting in either WWTR1-CAMTA1 or YAP1-TFE3 fusion. We sought to develop molecular and immunohistochemical (IHC) assays to aid in the diagnosis and characterization of EHE. Fifty-two formalin-fixed, paraffin-embedded (FFPE) cases diagnosed between 2002 and 2014 were retrieved from the pathology files of our institutions. Reverse transcription-polymerase chain reaction (RT-PCR) assays were optimized to detect WWTR1-CAMTA1 and YAP1-TFE3 fusion transcripts in FFPE tissue and transcription factor E3 (TFE3) protein accumulation was examined by immunohistochemistry (IHC). RNA was extracted from 33 adequate samples, with more recent cases providing a greater yield of high quality RNA. Fourteen of 18 informative cases were positive for WWTR1-CAMTA1 fusion transcripts, four of which showed higher-grade cytological features termed by some as 'malignant EHE'. Novel in-frame fusion transcripts were identified in four cases by direct sequencing. IHC revealed variable nuclear TFE3 staining in six of 17 cases; three with patchy staining showed WWTR1-CAMTA1 fusion. One of 18 informative cases was positive for YAP1-TFE3 fusion and showed strong nuclear TFE3 staining by IHC. This study confirms the high incidence of WWTR1-CAMTA1 and YAP1-TFE3 rearrangements in EHE and indicates that the staining pattern for TFE3 IHC is critical for specificity. © 2015 John Wiley & Sons Ltd.

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

PubMed

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.

Generation of the Fluorescent HMGB1-GFP Fusion Protein in Insect Cells and Evaluation of its Immunogenicity in Two Mice Models.

PubMed

Anvar, Ali; Vahabpour, Rouhollah; Salahshourifar, Iman; Bolhassani, Azam

2017-01-01

High mobility group box 1 (HMGB1) is a highly conserved protein present in the nuclei and cytoplasm of cells which has an important role as a mediator of inflammation in the extracellular environment. HMGB1 was identified as an innate adjuvant that induces immune responses against soluble antigens in vivo. Our goal is the generation of recombinant HMGB1-GFP fusion protein in insect cells for evaluation of immune responses in mouse model. In the current study, we used a baculovirus expression system for insect cells that was based on expression of HMGB1 with target gene (GFP), and purified the recombinant HMGB1- GFP fusion protein. We then demonstrated whether immunogenicity of GFP changes in the presence or absence of recombinant HMGB1 acting as an adjuvant in C57BL/6 and BALB/c mice. Our data showed that HMGB1 had a major influence on antibody immune responses induced by GFP in both animal models. The groups receiving HMGB1-GFP fusion protein showed total IgG and IgG2a responses significantly higher than IgG1 in BALB/c mice. Indeed, a mixed IgG1/IgG2a response was observed with high intensity toward IgG2a. In contrast, C57BL/6 mice immunized by HMGB1-GFP protein elicited the same levels of IgG1 and IgG2a. However, the levels of IgG2a and total IgG against the recombinant GFP (rGFP) in C57BL/6 mice were lower than those in BALB/c mice. We concluded that fusion of HMGB1 with GFP was immunologically more effective than GFP alone. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

PubMed

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.

The influence of monovalent cations on trimeric G protein G(i)1α activity in HEK293 cells stably expressing DOR-G(i)1α (Cys(351)-Ile(351)) fusion protein.

PubMed

Vošahlíková, M; Svoboda, P

2011-01-01

The effect of monovalent cations on trimeric G protein G(i)1α was measured at equimolar concentration of chloride anion in pertussis-toxin (PTX)-treated HEK293 cells stably expressing PTX-insensitive DOR- G(i)1α (Cys(351)-Ile(351)) fusion protein by high-affinity [(35)S]GTPgammaS binding assay. The high basal level of binding was detected in absence of DOR agonist and monovalent ions and this high level was inhibited with the order of: Na(+) > K(+) > Li(+). The first significant inhibition was detected at 1 mM NaCl. The inhibition by monovalent ions was reversed by increasing concentrations of DOR agonist DADLE. The maximum DADLE response was also highest for sodium and decreased in the order of: Na(+) > K(+) ~ Li(+). Our data indicate i) an inherently high activity of trimeric G protein G(i)1α when expressed within DOR- G(i)1α fusion protein and determined in the absence of monovalent cations, ii) preferential sensitivity of DOR- G(i)1alpha to sodium as far as maximum of agonist response is involved.

Crystal Structure of Dengue Type 1 Envelope Protein in the Postfusion Conformation and its Implication for Receptor Binding, Membrane Fusion and Antibody Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nayak, V.; Dessau, M; Kucera, K

Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flavivirusesmore » and are part of the pH sensor that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.« less

Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

PubMed Central

Smith, Everett Clinton; Culler, Megan R.; Hellman, Lance M.; Fried, Michael G.; Creamer, Trevor P.

2012-01-01

While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

Pretargeting with bispecific fusion proteins facilitates delivery of nanoparticles to tumor cells with distinct surface antigens.

PubMed

Yang, Qi; Parker, Christina L; Lin, Yukang; Press, Oliver W; Park, Steven I; Lai, Samuel K

2017-06-10

Tumor heterogeneity, which describes the genetically and phenotypically distinct subpopulations of tumor cells present within the same tumor or patient, presents a major challenge to targeted delivery of diagnostic and/or therapeutic agents. An ideal targeting strategy should deliver a given nanocarrier to the full diversity of cancer cells, which is difficult to achieve with conventional ligand-conjugated nanoparticles. We evaluated pretargeting (i.e., multistep targeting) as a strategy to facilitate nanoparticle delivery to multiple target cells by measuring the uptake of biotinylated nanoparticles by lymphoma cells with distinct surface antigens pretreated with different bispecific streptavidin-scFv fusion proteins. Fusion proteins targeting CD20 or tumor-associated glycoprotein 72 (TAG-72) mediated the specific in vitro uptake of 100nm biotin-functionalized nanoparticles by Raji and Jurkat lymphoma cells (CD20-positive and TAG-72-positive cells, respectively). Greater uptake was observed for pretargeted nanoparticles with increasing amounts of surface biotin, with 6- to 18-fold higher uptake vs. non-biotinylated nanoparticle and fusion protein controls. Fully biotin-modified particles remained resistant to cultured macrophage cell uptake, although they were still quickly cleared from systemic circulation in vivo (t 1/2 <1h). For single Raji tumor-bearing mice, pretargeting with CD20-specific FP significantly increased nanoparticle tumor targeting. In mice bearing both Raji and Jurkat tumors, pretargeting with both fusion proteins markedly increased nanoparticle targeting to both tumor types, compared to animals dosed with nanoparticles alone. These in vitro and in vivo observations support further evaluations of pretargeting fusion protein cocktails as a strategy to enhance nanoparticle delivery to a diverse array of molecularly distinct target cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

PubMed

Su, Chin-Fen; Kuo, I-Chun; Chen, Peng-Wen; Huang, Chiung-Hui; Seow, See Voon; Chua, Kaw Yan; Yu, Su-May

2012-02-01

Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

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

PubMed Central

Brimer, Nicole

2017-01-01

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. PMID:29281732

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

PubMed Central

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

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

PubMed

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.

Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

PubMed

Liu, J; Wang, Y; Szalay, A A; Escher, A

2000-01-01

We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells. Copyright 2000 John Wiley & Sons, Ltd.

Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

PubMed Central

Curto, M.-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M.-Henar

2014-01-01

The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell–cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell–cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell–cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

PubMed Central

Tsai, Wen-Yang; Hsieh, Szu-Chia; Lai, Chih-Yun; Lin, Hong-En; Nerurkar, Vivek R.; Wang, Wei-Kung

2012-01-01

Background The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines. Methodology/Principal Findings In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested. Conclusions/Significance A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response. PMID:23300717

Potential Pitfalls and Solutions for Use of Fluorescent Fusion Proteins to Study the Lysosome

PubMed Central

Huang, Ling; Pike, Douglas; Sleat, David E.; Nanda, Vikas; Lobel, Peter

2014-01-01

Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications. PMID:24586430

Impact of fluorescent protein fusions on the bacterial flagellar motor.

PubMed

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.

PubMed

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.

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

PubMed

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.

Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions.

PubMed

Hentz, N G; Daunert, S

1996-11-15

An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

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

PubMed Central

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

High level expression, purification and characterization of active fusion human C1q and tumor necrosis factor related protein 2 (hCTRP2) in Escherichia coli.

PubMed

Li, Hongbo; Gao, Xuefei; Zhou, Yi; Li, Na; Ge, Caozuo; Hui, Xiaoyan; Wang, Yu; Xu, Aimin; Jin, Shouguang; Wu, Donghai

2011-09-01

C1q and tumor necrosis factor related proteins (CTRPs) are a family of adiponectin paralogues. Among them, CTRP2 is the only CTRP protein that has been shown to possess similar biological activities as adiponectin. To further explore the physiological roles of human CTRP2 and its mechanisms of action, hCTRP2 gene was expressed in Escherichia coli and Pichia pastoris, respectively. In the P. pastoris expression system, recombinant hCTRP2 could be secreted into the culture medium under induction condition, however, the resultant recombinant protein was highly unstable, resulting two main degradation products with molecular masses of approximately 20 and 26 kDa, respectively. In the E. coli expression system, a large amount of soluble thioredoxin (Trx)-hCTRP2 fusion protein could be produced, which accounts about 42% of the total soluble bacterial proteins. The recombinant Trx-hCTRP2 fusion protein was purified to an approximately 95% purity using Ni-NTA affinity chromatography and Superdex G-75 column with a yield of about 15 mg/l protein from 1l bacterial culture. The purified recombinant Trx-hCTRP2 was shown to be active under in vitro assay conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

[Expression of the fusion protein CFP10-ESAT6 of Mycobacterium tuberculosis and the study of its immunogenicity].

PubMed

Wang, Xiao-ying; Bao, Lang; Zhao, Ming-cai; Zhang, Hui-dong; Long, Yang

2006-05-01

To express a recombinant fusion protein CFP10-ESAT6 of Mycobacterium tuberculosis, and obtain the polyclonal antibodies of this fusion protein by immune rabbit. The 630 bp cfpl0-esat6 fusion gene fragments were amplified from the genomic DNA of a Mycobacterium tuberculosis reference strain H37Rv and inserted into the expression plasmid pET32a (+) to generate the recombinant plasmid pET-cfp10-esat6. The recombinat expression plasmid was transformed into E. coli BL21 (DE3). The fused protein CFP10-ESAT6 with His-tag was expressed after inducing with IPTG and purified with affinity chromatography. This protein was used to immune the rabbit to obtained the polyclonal antibodies, and been analyzed with Western-blot and ELISA. The recombinant plasmid pET-cfp10-esat6 was success fully constructed, the recombinant fusion protein CFP10-ESAT6 could be expressed at relatively high levels, and the polyclonal antibodies of fusion protein were obtained. The successful construction and expression of the recombinant fusion protein CFP10-ESAT6 and the obtained polyclonal antibodies will be very helpful for the development of new anti-tuberculosis vaccine and the clinical serologic diagnosis.

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

PubMed

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.

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity.

PubMed

Lee, Jinwoo; Nyenhuis, David A; Nelson, Elizabeth A; Cafiso, David S; White, Judith M; Tamm, Lukas K

2017-09-19

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.

Membrane proteins in human erythrocytes during cell fusion induced by oleoylglycerol

PubMed Central

Quirk, Susan J.; Ahkong, Quet Fah; Botham, Gaynor M.; Vos, Jan; Lucy, Jack A.

1978-01-01

1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca2+ used, at least 80% of the cells fused after 30min at 37°C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in `ghosts' prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca2+, although some cells fused when no exogenous Ca2+ was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of `ghosts' prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1–2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca2+ into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed. ImagesPLATE 4PLATE 1PLATE 2PLATE 3 PMID:728105

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Tae-Joon; Hahn, Ji-Sook

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 expressionmore » 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.« less

Fusion proteins as alternate crystallization paths to difficult structure problems

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; Rueker, Florian; Ho, Joseph X.; Lim, Kap; Keeling, Kim; Gilliland, Gary; Ji, Xinhua

1994-01-01

The three-dimensional structure of a peptide fusion product with glutathione transferase from Schistosoma japonicum (SjGST) has been solved by crystallographic methods to 2.5 A resolution. Peptides or proteins can be fused to SjGST and expressed in a plasmid for rapid synthesis in Escherichia coli. Fusion proteins created by this commercial method can be purified rapidly by chromatography on immobilized glutathione. The potential utility of using SjGST fusion proteins as alternate paths to the crystallization and structure determination of proteins is demonstrated.

Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions

PubMed Central

2015-01-01

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. PMID:25265085

Enhance tumor radiosensitivity by intracellular delivery of eukaryotic translation initiation factor 4E binding proteins.

PubMed

Tian, Shuang; Li, Xiu-Li; Shi, Mei; Yao, Yuan-Qing; Li, Li-Wen; Xin, Xiao-Yan

2011-02-01

PTEN (phosphatase and tensin homologue deleted on chromosome ten)/PI3K (phosphatidylinositol 3-kinase)/Akt/mTOR (mammalian target of rapamycin) signaling pathway, which is commonly dysregulated in a broad array of human malignancies, controls the assembly of eukaryotic translation initiation factor 4F (eIF4F) complex through regulation of eIF4E binding proteins (4E-BPs) phosphorylation. And accumulated data over the past two decades implicated eIF4F complex as one of the promising targets for anticancer therapy. It has been confirmed that the translation initiation of mRNA coding for hypoxia-inducible factor-1α (HIF-1α) and survivin, which had been considered as the two major determinants of tumor radiosensitivity, are both controlled by eIF4F complex. Also, eIF4F complex controls the expression of VEGF and bFGF, the two well-known pro-angiogenic factors involved in developing radioresistance. Therefore eIF4F complex plays a pivotal role in regulation of radiosensitivity. In this article, we postulate that cell-permeable, phosphorylation-defective 4E-BP fusion proteins, which could be prepared by substituting the mTOR recognition motif located in N-terminal of 4E-BPs with protein transduction domain from HIV-1 TAT, HSV-1 VP22 or PTD4, could not only inhibit tumor growth but also enhance tumor response to radiation therapy through disruption of eIF4F complex assembly. In our opinion, the recombinant fusion proteins are superior to mTOR inhibitors for they do not cause immunosuppression, do not lead to Akt activation, and could be easily prepared by prokaryotic expression. If the hypothesis was proved to be practical, the cell-permeable, phosphorylation-defective 4E-BP fusion proteins would be widely used in clinical settings to improve tumor response to radiotherapy in the near future. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

PubMed Central

Clifton, Matthew C.; Dranow, David M.; Leed, Alison; Fulroth, Ben; Fairman, James W.; Abendroth, Jan; Atkins, Kateri A.; Wallace, Ellen; Fan, Dazhong; Xu, Guoping; Ni, Z. J.; Daniels, Doug; Van Drie, John; Wei, Guo; Burgin, Alex B.; Golub, Todd R.; Hubbard, Brian K.; Serrano-Wu, Michael H.

2015-01-01

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. PMID:25909780

Viral membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 formedmore » 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.« less

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

PubMed

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.

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

PubMed

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.

Elimination of established murine colon carcinoma metastases by antibody-interleukin 2 fusion protein therapy.

PubMed

Xiang, R; Lode, H N; Dolman, C S; Dreier, T; Varki, N M; Qian, X; Lo, K M; Lan, Y; Super, M; Gillies, S D; Reisfeld, R A

1997-11-01

A recombinant humanized antibody-interleukin 2 fusion protein (huKS1/4-IL-2) was used to direct IL-2 to the tumor microenvironment and elicit a T cell-mediated eradication of established pulmonary and hepatic CT26-KSA colon carcinoma metastases in syngeneic BALB/c mice. This antitumor effect was specific because a fusion protein, which was nonreactive with these tumor cells, failed to exert any such effect. The efficacy of the huKS1/4-IL-2 fusion protein in eliminating metastases was documented because mixtures of monoclonal antibody huKS1/4 with recombinant human IL-2 were ineffective and, at best, only partially reduced tumor load. Two lines of evidence indicated the eradication of metastases and the absence of minimal residual disease in animals treated with the fusion protein: first, the lack of detection of CT26-KSA cells by reverse transcription-PCR, which can detect one tumor cell in 10(6) liver cells; and second, the tripling of life span. The effector mechanism involved in this tumor eradication is dependent on T cells because the IL-2-directed therapy is ineffective in T cell-deficient SCID mice. The essential effector cells were further characterized as CD8+ T cells by in vivo depletion studies. Such T cells, isolated from tumor-bearing mice after fusion protein therapy, elicited MHC class I-restricted cytotoxicity in vitro against colon carcinoma target cells. Taken together, these data indicate that fusion protein-directed IL-2 therapy induces a T cell-dependent host immune response capable of eradicating established colon cancer metastases in an animal tumor model.

Full-Length Trimeric Influenza Virus Hemagglutinin II Membrane Fusion Protein and Shorter Constructs Lacking the Fusion Peptide or Transmembrane Domain: Hyperthermostability of the Full-Length Protein and the Soluble Ectodomain and Fusion Peptide Make Significant Contributions to Fusion of Membrane Vesicles†

PubMed Central

Ratnayake, Punsisi U.; Ekanayaka, E. A. Prabodha; Komanduru, Sweta S.; Weliky, David P.

2015-01-01

Influenza virus is a Class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5–6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ~25, ~160, ~25, and ~10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP + SE, and SHA2-TM ≡ SE + TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm > 90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM. PMID:26297995

Full-length trimeric influenza virus hemagglutinin II membrane fusion protein and shorter constructs lacking the fusion peptide or transmembrane domain: Hyperthermostability of the full-length protein and the soluble ectodomain and fusion peptide make significant contributions to fusion of membrane vesicles.

PubMed

Ratnayake, Punsisi U; Prabodha Ekanayaka, E A; Komanduru, Sweta S; Weliky, David P

2016-01-01

Influenza virus is a class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5-6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ∼ 25, ∼ 160, ∼ 25, and ∼ 10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP+SE, and SHA2-TM ≡ SE+TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm>90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM. Copyright © 2015 Elsevier Inc. All rights reserved.

The Multifaceted Role of SNARE Proteins in Membrane Fusion

PubMed Central

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. PMID:28163686

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

PubMed

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.

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.

PubMed

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.

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

PubMed

Corcoran, Jennifer A; Salsman, Jayme; de Antueno, Roberto; Touhami, Ahmed; Jericho, Manfred H; Clancy, Eileen K; Duncan, Roy

2006-10-20

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Expression of chimeric ras protein with OmpF signal peptide in Escherichia coli: localization of OmpF fusion protein in the inner membrane.

PubMed

Yamamoto, T; Okawa, N; Endo, T; Kaji, A

1991-08-01

The ras gene was fused with the DNA sequence of OmpF signal peptide or with the DNA sequence of OmpF signal peptide plus the amino terminal portion of the OmpF gene. They were placed in plasmids together with the bacteriophage lambda PL promoter. These plasmids were introduced into Escherichia coli strain K-12 and the OmpF signal peptide fusion proteins were expressed. These fusion proteins were identified as 29.0 and 30.0 kDa proteins. However, processed products of these proteins were not found in the extract. The fusion proteins were localized mostly in the cytoplasm and the inner membrane, but none of them was secreted into the periplasmic space. On the other hand, the ras protein alone was found in the cytoplasm and not in the inner membrane. Viable counts of E. coli harbouring these plasmids decreased when these fused proteins were induced. Induction of the ras protein alone did not harm cells. These observations suggest that insertion of the heterologous proteins into the inner membrane may cause the bactericidal effect.

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

PubMed

Jossé, Lyne; Xie, Jianling; Proud, Christopher G; Smales, C Mark

2016-12-15

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1. © 2016 The Author(s).

Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

PubMed

Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

2013-12-13

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

NASA Astrophysics Data System (ADS)

Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

1990-04-01

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

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

NASA Technical Reports Server (NTRS)

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.

Conserved Glycine Residues in the Fusion Peptide of the Paramyxovirus Fusion Protein Regulate Activation of the Native State

PubMed Central

Russell, Charles J.; Jardetzky, Theodore S.; Lamb, Robert A.

2004-01-01

Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation. PMID:15564482

Analysis of Snail1 function and regulation by Twist1 in palatal fusion.

PubMed

Yu, Wenli; Zhang, Yanping; Ruest, L Bruno; Svoboda, Kathy K H

2013-01-01

Palatal fusion is a tightly controlled process which comprises multiple cellular events, including cell movement and differentiation. Midline epithelial seam (MES) degradation is essential to palatal fusion. In this study, we analyzed the function of Snail1 during the degradation of the MES. We also analyzed the mechanism regulating the expression of the Snail1 gene in palatal shelves. Palatal explants treated with Snail1 siRNA did not degrade the MES and E-cadherin was not repressed leading to failure of palatal fusion. Transforming growth factor beta 3 (Tgfβ3) regulated Snail1 mRNA, as Snail1 expression decreased in response to Tgfβ3 neutralizing antibody and a PI-3 kinase (PI3K) inhibitor. Twist1, in collaboration with E2A factors, regulated the expression of Snail1. Twist1/E47 dimers bond to the Snail1 promoter to activate expression. Without E47, Twist1 repressed Snail1 expression. These results support the hypothesis that Tgfβ3 may signal through Twist1 and then Snail1 to downregulate E-cadherin expression during palatal fusion.

Targeting of Cytolytic T-Cells for Breast Cancer Therapy Using Novel-Fusion Proteins

DTIC Science & Technology

1999-07-01

1 construct was subsequently subcloned into the Pichia pastoris expression plasmid pPICZcxB (Invitrogen) which contains the alcohol oxidase promoter...breast carcinomas, and the extracellular domain of B7.2 (CD86). This fusion protein was expressed and purified from Pichia pastoris, shown to retain...year’s report, the hB7.2/B1 chimeric fusion protein produced in Pichia pastoris, was shown to bind to both recombinant and cell surface tumor marker erbB

Development of a homogeneous immunoassay system using protein A fusion fragmented Renilla luciferase.

PubMed

Mie, Masayasu; Thuy, Ngo Phan Bich; Kobatake, Eiry

2012-03-07

A homogeneous immunoassay system was developed using fragmented Renilla luciferase (Rluc). The B domain of protein A was fused to two Rluc fragments. When complexes between an antibody and fragmented Rluc fusion proteins bind to target molecules, the Rluc fragments come into close proximity and the luminescence activity of fragmented Rluc is restored by complementation. As proof-of-principle, this fragmented Rluc system was used to detect E. coli homogeneously using an anti-E. coli antibody.

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

PubMed

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.

Function of fusion regulatory proteins (FRPs) in immune cells and virus-infected cells.

PubMed

Tsurudome, M; Ito, Y

2000-01-01

Two molecules that regulate cell fusion have been identified and designated fusion regulatory protein-1 (FRP-1) and FRP-2. FRP-1 is a complex composed of a glycosylated heavy chain and a nonglycosylated light chain that are disulfide linked. FRP-1 heavy chain is identical to 4F2/CD98 heavy chain, whereas FRP-2 is identical to integrin alpha3 subunit. The FRP-1 heavy chain is a multifunctional molecule: that is, fusion regulator, amino acid transporter, integrin regulator, comitogenic factor, Na+-Ca2+ exchanger, oncogenic protein, and so on. Several aspects of the structure and function of the FRP-1 system are reviewed: fusion regulatory molecular mechanisms, cross-talk between the FRP-1 and integrin, the FRP-1 system as amino acid transporter, and FRP-1-mediated T-cell activation. The FRP-1 system is involved in virus-mediated cell fusion and multinucleated giant cell formation of blood monocytes. Monoclonal antibodies against human FRP-1 heavy chain induce polykaryocytes that have properties as osteoclasts. Multiple steps participate in molecular mechanisms regulating cell fusion. The FRP-1 heavy chain supports amino acid transport activity and the FRP-1 light chains have recently been cloned as amino acid transporters that require association with the heavy chain to exhibit their activity. Novel pathways for monocyte-dependent regulation of T-cell activation have recently been found that are mediated by the FRP-1 system. In conclusion, the FRP-1 molecules are essential factors for basic cellular functions.

Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro

PubMed Central

Webb, Stacy R.; Smith, Stacy E.; Fried, Michael G.

2018-01-01

ABSTRACT Enveloped viruses require viral fusion proteins to promote fusion of the viral envelope with a target cell membrane. To drive fusion, these proteins undergo large conformational changes that must occur at the right place and at the right time. Understanding the elements which control the stability of the prefusion state and the initiation of conformational changes is key to understanding the function of these important proteins. The construction of mutations in the fusion protein transmembrane domains (TMDs) or the replacement of these domains with lipid anchors has implicated the TMD in the fusion process. However, the structural and molecular details of the role of the TMD in these fusion events remain unclear. Previously, we demonstrated that isolated paramyxovirus fusion protein TMDs associate in a monomer-trimer equilibrium, using sedimentation equilibrium analytical ultracentrifugation. Using a similar approach, the work presented here indicates that trimeric interactions also occur between the fusion protein TMDs of Ebola virus, influenza virus, severe acute respiratory syndrome coronavirus (SARS CoV), and rabies virus. Our results suggest that TM-TM interactions are important in the fusion protein function of diverse viral families. IMPORTANCE Many important human pathogens are enveloped viruses that utilize membrane-bound glycoproteins to mediate viral entry. Factors that contribute to the stability of these glycoproteins have been identified in the ectodomain of several viral fusion proteins, including residues within the soluble ectodomain. Although it is often thought to simply act as an anchor, the transmembrane domain of viral fusion proteins has been implicated in protein stability and function as well. Here, using a biophysical approach, we demonstrated that the fusion protein transmembrane domains of several deadly pathogens—Ebola virus, influenza virus, SARS CoV, and rabies virus—self-associate. This observation across various viral

Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

PubMed Central

Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

2016-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

Transforming properties of the Huntingtin interacting protein 1/ platelet-derived growth factor beta receptor fusion protein.

PubMed

Ross, T S; Gilliland, D G

1999-08-06

We have previously reported that the Huntingtin interacting protein 1 (HIP1) gene is fused to the platelet-derived growth factor beta receptor (PDGFbetaR) gene in a patient with chronic myelomonocytic leukemia. We now show that HIP1/PDGFbetaR oligomerizes, is constitutively tyrosine-phosphorylated, and transforms the murine hematopoietic cell line, Ba/F3, to interleukin-3-independent growth. A kinase-inactive mutant is neither tyrosine-phosphorylated nor able to transform Ba/F3 cells. Oligomerization and kinase activation required the 55-amino acid carboxyl-terminal TALIN homology region but not the leucine zipper domain. Tyrosine phosphorylation of a 130-kDa protein and STAT5 correlates with transformation in cells expressing HIP1/PDGFbetaR and related mutants. A deletion mutant fusion protein that contains only the TALIN homology region of HIP1 fused to PDGFbetaR is incapable of transforming Ba/F3 cells and does not tyrosine-phosphorylate p130 or STAT5, although it is itself constitutively tyrosine-phosphorylated. We have also analyzed cells expressing Tyr --> Phe mutants of HIP1/PDGFbetaR in the known PDGFbetaR SH2 docking sites and report that none of these sites are necessary for STAT5 activation, p130 phosphorylation, or Ba/F3 transformation. The correlation of factor-independent growth of hematopoietic cells with p130 and STAT5 phosphorylation/activation in both the HIP1/PDGFbetaR Tyr --> Phe and deletion mutational variants suggests that both STAT5 and p130 are important for transformation mediated by HIP1/PDGFbetaR.

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

PubMed

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.

Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

PubMed Central

Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

2016-01-01

Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

PubMed Central

Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

2011-01-01

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader.

PubMed

Duellman, Tyler; Burnett, John; Yang, Jay

2015-03-15

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9-mCherry fusion protein was detected 6h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9-mCherry secretion, demonstrating the utility of this method in a biological experiment. Copyright © 2014 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ebrahimi, Seyyed Mahmoud, E-mail: smebrahimi@shirazu.ac.ir; Research Center of Virus and Vaccine, Baqiyatallah University of Medical Science, P.O.Box 14155-3651, Tehran; Dabaghian, Mehran

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 M2more » (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.« less

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

PubMed

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.

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

PubMed Central

Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

2014-01-01

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

[Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

PubMed

Song, Xiaoli; Liu, Xiaoyun; Cai, Lei; Wu, Jianwei; Wang, Jihua

2015-12-01

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

Subcellular localization of transiently expressed fluorescent fusion proteins.

PubMed

Collings, David A

2013-01-01

The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

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

PubMed

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.

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

PubMed Central

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

2013-01-01

It is increasingly recognized that chimeric RNAs may exert a novel layer of cellular complexity that contributes to oncogenesis and cancer progression, and could be utilized as molecular biomarkers and therapeutic targets. To date yet no fusion chimeric RNAs have been identified in esophageal cancer, the 6th most frequent cause of cancer death in the world. While analyzing the expression of 32 recurrent cancer chimeric RNAs in esophageal squamous cell carcinoma (ESCC) from patients and cancer cell lines, we identified GOLM1-MAK10, as a highly cancer-enriched chimeric RNA in ESCC. In situ hybridization revealed that the expression of the chimera is largely restricted to cancer cells in patient tumors, and nearly undetectable in non-neoplastic esophageal tissue from normal subjects. The aberrant chimera closely correlated with histologic differentiation and lymph node metastasis. Furthermore, we demonstrate that chimera GOLM1-MAK10 encodes a secreted fusion protein. Mechanistic studies reveal that GOLM1-MAK10 is likely derived from transcription read-through/splicing rather than being generated from a fusion gene. Collectively, these findings provide novel insights into the molecular mechanism involved in ESCC and provide a novel potential target for future therapies. The secreted fusion protein translated from GOLM1-MAK10 could also serve as a unique protein signature detectable by standard non-invasive assays. These observations are critical as there is no clinically useful molecular signature available for detecting this deadly disease or monitoring the treatment response. PMID:24243830

Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein.

PubMed

Kaushik, Himani; Deshmukh, Sachin; Mathur, Deepika Dayal; Tiwari, Archana; Garg, Lalit C

2013-01-01

Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM1 ganglioside receptor of LTB. The rLTB.Etx40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens. aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli.

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

PubMed

Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

2011-05-01

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied

Structure of the Newcastle disease virus F protein in the post-fusion conformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swanson, Kurt; Wen, Xiaolin; Leser, George P.

2010-11-17

The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure containsmore » heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.« less

Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yechun; Yi, Hankuil; Wang, Melissa

2012-10-24

To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the firstmore » molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.« less

Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

PubMed Central

Ader, Nadine; Brindley, Melinda; Avila, Mislay; Örvell, Claes; Horvat, Branka; Hiltensperger, Georg; Schneider-Schaulies, Jürgen; Vandevelde, Marc; Zurbriggen, Andreas; Plemper, Richard K.

2013-01-01

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering. PMID:23077316

Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

PubMed

Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

2012-10-09

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein

PubMed Central

Welch, Brett D.; Liu, Yuanyuan; Kors, Christopher A.; Leser, George P.; Jardetzky, Theodore S.; Lamb, Robert A.

2012-01-01

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein. PMID:23012473

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella typhimurium.

PubMed

Bargieri, Daniel Y; Leite, Juliana A; Lopes, Stefanie C P; Sbrogio-Almeida, Maria Elisabete; Braga, Catarina J M; Ferreira, Luis C S; Soares, Irene S; Costa, Fabio T M; Rodrigues, Mauricio M

2010-04-01

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His(6)FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund's adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP1(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Elastin-like-polypeptide based fusion proteins for osteogenic factor delivery in bone healing.

PubMed

McCarthy, Bryce; Yuan, Yuan; Koria, Piyush

2016-07-08

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein-2 (BMP-2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP-2 is necessary, here we demonstrate the viability of an elastin-like peptide (ELP) fusion protein containing BMP-2 for delivery of the BMP-2. This fusion protein retains the performance characteristics of both the BMP-2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self-aggregating nanoparticles at human-body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage-efficient and location-precise noncytotoxic delivery vehicle for BMP-2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029-1037, 2016. © 2016 American Institute of Chemical Engineers.

Identification of novel target sites and an inhibitor of the dengue virus E protein.

PubMed

Yennamalli, Ragothaman; Subbarao, Naidu; Kampmann, Thorsten; McGeary, Ross P; Young, Paul R; Kobe, Bostjan

2009-06-01

Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally diverse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC(50) in the micromolar range against dengue virus type 2.

Identification of novel target sites and an inhibitor of the dengue virus E protein

NASA Astrophysics Data System (ADS)

Yennamalli, Ragothaman; Subbarao, Naidu; Kampmann, Thorsten; McGeary, Ross P.; Young, Paul R.; Kobe, Bostjan

2009-06-01

Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally diverse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC50 in the micromolar range against dengue virus type 2.

Fusion peptides from oncogenic chimeric proteins as putative specific biomarkers of cancer.

PubMed

Conlon, Kevin P; Basrur, Venkatesha; Rolland, Delphine; Wolfe, Thomas; Nesvizhskii, Alexey I; MacCoss, Michael J; Lim, Megan S; Elenitoba-Johnson, Kojo S J

2013-10-01

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

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

PubMed

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.

Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

PubMed

Khalid, Ruqyya; Afzal, Madeeha; Khurshid, Sana; Paracha, Rehan Zafar; Khan, Imran H; Akhtar, Muhammad Waheed

Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and β-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli.

Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

PubMed

Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

2017-01-01

Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

[Construction and expression of a eukaryotic expression vector containing human CR2-Fc fusion protein].

PubMed

Li, Xinxin; Wu, Zhihao; Zhang, Chuanfu; Jia, Leili; Song, Hongbin; Xu, Yuanyong

2014-01-01

To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.

Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

PubMed

Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

2013-06-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

PubMed

Li, Zhuo; Hung, Cher; Paterson, Reay G; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J; Lamb, Robert A; He, Biao

2015-10-06

Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

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.

PubMed

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.

Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit are Critical for its Fusion Activity.

PubMed

Hicks, Stephanie N; Chaiwatpongsakorn, Supranee; Costello, Heather M; McLellan, Jason S; Ray, William; Peeples, Mark E

2018-05-09

The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both viral-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F 1 and F 2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F 1 subunit to refold dramatically while F 2 changes minimally. Hypothesizing that the most likely site for interaction with a target-cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F 2 by scanning mutagenesis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting they are involved in refolding. Alanine mutation at Ile64, also on the ascent to the apex, and Lys75, did not prevent fusion following forced triggering, suggesting they are not involved in refolding and may instead be involved in the natural triggering of the F protein. IMPORTANCE RSV infects virtually every child by the age of 3, causing nearly 33 million acute lower respiratory infections (ALRI) worldwide each year in children younger than 5 (Nair H, et al. 2010. Lancet 375:1545-55). RSV is also the second leading cause of respiratory related death in the elderly (Falsey AR, Walsh EE. 2005. Drugs Aging 22:577-87; Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE

The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein.

PubMed

Fédry, Juliette; Liu, Yanjie; Péhau-Arnaudet, Gérard; Pei, Jimin; Li, Wenhao; Tortorici, M Alejandra; Traincard, François; Meola, Annalisa; Bricogne, Gérard; Grishin, Nick V; Snell, William J; Rey, Félix A; Krey, Thomas

2017-02-23

Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

PubMed

Heine, Thomas; Tucker, Kathryn; Okonkwo, Nonye; Assefa, Berhanegebriel; Conrad, Catleen; Scholtissek, Anika; Schlömann, Michael; Gassner, George; Tischler, Dirk

2017-04-01

The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s -1 . This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

Distinct roles for key karyogamy proteins during yeast nuclear fusion.

PubMed

Melloy, Patricia; Shen, Shu; White, Erin; Rose, Mark D

2009-09-01

During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.

Impairment of autophagosome-lysosome fusion in the buff mutant mice with the VPS33AD251E mutation

PubMed Central

Zhen, Yuanli; Li, Wei

2015-01-01

The HOPS (homotypic fusion and protein sorting) complex functions in endocytic and autophagic pathways in both lower eukaryotes and mammalian cells through its involvement in fusion events between endosomes and lysosomes or autophagosomes and lysosomes. However, the differential molecular mechanisms underlying these fusion processes are largely unknown. Buff (bf) is a mouse mutant that carries an Asp251-to-Glu point mutation (D251E) in the VPS33A protein, a tethering protein and a core subunit of the HOPS complex. Bf mice showed impaired spontaneous locomotor activity, motor learning, and autophagic activity. Although the gross anatomy of the brain was apparently normal, the number of Purkinje cells was significantly reduced. Furthermore, we found that fusion between autophagosomes and lysosomes was defective in bf cells without compromising the endocytic pathway. The direct association of mutant VPS33AD251E with the autophagic SNARE complex, STX17 (syntaxin 17)-VAMP8-SNAP29, was enhanced. In addition, the VPS33AD251E mutation enhanced interactions with other HOPS subunits, namely VPS41, VPS39, VPS18, and VPS11, except for VPS16. Reduction of the interactions between VPS33AY440D and several other HOPS subunits led to decreased association with STX17. These results suggest that the VPS33AD251E mutation plays dual roles by increasing the HOPS complex assembly and its association with the autophagic SNARE complex, which selectively affects the autophagosome-lysosome fusion that impairs basal autophagic activity and induces Purkinje cell loss. PMID:26259518

In vitro translocation experiments with RxLR-reporter fusion proteins of Avr1b from Phytophthora sojae and AVR3a from Phytophthora infestans fail to demonstrate specific autonomous uptake in plant and animal cells.

PubMed

Wawra, Stephan; Djamei, Armin; Albert, Isabell; Nürnberger, Thorsten; Kahmann, Regine; van West, Pieter

2013-05-01

Plant-pathogenic oomycetes have a large set of secreted effectors that can be translocated into their host cells during infection. One group of these effectors are the RxLR effectors for which it has been shown, in a few cases, that the RxLR motif is important for their translocation. It has been suggested that the RxLR-leader sequences alone are enough to translocate the respective effectors into eukaryotic cells through binding to surface-exposed phosphoinositol-3-phosphate. These conclusions were primary based on translocation experiments conducted with recombinant fusion proteins whereby the RxLR leader of RxLR effectors (i.e., Avr1b from Phytophthora sojae) were fused to the green fluorescent protein reporter-protein. However, we failed to observe specific cellular uptake for a comparable fusion protein where the RxLR leader of the P. infestans AVR3a was fused to monomeric red fluorescent protein. Therefore, we reexamined the ability of the reported P. sojae AVR1b RxLR leader to enter eukaryotic cells. Different relevant experiments were performed in three independent laboratories, using fluorescent reporter fusion constructs of AVR3a and Avr1b proteins in a side-by-side comparative study on plant tissue and human and animal cells. We report that we were unable to obtain conclusive evidence for specific RxLR-mediated translocation.

A fusion-protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

PubMed

Hu, Jia; Chen, Xiang; Zhang, Xuhua; Yuan, Xiaopeng; Yang, Mingjuan; Dai, Hui; Yang, Wei; Zhou, Qinghua; Wen, Weihong; Wang, Qirui; Qin, Weijun; Zhao, Aizhi

2018-05-01

A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (V H ) and light (V L ) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development. © 2018 The Protein Society.

Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

PubMed

de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

2013-04-07

We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

PubMed

Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

2015-07-01

Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.

Macrophage Fusion Is Controlled by the Cytoplasmic Protein Tyrosine Phosphatase PTP-PEST/PTPN12

PubMed Central

Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean

2013-01-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading. PMID:23589331

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

NASA Astrophysics Data System (ADS)

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.

TRAIL-CM4 fusion protein shows in vitro antibacterial activity and a stronger antitumor activity than solo TRAIL protein.

PubMed

Sang, Ming; Zhang, Jiaxin; Li, Bin; Chen, Yuqing

2016-06-01

A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan blue staining and MTT analyses showed that, similar to a TRAIL positive control, Jurkat cell proliferation was significantly inhibited. Flow cytometry analyses using Annexin V-fluorescein revealed that Jurkat cells treated with the TRAIL-CM4 fusion protein exhibited increased apoptosis. Laser confocal microscopy showed that APB-CM4 and the fusion protein TRAIL-CM4 can bind to Jurkat cell membranes and initiate their destruction. ABP-CM4 enhances the antitumor activity of TRAIL by targeting and damaging the tumor cell membrane. In antibacterial experiments, agar well diffusion and bacterial growth inhibition curve assays revealed concentration-dependent TRAIL-CM4 antibacterial activity against Escherichia coli K12D31. The expressed TRAIL-CM4 fusion protein exhibited enhanced antitumor and antibacterial activities. Fusion protein expression allowed the two different proteins to function in combination. Copyright © 2016 Elsevier Inc. All rights reserved.

Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

PubMed

Liu, Songling; Premont, Richard T; Rockey, Don C

2014-06-27

Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteins on exocytic vesicles mediate calcium-triggered fusion.

PubMed Central

Vogel, S S; Zimmerberg, J

1992-01-01

In many exocytic systems, micromolar concentrations of intracellular Ca2+ trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with greater than or equal to 10 microM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca(2+)-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous. Images PMID:1584814

Generation of fusion protein EGFRvIII-HBcAg and its anti-tumor effect in vivo

PubMed Central

Duan, Xiao-yi; Han, Dong-gang; Zhang, Ming-xin; Wang, Jian-sheng

2009-01-01

The epidermal growth factor receptor variant III (EGFRvIII) is the most common variation of EGFR. Because it shows a high frequency in several different types of tumor and has not been detected in normal tissues, it is an ideal target for tumor specific therapy. In this study, we prepared EGFRvIII-HBcAg fusion protein. After immunization with fusion protein, HBcAg or PBS, the titers of antibody in BALB/c mice immunized with fusion protein reached 2.75 × 105. Western blot analysis demonstrated that the fusion protein had specific antigenicity against anti-EGFRvIII antibody. Further observation showed fusion protein induced a high frequency of IFN-γ-secreting lymphocytes. CD4+T cells rather than CD8+T cells were associated with the production of IFN-γ. Using Renca-vIII(+) cell as specific stimulator, we observed remarkable cytotoxic activity in splenocytes from mice immunized with fusion protein. Mice were challenged with Renca-vIII(+) cells after five times immunization. In fusion protein group, three of ten mice failed to develop tumor and all survived at the end of the research. The weight of tumors in fusion protein were obviously lighter than that in other two groups (t = 4.73, P = 0.044;t = 6.89, P = 0.040). These findings demonstrated that EGFRvIII-HBcAg fusion protein triggered protective responses against tumor expressing EGFRvIII. PMID:19788747

HaloTag technology for specific and covalent labeling of fusion proteins.

PubMed

Benink, Hélène A; Urh, Marjeta

2015-01-01

Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions.

Recombinant fusion protein and DNA vaccines against foot and mouth disease virus infection in guinea pig and swine.

PubMed

Huang, H; Yang, Z; Xu, Q; Sheng, Z; Xie, Y; Yan, W; You, Y; Sun, L; Zheng, Z

1999-01-01

In this study, we provide evidence that a recombinant fusion protein containing beta-galactosidase and a tandem repeat peptide of immunogenic dominant epitope of foot-and-mouth disease virus (FMDV) VP1 protein elicits high levels of neutralizing antibody and protects both guinea pigs and swine against infection. Vaccination with this fusion protein induced a FMDV-specific proliferative T-cell response and a neutralizing antibody response. The immunized guinea pigs and swine were protected against FMD type O virus infection. Two DNA plasmids expressing genes of foot-and-mouth disease were constructed. Both plasmids pBO1 and pCO1 contain a signal sequence of the swine immunoglobulin G (IgG) gene and fusion protein gene of pXZ84. The signal sequence and fusion protein gene were under the control of a metallothionein promoter in the case of the pBO1 plasmid and under the control of a cytomegalovirus immediate early promoter in the case of pCO1 plasmid. When pBO1 and pCO1 were inoculated intramuscularly into guinea pigs, both plasmids elicited a neutralizing antibody response and spleen cell proliferation increased following stimulation with FMDV antigen, but animals were not protected from viral challenge.

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

PubMed

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.

Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system.

PubMed

Guo, Jun-Qing; Li, Qing-Mei; Zhou, Ji-Yong; Zhang, Gai-Ping; Yang, Yan-Yan; Xing, Guang-Xu; Zhao, Dong; You, Shang-You; Zhang, Chu-Yu

2006-01-01

A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.

Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins.

PubMed

Inobe, Tomonao; Nukina, Nobuyuki

2016-07-01

Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addition of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biological applications. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a.

PubMed

Miranda, Tina Branscombe; Webb, Kristofor J; Edberg, Dale D; Reeves, Raymond; Clarke, Steven

2005-10-28

The HMGA family proteins HMGA1a and HMGA1b are nuclear nonhistone species implicated in a wide range of cellular processes including inducible gene transcription, modulation of chromosome structure through nucleosome and chromosome remodeling, and neoplastic transformation. HMGA proteins are highly modified, and changes in their phosphorylation states have been correlated with the phase of the cell cycle and changes in their transcriptional activity. HMGA1a is also methylated in the first DNA-binding AT-hook at Arg25 and other sites, although the enzyme or enzymes responsible have not been identified. We demonstrate here that a GST fusion of protein arginine methyltransferase 6 (PRMT6) specifically methylates full-length recombinant HMGA1a protein in vitro. Although GST fusions of PRMT1 and PRMT3 were also capable of methylating the full-length HMGA1a polypeptide, they recognize its proteolytic degradation products much better. GST fusions of PRMT4 or PRMT7 were unable to methylate the full-length protein or its degradation products. We conclude that PRMT6 is a good candidate for the endogenous enzyme responsible for HGMA1a methylation.

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

PubMed

Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

2018-02-01

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

Epididymal secreted protein Crisp-1 and sperm function.

PubMed

Roberts, Kenneth P; Ensrud, Kathy M; Wooters, Joseph L; Nolan, Michael A; Johnston, Daniel S; Hamilton, David W

2006-05-16

Crisp-1 is a member of the cysteine-rich secretory protein family. This family of proteins is characterized by the presence of 16 conserved cysteine residues, the characteristic from which the family name is derived. Members of the Crisp protein family are found in the secretions of the reproductive tract and salivary glands, including venom toxins from several species of snakes and lizards. The Crisp proteins are modular, each containing an amino terminal pathogenesis-related (PR)-like domain and a carboxyl terminal cysteine-rich domain (CRD) connected by a hinge region. Sequence and structural similarities to proteins with known functions suggest that the Crisp family of proteins may act by regulating cellular ion channels. Rat Crisp-1 is synthesized as two distinct isoforms (referred to as Proteins D and E) by the epididymal epithelium and both are secreted into the luminal fluid where they interact with spermatozoa. Our laboratory has correlated Crisp-1 binding to sperm with inhibiting the signaling cascades that initiate capacitation while others have shown that blocking Crisp-1 binding sites on oocytes interferes with sperm-egg fusion. We hypothesize that the D and E populations of rat Crisp-1 have different interactions with sperm that modulate these distinct biological activities. Through tandem mass spectrometry (MS/MS) and monosaccharide composition analyses, we have identified at least one difference between the D and E forms as an additional single O-linked N-acetyl galactosamine on an amino terminal threonine residue in Protein E. This post-translational modification appears to account for the unique 'E' epitope bound by monoclonal antibody 4E9 developed in our laboratory, and may also lead to differential processing and localization of Protein E on sperm, when compared to Protein D. These findings are the first step in distinguishing the molecular basis of the biological activities of the D and E forms of rat Crisp-1. The epididymal

Conditional and constitutive expression of a Tbx1-GFP fusion protein in mice

PubMed Central

2013-01-01

Background Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.2 deletion syndrome (22q11DS). This region includes TBX1, a T-box transcription factor gene that contributes to the etiology of 22q11DS. The requirement for TBX1 in mammalian development is dosage-sensitive, such that loss-of-function (LOF) and gain-of-function (GOF) of TBX1 in both mice and humans results in disease relevant congenital malformations. Results To further gain insight into the role of Tbx1 in development, we have targeted the Rosa26 locus to generate a new GOF mouse model in which a Tbx1-GFP fusion protein is expressed conditionally using the Cre/LoxP system. Tbx1-GFP expression is driven by the endogenous Rosa26 promoter resulting in ectopic and persistent expression. Tbx1 is pivotal for proper ear and heart development; ectopic activation of Tbx1-GFP in the otic vesicle by Pax2-Cre and Foxg1-Cre represses neurogenesis and produces morphological defects of the inner ear. Overexpression of a single copy of Tbx1-GFP using Tbx1Cre/+ was viable, while overexpression of both copies resulted in neonatal lethality with cardiac outflow tract defects. We have partially rescued inner ear and heart anomalies in Tbx1Cre/- null embryos by expression of Tbx1-GFP. Conclusions We have generated a new mouse model to conditionally overexpress a GFP-tagged Tbx1 protein in vivo. This provides a useful tool to investigate in vivo direct downstream targets and protein binding partners of Tbx1. PMID:23971992

Radar E-O image fusion

NASA Technical Reports Server (NTRS)

Oneil, William F.

1993-01-01

The fusion of radar and electro-optic (E-O) sensor images presents unique challenges. The two sensors measure different properties of the real three-dimensional (3-D) world. Forming the sensor outputs into a common format does not mask these differences. In this paper, the conditions under which fusion of the two sensor signals is possible are explored. The program currently planned to investigate this problem is briefly discussed.

A phorbol ester-binding protein is required downstream of Rab5 in endosome fusion.

PubMed

Aballay, A; Barbieri, M A; Colombo, M I; Arenas, G N; Stahl, P D; Mayorga, L S

1998-12-28

Previous observations indicate that a zinc and phorbol ester binding factor is necessary for endosome fusion. To further characterize the role of this factor in the process, we used an in vitro endosome fusion assay supplemented with recombinant Rab5 proteins. Both zinc depletion and addition of calphostin C, an inhibitor of protein kinase C, inhibited endosome fusion in the presence of active Rab5. Addition of the phorbol ester PMA (phorbol 12-myristate 13-acetate) reversed the inhibition of endosome fusion caused by a Rab5 negative mutant. Moreover, PMA stimulated fusion in the presence of Rab5 immunodepleted cytosol. These results suggest that the phorbol ester binding protein is acting downstream of Rab5 in endosome fusion.

[Construction, expression and characterization of the fusion gene of super-antigen SEA and single chain Fv of the ND-1 monoclonal antibody against human colorectal cancer].

PubMed

Chen, Hang; Li, Li; Fang, Jin

2012-04-01

To construct and express the recombinant ND-1-scFv/SEA, a fusion protein of superantigen (staphylococcal enterotoxinA, SEA) and single-chain variable fragment of monoclonal antibody ND-1 against human clolorectal carcinoma, and to enhance the targeted killing effect of SEA. The expression of the fusion protein was induced in E.coli M15 by IPTG. Ni-NTA resin affinity chromatography was used to separate and purify the expressed product. The specific binding activity of the purified ND-1-scFv/SEA protein was examined by indirect immunofluorescence assay and the targeted-cytotoxicity was determined using MTT assay. The expressing vector of fusion gene ND-1scFv/SEA was constructed successfully. ND-1-scFv/SEA protein retained a high binding affinity to antigen-positive human colorectal cancer cell CCL-187 and had a stronger capability to activate PBMC and kill the target cells compared to SEA alone, with a killing rate of 91% at 4 μg/mL. ND-1-scFv/SEA fusion protein could specifically target colorectal cancer cell, enhance the activity of kill tumor cell and has potential applications in the targeted therapy of colorectal cancer.

Fusion of NUP98 and the SET binding protein 1 (SETBP1) gene in a paediatric acute T cell lymphoblastic leukaemia with t(11;18)(p15;q12).

PubMed

Panagopoulos, Ioannis; Kerndrup, Gitte; Carlsen, Niels; Strömbeck, Bodil; Isaksson, Margareth; Johansson, Bertil

2007-01-01

Three NUP98 chimaeras have previously been reported in T cell acute lymphoblastic leukaemia (T-ALL): NUP98/ADD3, NUP98/CCDC28A, and NUP98/RAP1GDS1. We report a T-ALL with t(11;18)(p15;q12) resulting in a novel NUP98 fusion. Fluorescent in situ hybridisation showed NUP98 and SET binding protein 1(SETBP1) fusion signals; other analyses showed that exon 12 of NUP98 was fused in-frame with exon 5 of SETBP1. Nested polymerase chain reaction did not amplify the reciprocal SETBP1/NUP98, suggesting that NUP98/SETBP1 transcript is pathogenetically important. SETBP1 has previously not been implicated in leukaemias; however, it encodes a protein that specifically interacts with SET, fused to NUP214 in a case of acute undifferentiated leukaemia.

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

NASA Astrophysics Data System (ADS)

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.

Active sites prediction and binding analysis E1-E2 protein human papillomavirus with biphenylsulfonacetic acid

NASA Astrophysics Data System (ADS)

Iryani, I.; Amelia, F.; Iswendi, I.

2018-04-01

Cervix cancer triggered by Human papillomavirus infection is the second cause to woman death in worldwide. The binding site of E1-E2 protein of HPV 16 is not known from a 3-D structure yet, so in this study we address this issue to study the structure of E1-E2 protein from Human papillomavirus type 16 and to find its potential binding sites using biphenylsulfonacetic acid as inhibitor. Swiss model was used for 3D structure prediction and PDB: 2V9P (E1 protein) and 2NNU (E2 protein) having 52.32% and 100% identity respectively was selected as a template. The 3D model structure developed of E1 and E2 in the core and allowed regions were 99.2% and 99.5%. The ligand binding sites were predicted using online server meta pocket 2.0 and MOE 2009.10 was used for docking. E1-and E2 protein of HPV-16 has three potential binding site that can interact with the inhibitors. The Docking biphenylsulfonacetic acid using these binding sites shows that ligand interact with the protein through hydrogen bonds on Lys 403, Arg 410, His 551 in the first pocket, on Tyr 32, Leu 99 in the second pocket, and Lys 558m Lys 517 in the third pocket.

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

PubMed Central

Ko, Young-Joon; Lee, Miriam; Kang, KyeongJin; Song, Woo Keun; Jun, Youngsoo

2014-01-01

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms. PMID:24821814

Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

PubMed Central

Nakahashi, Mito; Matsushima, Yoshiaki; Ito, Morihiro; Nishio, Machiko; Kawano, Mitsuo; Komada, Hiroshi; Nosaka, Tetsuya

2013-01-01

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity. PMID:23698295

The TEL-AML1 fusion protein of acute lymphoblastic leukemia modulates IRF3 activity during early B-cell differentiation.

PubMed

de Laurentiis, A; Hiscott, J; Alcalay, M

2015-12-03

The t(12;21) translocation is the most common genetic rearrangement in childhood acute lymphoblastic leukemia (ALL) and gives rise to the TEL-AML1 fusion gene. Many studies on TEL-AML1 describe specific properties of the fusion protein, but a thorough understanding of its function is lacking. We exploited a pluripotent hematopoietic stem/progenitor cell line, EML1, and generated a cell line (EML-TA) stably expressing the TEL-AML1 fusion protein. EML1 cells differentiate to mature B-cells following treatment with IL7; whereas EML-TA display an impaired differentiation capacity and remain blocked at an early stage of maturation. Global gene expression profiling of EML1 cells at different stages of B-lymphoid differentiation, compared with EML-TA, identified the interferon (IFN)α/β pathway as a primary target of repression by TEL-AML1. In particular, expression and phosphorylation of interferon-regulatory factor 3 (IRF3) was decreased in EML-TA cells; strikingly, stable expression of IRF3 restored the capacity of EML-TA cells to differentiate into mature B-cells. Similarly, IRF3 silencing in EML1 cells by siRNA was sufficient to block B-lymphoid differentiation. The ability of TEL-AML1 to block B-cell differentiation and downregulate the IRF3-IFNα/β pathway was confirmed in mouse and human primary hematopoietic precursor cells (Lin- and CD34+ cells, respectively), and in a patient-derived cell line expressing TEL-AML1 (REH). Furthermore, treatment of TEL-AML1 expressing cells with IFNα/β was sufficient to overcome the maturation block. Our data provide new insight on TEL-AML1 function and may offer a new therapeutic opportunity for B-ALL.

Dual Split Protein-Based Fusion Assay Reveals that Mutations to Herpes Simplex Virus (HSV) Glycoprotein gB Alter the Kinetics of Cell-Cell Fusion Induced by HSV Entry Glycoproteins

PubMed Central

Atanasiu, Doina; Saw, Wan Ting; Gallagher, John R.; Hannah, Brian P.; Matsuda, Zene; Whitbeck, J. Charles; Cohen, Gary H.

2013-01-01

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, “slow and fast,” emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a “hair trigger.” Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion. PMID:23946457

Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein

PubMed Central

Meulendyke, Kelly Ann; Wurth, Mark Allen; McCann, Richard O.; Dutch, Rebecca Ellis

2005-01-01

The Hendra virus fusion (F) protein is synthesized as a precursor protein, F0, which is proteolytically processed to the mature form, F1+F2. Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca2+, and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXΦ. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins. PMID:16188966

Structure and Function Study of HIV and Influenza Fusion Proteins

NASA Astrophysics Data System (ADS)

Liang, Shuang

Human immunodeficiency virus (HIV) and influenza virus are membrane-enveloped viruses causing acquired immunodeficiency syndrome (AIDS) and flu. The initial step of HIV and influenza virus infection is fusion between viral and host cell membrane catalyzed by the viral fusion protein gp41 and hemagglutinin (HA) respectively. However, the structure of gp41 and HA as well as the infection mechanism are still not fully understood. This work addresses (1) full length gp41 ectodomain and TM domain structure and function and (2) IFP membrane location and IFP-membrane interaction. My studies of gp41 protein and IFP can provide better understanding of the membrane fusion mechanism and may aid development of anti-viral therapeutics and vaccine. The full length ectodomain and transmembrane domain of gp41 and shorter constructs were expressed, purified and solubilized at physiology conditions. The constructs adopt overall alpha helical structure in SDS and DPC detergents, and showed hyperthermostability with Tm > 90 °C. The oligomeric states of these proteins vary in different detergent buffer: predominant trimer for all constructs and some hexamer fraction for HM and HM_TM protein in SDS at pH 7.4; and mixtures of monomer, trimer, and higher-order oligomer protein in DPC at pH 4.0 and 7.4. Substantial protein-induced vesicle fusion was observed, including fusion of neutral vesicles at neutral pH, which are the conditions similar HIV/cell fusion. Vesicle fusion by a gp41 ectodomain construct has rarely been observed under these conditions, and is aided by inclusion of both the FP and TM, and by protein which is predominantly trimer rather than monomer. Current data was integrated with existing data, and a structural model was proposed. Secondary structure and conformation of IFP is a helix-turn-helix structure in membrane. However, there has been arguments about the IFP membrane location. 13C-2H REDOR solid-state NMR is used to solve this problem. The IFP adopts major alpha

Construction of membrane-anchoring fusion protein of Thermococcus kodakaraensis glycerol kinase and its application to repetitive batchwise reactions.

PubMed

Restiawaty, Elvi; Honda, Kohsuke; Okano, Kenji; Hirota, Ryuichi; Omasa, Takeshi; Kuroda, Akio; Ohtake, Hisao

2012-04-01

We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Thermus thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70°C. In this study, TkGK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70°C for 2h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. coli cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport

PubMed Central

Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

2015-01-01

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. PMID:25657322

Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

PubMed

Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

2015-10-08

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

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

PubMed

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.

A nonallergenic birch pollen allergy vaccine consisting of hepatitis PreS-fused Bet v 1 peptides focuses blocking IgG toward IgE epitopes and shifts immune responses to a tolerogenic and Th1 phenotype.

PubMed

Marth, Katharina; Breyer, Isabella; Focke-Tejkl, Margarete; Blatt, Katharina; Shamji, Mohamed H; Layhadi, Janice; Gieras, Anna; Swoboda, Ines; Zafred, Domen; Keller, Walter; Valent, Peter; Durham, Stephen R; Valenta, Rudolf

2013-04-01

Allergen-specific immunotherapy is the only allergen-specific and disease-modifying treatment for allergy. The construction and characterization of a vaccine for birch pollen allergy is reported. Two nonallergenic peptides, PA and PB, derived from the IgE-reactive areas of the major birch pollen allergen Bet v 1 were fused to the hepatitis B surface protein, PreS, in four recombinant fusion proteins containing different numbers and combinations of the peptides. Fusion proteins expressed in Escherichia coli and purified to homogeneity showed a lack of IgE reactivity and allergenic activity when tested with sera and basophils from patients allergic to birch pollen. Compared to Bet v 1 allergen, peptides PA and PB showed reduced T cell activation in PBMCs from allergic patients, whereas PreS fusion proteins induced less IL-5 and more IL-10 and IFN-γ. Immunization of rabbits with the fusion proteins, in particular with a PreS fusion protein 2PAPB-PreS, containing two copies of each peptide, induced high levels of IgG Abs against the major IgE-reactive site on Bet v 1 and related allergens. These IgG Abs inhibited allergic patients' IgE binding to Bet v 1 better than did IgG induced by immunization with complete Bet v 1. Furthermore, 2PAPB-PreS-induced IgG inhibited Bet v 1-induced basophil activation in allergic patients and CD23-facilitated allergen presentation. Our study exemplifies novel beneficial features for a PreS carrier-based peptide vaccine for birch pollen, which, in addition to the established reduction in allergenic activity, include the enhanced focusing of blocking Ab responses toward IgE epitopes, immunomodulatory activity, and reduction of CD23-facilitated allergen presentation.

A Nonallergenic Birch Pollen Allergy Vaccine Consisting of Hepatitis PreS–Fused Bet v 1 Peptides Focuses Blocking IgG toward IgE Epitopes and Shifts Immune Responses to a Tolerogenic and Th1 Phenotype

PubMed Central

Marth, Katharina; Breyer, Isabella; Focke-Tejkl, Margarete; Blatt, Katharina; Shamji, Mohamed H.; Layhadi, Janice; Gieras, Anna; Swoboda, Ines; Zafred, Domen; Keller, Walter; Valent, Peter; Durham, Stephen R.; Valenta, Rudolf

2014-01-01

Allergen-specific immunotherapy is the only allergen-specific and disease-modifying treatment for allergy. The construction and characterization of a vaccine for birch pollen allergy is reported. Two nonallergenic peptides, PA and PB, derived from the IgE-reactive areas of the major birch pollen allergen Bet v 1 were fused to the hepatitis B surface protein, PreS, in four recombinant fusion proteins containing different numbers and combinations of the peptides. Fusion proteins expressed in Escherichia coli and purified to homogeneity showed a lack of IgE reactivity and allergenic activity when tested with sera and basophils from patients allergic to birch pollen. Compared to Bet v 1 allergen, peptides PA and PB showed reduced T cell activation in PBMCs from allergic patients, whereas PreS fusion proteins induced less IL-5 and more IL-10 and IFN-γ. Immunization of rabbits with the fusion proteins, in particular with a PreS fusion protein 2PAPB-PreS, containing two copies of each peptide, induced high levels of IgG Abs against the major IgE-reactive site on Bet v 1 and related allergens. These IgG Abs inhibited allergic patients’ IgE binding to Bet v 1 better than did IgG induced by immunization with complete Bet v 1. Furthermore, 2PAPB-PreS–induced IgG inhibited Bet v 1–induced basophil activation in allergic patients and CD23-facilitated allergen presentation. Our study exemplifies novel beneficial features for a PreS carrier–based peptide vaccine for birch pollen, which, in addition to the established reduction in allergenic activity, include the enhanced focusing of blocking Ab responses toward IgE epitopes, immunomodulatory activity, and reduction of CD23-facilitated allergen presentation. PMID:23440415

Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

PubMed Central

Shirogane, Yuta; Suzuki, Satoshi O.; Ikegame, Satoshi; Koga, Ritsuko

2013-01-01

Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein. PMID:23255801

Eradication of large tumors expressing human papillomavirus E7 protein by therapeutic vaccination with E7 fused to the extra domain a from fibronectin.

PubMed

Mansilla, Cristina; Berraondo, Pedro; Durantez, Maika; Martínez, Marta; Casares, Noelia; Arribillaga, Laura; Rudilla, Francesc; Fioravanti, Jessica; Lozano, Teresa; Villanueva, Lorea; Sarobe, Pablo; Borrás, Francisco; Leclerc, Claude; Prieto, Jesús; Lasarte, Juan José

2012-08-01

Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8(+) T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 5-7 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma. Copyright © 2011 UICC.

Membrane fusion and exocytosis.

PubMed

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals

PubMed Central

Paulmurugan, Ramasamy; Gambhir, Sanjiv S.

2014-01-01

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule–mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction–mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGS-FACGSLSCGSF. A 9 ± 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation. PMID:16103094

Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

PubMed

Paulmurugan, Ramasamy; Gambhir, Sanjiv S

2005-08-15

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans.

PubMed

Mao, Yuxin; Zhang, Zimei; Wong, Brian

2003-12-01

Glycophosphatidylinositol (GPI)-anchored proteins account for 26-35% of the Candida albicans cell wall. To understand the signals that regulate these proteins' cell surface localization, green fluorescent protein (GFP) was fused to the N- and C-termini of the C. albicans cell wall proteins (CWPs) Hwp1p, Als3p and Rbt5p. C. albicans expressing all three fusion proteins were fluorescent at the cell surface. GFP was released from membrane fractions by PI-PLC and from cell walls by beta-glucanase, which implied that GFP was GPI-anchored to the plasma membrane and then covalently attached to cell wall glucans. Twenty and 25 amino acids, respectively, from the N- and C-termini of Hwp1p were sufficient to target GFP to the cell surface. C-terminal substitutions that are permitted by the omega rules (G613D, G613N, G613S, G613A, G615S) did not interfere with GFP localization, whereas some non-permitted substitutions (G613E, G613Q, G613R, G613T and G615Q) caused GFP to accumulate in intracellular ER-like structures and others (G615C, G613N/G615C and G613D/G615C) did not. These results imply that (i) GFP fusions can be used to analyse the N- and C-terminal signal peptides of GPI-anchored CWPs, (ii) the omega amino acid in Hwp1p is G613, and (iii) C can function at the omega+2 position in C. albicans GPI-anchored proteins.

Crystal Structure of the Minimalist Max-E47 Protein Chimera

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahmadpour, Faraz; Ghirlando, Rodolfo; De Jong, Antonia T.

Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5'-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47more » dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts.« less

Fusion protein based on Grb2-SH2 domain for cancer therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, Yuriko; Graduate School of Pharmaceutical Sciences, Chiba University; Furukawa, Takako, E-mail: tfuru@nirs.go.jp

2010-08-20

Research highlights: {yields} Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. {yields} We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. {yields} The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. {yields} TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylatedmore » EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.« less

Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: Implication for optional anti-PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients.

PubMed

Hong, Shaodong; Chen, Nan; Fang, Wenfeng; Zhan, Jianhua; Liu, Qing; Kang, Shiyang; He, Xiaobo; Liu, Lin; Zhou, Ting; Huang, Jiaxing; Chen, Ying; Qin, Tao; Zhang, Yaxiong; Ma, Yuxiang; Yang, Yunpeng; Zhao, Yuanyuan; Huang, Yan; Zhang, Li

2016-03-01

Driver mutations were reported to upregulate programmed death-ligand 1 (PD-L1) expression. However, how PD-L1 expression and immune function was affected by ALK-TKIs and anti-PD-1/PD-L1 treatment in ALK positive non-small-cell lung cancer (NSCLC) remains poorly understood. In the present study, western-blot, real-time PCR, flow cytometry and immunofluorescence were employed to explore how PD-L1 was regulated by ALK fusion protein. ALK-TKIs and relevant inhibitors were used to identify the downstream signaling pathways involved in PD-L1 regulation. Cell apoptosis, viability and Elisa test were used to study the immune suppression by ALK activation and immune reactivation by ALK-TKIs and/or PD-1 blocking in tumor cells and DC-CIK cells co-culture system. We found that PD-L1 expression was associated with EGFR mutations and ALK fusion genes in NSCLC cell lines. Over-expression of ALK fusion protein increased PD-L1 expression. PD-L1 mediated by ALK fusion protein increased the apoptosis of T cells in tumor cells and DC-CIK cells co-culture system. Inhibiting ALK by sensitive TKIs could enhance the production of IFNγ. Anti-PD-1 antibody was effective in both crizotinib sensitive and resistant NSCLC cells. Synergistic tumor killing effects were not observed with ALK-TKIs and anti-PD-1 antibody combination in co-culture system. ALK-TKIs not only directly inhibited tumor viability but also indirectly enhanced the antitumor immunity via the downregulation of PD-L1. Anti-PD-1/PD-L1 antibodies could be an optional therapy for crizotinib sensitive, especially crizotinib resistant NSCLC patients with ALK fusion gene. Combination of ALK-TKIs and anti-PD-1/PD-L1 antibodies treatment for ALK positive NSCLC warrants more data before moving into clinical practice.

Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: Implication for optional anti-PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients

PubMed Central

Hong, Shaodong; Chen, Nan; Fang, Wenfeng; Zhan, Jianhua; Liu, Qing; Kang, Shiyang; He, Xiaobo; Liu, Lin; Zhou, Ting; Huang, Jiaxing; Chen, Ying; Qin, Tao; Zhang, Yaxiong; Ma, Yuxiang; Yang, Yunpeng; Zhao, Yuanyuan; Huang, Yan; Zhang, Li

2016-01-01

ABSTRACT Driver mutations were reported to upregulate programmed death-ligand 1 (PD-L1) expression. However, how PD-L1 expression and immune function was affected by ALK-TKIs and anti-PD-1/PD-L1 treatment in ALK positive non-small-cell lung cancer (NSCLC) remains poorly understood. In the present study, western-blot, real-time PCR, flow cytometry and immunofluorescence were employed to explore how PD-L1 was regulated by ALK fusion protein. ALK-TKIs and relevant inhibitors were used to identify the downstream signaling pathways involved in PD-L1 regulation. Cell apoptosis, viability and Elisa test were used to study the immune suppression by ALK activation and immune reactivation by ALK-TKIs and/or PD-1 blocking in tumor cells and DC-CIK cells co-culture system. We found that PD-L1 expression was associated with EGFR mutations and ALK fusion genes in NSCLC cell lines. Over-expression of ALK fusion protein increased PD-L1 expression. PD-L1 mediated by ALK fusion protein increased the apoptosis of T cells in tumor cells and DC-CIK cells co-culture system. Inhibiting ALK by sensitive TKIs could enhance the production of IFNγ. Anti-PD-1 antibody was effective in both crizotinib sensitive and resistant NSCLC cells. Synergistic tumor killing effects were not observed with ALK-TKIs and anti-PD-1 antibody combination in co-culture system. ALK-TKIs not only directly inhibited tumor viability but also indirectly enhanced the antitumor immunity via the downregulation of PD-L1. Anti-PD-1/PD-L1 antibodies could be an optional therapy for crizotinib sensitive, especially crizotinib resistant NSCLC patients with ALK fusion gene. Combination of ALK-TKIs and anti-PD-1/PD-L1 antibodies treatment for ALK positive NSCLC warrants more data before moving into clinical practice. PMID:27141355

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

PubMed Central

Wu, Zhenyong; Thiyagarajan, Sathish; O’Shaughnessy, Ben; Karatekin, Erdem

2017-01-01

Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically

Identification of a novel FN1-FGFR1 genetic fusion as a frequent event in phosphaturic mesenchymal tumour.

PubMed

Lee, Jen-Chieh; Jeng, Yung-Ming; Su, Sheng-Yao; Wu, Chen-Tu; Tsai, Keh-Sung; Lee, Cheng-Han; Lin, Chung-Yen; Carter, Jodi M; Huang, Jenq-Wen; Chen, Shu-Hwa; Shih, Shyang-Rong; Mariño-Enríquez, Adrián; Chen, Chih-Chi; Folpe, Andrew L; Chang, Yih-Leong; Liang, Cher-Wei

2015-03-01

Phosphaturic mesenchymal tumours (PMTs) are uncommon soft tissue and bone tumours that typically cause hypophosphataemia and tumour-induced osteomalacia (TIO) through secretion of phosphatonins including fibroblast growth factor 23 (FGF23). PMT has recently been accepted by the World Health Organization as a formal tumour entity. The genetic basis and oncogenic pathways underlying its tumourigenesis remain obscure. In this study, we identified a novel FN1-FGFR1 fusion gene in three out of four PMTs by next-generation RNA sequencing. The fusion transcripts and proteins were subsequently confirmed with RT-PCR and western blotting. Fluorescence in situ hybridization analysis showed six cases with FN1-FGFR1 fusion out of an additional 11 PMTs. Overall, nine out of 15 PMTs (60%) harboured this fusion. The FN1 gene possibly provides its constitutively active promoter and the encoded protein's oligomerization domains to overexpress and facilitate the activation of the FGFR1 kinase domain. Interestingly, unlike the prototypical leukaemia-inducing FGFR1 fusion genes, which are ligand-independent, the FN1-FGFR1 chimeric protein was predicted to preserve its ligand-binding domains, suggesting an advantage of the presence of its ligands (such as FGF23 secreted at high levels by the tumour) in the activation of the chimeric receptor tyrosine kinase, thus effecting an autocrine or a paracrine mechanism of tumourigenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

RSPO fusion transcripts in colorectal cancer in Japanese population.

PubMed

Shinmura, Kazuya; Kahyo, Tomoaki; Kato, Hisami; Igarashi, Hisaki; Matsuura, Shun; Nakamura, Satoki; Kurachi, Kiyotaka; Nakamura, Toshio; Ogawa, Hiroshi; Funai, Kazuhito; Tanahashi, Masayuki; Niwa, Hiroshi; Sugimura, Haruhiko

2014-08-01

R-spondin (RSPO) gene fusions have recently been discovered in a subset of human colorectal cancer (CRC) in the U.S. population; however, whether the fusion is recurrent in CRC arising in patients from the other demographic areas and whether it is specific for CRC remain uncertain. In this study, we examined 75 primary CRCs and 121 primary lung cancers in the Japanese population for EIF3E-RSPO2 and PTPRK-RSPO3 fusion transcripts using RT-PCR and subsequent sequencing analyses. Although the expression of EIF3E-RSPO2 and PTPRK-RSPO3 was not detected in any of the lung carcinomas, RSPO fusions were detected in three (4%) of the 75 CRCs. Two CRCs contained EIF3E-RSPO2 fusion transcripts, and another CRC contained PTPRK-RSPO3 fusion transcripts. Interestingly, in one of the two EIF3E-RSPO2 fusion-positive CRCs, a novel fusion variant form of EIF3E-RSPO2 was identified: exon 1 of EIF3E was connected to exon 2 of RSPO2 by a 351-bp insertion. A quantitative RT-PCR analysis revealed that RSPO mRNA expression was upregulated in the three CRCs containing RSPO fusion transcripts, while it was downregulated in nearly all of the other CRCs. An immunohistochemical analysis and a mutational analysis revealed that the RSPO fusion-containing CRC had a CDX2 cell lineage, was positive for mismatch repair protein expression, and had the wild-type APC allele. Finally, the forced expression of RSPO fusion proteins were shown to endow colorectal cells with an increased growth ability. These results suggest that the expression of RSPO fusion transcripts is related to a subset of CRCs arising in the Japanese population.