Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies

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

Peters, J.; Peters, M.; Lottspeich, F.

1987-11-01

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate (HPI))-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and M/sub r/ estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%)more » of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.« less

Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides

PubMed Central

Santona, Antonella; Carta, Franco; Fraghí, Peppinetta; Turrini, Franco

2002-01-01

As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection. PMID:11748179

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

PubMed

Schmid-Siegert, Emanuel; Richard, Sophie; Luraschi, Amanda; Mühlethaler, Konrad; Pagni, Marco; Hauser, Philippe M

2017-11-07

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. IMPORTANCE Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens. Copyright © 2017 Schmid-Siegert et al.

Construction of a cell-surface display system based on the N-terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins.

PubMed

Bao, S; Yu, S; Guo, X; Zhang, F; Sun, Y; Tan, L; Duan, Y; Lu, F; Qiu, X; Ding, C

2015-07-01

To construct and demonstrate a surface display system that could be used to identify mycoplasma adhesion proteins. Using the N-terminal domain of InaZ (InaZN) as the anchoring motif and the enhanced green fluorescent protein (EGFP) as the reporter, the surface display system pET-InaZN-EGFP was constructed. Then, the mgc2 gene which encodes an adhesin and the holB gene which encodes DNA polymerase III subunit delta' (nonadhesin, negative control) of Mycoplasma gallisepticum were cloned into the pET-InaZN-EGFP respectively. The fusion proteins were expressed in Escherichia coli BL21 (DE3). The distribution of the fusion proteins in E. coli cells was determined using SDS-PAGE followed by Western blotting, based on cell fractionation. Escherichia coli cell surface display of the fusion protein was confirmed by immunofluorescence microscopy. The results indicated that the fusion proteins were not only anchored to the outer membrane fraction but also were successfully displayed on the surface of E. coli cells. Adhesion analysis of E. coli harbouring InaZN-EGFP-mgc2 to host cells showed that the MGC2-positive E. coli cells can effectively adhere to the surfaces of DF-1 cells. A surface display system using the InaZN as the anchoring motif and EGFP as the reporter was developed to identify putative adhesins of mycoplasma. Results indicated that adhesion by the cytadhesin-like protein MGC2 of mycoplasma can be reproduced using this surface display system. This is the first construction of surface display system which could be used to identify the adhesion proteins of mycoplasma. The method developed in this study can even be used to select and identify the adhesion proteins of other pathogens. © 2015 The Society for Applied Microbiology.

A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection in the Drosophila neuromuscular system

PubMed Central

Kurusu, Mitsuhiko; Cording, Amy; Taniguchi, Misako; Menon, Kaushiki; Suzuki, Emiko; Zinn, Kai

2008-01-01

Summary In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron. PMID:18817735

Binding ligand prediction for proteins using partial matching of local surface patches.

PubMed

Sael, Lee; Kihara, Daisuke

2010-01-01

Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

Binding Ligand Prediction for Proteins Using Partial Matching of Local Surface Patches

PubMed Central

Sael, Lee; Kihara, Daisuke

2010-01-01

Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group. PMID:21614188

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

PubMed

Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Molecular Characterization of Mosquitocidal Toxin (Surface Layer Protein, SLP) from Bacillus cereus VCRC B540.

PubMed

Mani, Chinnasamy; Selvakumari, Jeyaperumal; Han, YeonSoo; Jo, YongHun; Thirugnanasambantham, Krishnaraj; Sundarapandian, Somaiah; Poopathi, Subbiah

2018-04-01

A marine Bacillus cereus (VCRC B540) with mosquitocidal effect was recently reported from red snapper fish (Lutjanus sanguineous) gut and surface layer protein (S-layer protein, SLP) was reported to be mosquito larvicidal factor. In this present study, the gene encoding the surface layer protein was amplified from the genomic DNA and functionally characterized. Amplification of SLP-encoding gene revealed 1,518 bp PCR product, and analysis of the sequence revealed the presence of 1482 bp open reading frame with coding capacity for a polypeptide of 493 amino acids. Phylogenetic analysis revealed with homology among closely related Bacillus cereus groups of organisms as well as Bacillus strains. Removal of nucleotides encoding signaling peptide revealed the functional cloning fragment of length 1398 bp. Theoretical molecular weight (51.7 kDa) and isoelectric point (5.99) of the deduced functional SLP protein were predicted using ProtParam. The amplified PCR product was cloned into a plasmid vector (pGEM-T), and the open reading frame free off signaling peptide was subsequently cloned inpET-28a(+) and expressed in Escherichia coli BL21 (DE3). The isopropyl-β-D-thiogalactopyranoside (IPTG)-induced recombinant SLP was confirmed using western blotting, and functional SLP revealed mosquito larvicidal property. Therefore, the major findings revealed that SLP is a factor responsible for mosquitocidal activity, and the molecular characterization of this toxin was extensively studied.

Protection of Mice against Plasmodium yoelii Sporozoite Challenge with P. yoelii Merozoite Surface Protein 1 DNA Vaccines

PubMed Central

Becker, Sylvia I.; Wang, Ruobing; Hedstrom, Richard C.; Aguiar, Joao C.; Jones, Trevor R.; Hoffman, Stephen L.; Gardner, Malcolm J.

1998-01-01

Immunization of mice with DNA vaccines encoding the full-length form and C and N termini of Plasmodium yoelii merozoite surface protein 1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titers after challenge. In C57BL/6 mice, two DNA vaccines provided protection comparable to that of recombinant protein consisting of the C terminus in Freund’s adjuvant. PMID:9632624

Dynamics of Agglutinin-Like Sequence (ALS) Protein Localization on the Surface of Candida Albicans

ERIC Educational Resources Information Center

Coleman, David Andrew

2009-01-01

The ALS gene family encodes large cell-surface glycoproteins associated with "C. albicans" pathogenesis. Als proteins are thought to act as adhesin molecules binding to host tissues. Wide variation in expression levels among the ALS genes exists and is related to cell morphology and environmental conditions. "ALS1," "ALS3," and "ALS4" are three of…

Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.

PubMed

Bartl, S; Weissman, I L

1994-01-04

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.

Identification and Characterization of Novel Surface Proteins in Lactobacillus johnsonii and Lactobacillus gasseri

PubMed Central

Ventura, Marco; Jankovic, Ivana; Walker, D. Carey; Pridmore, R. David; Zink, Ralf

2002-01-01

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family. PMID:12450842

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

PubMed

Balachandran, Manasi; Giannone, Richard J; Bemis, David A; Kania, Stephen A

2017-01-01

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

PubMed Central

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.

2017-01-01

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models. PMID:28859130

Genetically encoded pH sensor for tracking surface proteins through endocytosis.

PubMed

Grover, Anmol; Schmidt, Brigitte F; Salter, Russell D; Watkins, Simon C; Waggoner, Alan S; Bruchez, Marcel P

2012-05-14

Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

PubMed Central

Shi, Huazhong; Kim, YongSig; Guo, Yan; Stevenson, Becky; Zhu, Jian-Kang

2003-01-01

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf. PMID:12509519

Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.

PubMed

Singh, Vijay; Wang, Shenliang; Chan, Ke Min; Clark, Spencer A; Kool, Eric T

2013-04-24

Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

PubMed Central

Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

2005-01-01

Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

PubMed

Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

2003-08-01

Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.

Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics

PubMed Central

2010-01-01

Background Trichomonas vaginalis is the most common non-viral human sexually transmitted pathogen and importantly, contributes to facilitating the spread of HIV. Yet very little is known about its surface and secreted proteins mediating interactions with, and permitting the invasion and colonisation of, the host mucosa. Initial annotations of T. vaginalis genome identified a plethora of candidate extracellular proteins. Results Data mining of the T. vaginalis genome identified 911 BspA-like entries (TvBspA) sharing TpLRR-like leucine-rich repeats, which represent the largest gene family encoding potential extracellular proteins for the pathogen. A broad range of microorganisms encoding BspA-like proteins was identified and these are mainly known to live on mucosal surfaces, among these T. vaginalis is endowed with the largest gene family. Over 190 TvBspA proteins with inferred transmembrane domains were characterised by a considerable structural diversity between their TpLRR and other types of repetitive sequences and two subfamilies possessed distinct classic sorting signal motifs for endocytosis. One TvBspA subfamily also shared a glycine-rich protein domain with proteins from Clostridium difficile pathogenic strains and C. difficile phages. Consistent with the hypothesis that TvBspA protein structural diversity implies diverse roles, we demonstrated for several TvBspA genes differential expression at the transcript level in different growth conditions. Identified variants of repetitive segments between several TvBspA paralogues and orthologues from two clinical isolates were also consistent with TpLRR and other repetitive sequences to be functionally important. For one TvBspA protein cell surface expression and antibody responses by both female and male T. vaginalis infected patients were also demonstrated. Conclusions The biased mucosal habitat for microbial species encoding BspA-like proteins, the characterisation of a vast structural diversity for the TvBspA proteins, differential expression of a subset of TvBspA genes and the cellular localisation and immunological data for one TvBspA; all point to the importance of the TvBspA proteins to various aspects of T. vaginalis pathobiology at the host-pathogen interface. PMID:20144183

A secretome view of colonisation factors in Shiga toxin-encoding Escherichia coli (STEC): from enterohaemorrhagic E. coli (EHEC) to related enteropathotypes.

PubMed

Monteiro, Ricardo; Ageorges, Valentin; Rojas-Lopez, Maricarmen; Schmidt, Herbert; Weiss, Agnes; Bertin, Yolande; Forano, Evelyne; Jubelin, Grégory; Henderson, Ian R; Livrelli, Valérie; Gobert, Alain P; Rosini, Roberto; Soriani, Marco; Desvaux, Mickaël

2016-08-01

Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli, enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E. coli, hybrids with enteroaggregative E. coli have recently emerged. Of note, some enteroinvasive E. coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

PubMed Central

Galloway-Peña, Jessica R.; Liang, Xiaowen; Singh, Kavindra V.; Yadav, Puja; Chang, Chungyu; La Rosa, Sabina Leanti; Shelburne, Samuel; Ton-That, Hung; Höök, Magnus

2014-01-01

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. PMID:25512313

CLONING AND CHARACTERIZATION OF CDNA ENCODING GIARDIA LAMBLIA d-GIARDIN

USDA-ARS?s Scientific Manuscript database

A cDNA coding for d-giardin was cloned from Giardia lamblia trophozoites in order to localize the protein and study its function in mediating surface attachment. Recombinant d-giardin antigen was produced in Escherichia coli as a poly-histidine fusion protein and was purified by affinity chromatogr...

Structure of the Elastin-Contractile Units in the Thoracic Aorta and How Genes That Cause Thoracic Aortic Aneurysms and Dissections Disrupt This Structure.

PubMed

Karimi, Ashkan; Milewicz, Dianna M

2016-01-01

The medial layer of the aorta confers elasticity and strength to the aortic wall and is composed of alternating layers of smooth muscle cells (SMCs) and elastic fibres. The SMC elastin-contractile unit is a structural unit that links the elastin fibres to the SMCs and is characterized by the following: (1) layers of elastin fibres that are surrounded by microfibrils; (2) microfibrils that bind to the integrin receptors in focal adhesions on the cell surface of the SMCs; and (3) SMC contractile filaments that are linked to the focal adhesions on the inner side of the membrane. The genes that are altered to cause thoracic aortic aneurysms and aortic dissections encode proteins involved in the structure or function of the SMC elastin-contractile unit. Included in this gene list are the genes encoding protein that are structural components of elastin fibres and microfibrils, FBN1, MFAP5, ELN, and FBLN4. Also included are genes that encode structural proteins in the SMC contractile unit, including ACTA2, which encodes SMC-specific α-actin and MYH11, which encodes SMC-specific myosin heavy chain, along with MYLK and PRKG1, which encode kinases that control SMC contraction. Finally, mutations in the gene encoding the protein linking integrin receptors to the contractile filaments, FLNA, also predispose to thoracic aortic disease. Thus, these data suggest that functional SMC elastin-contractile units are important for maintaining the structural integrity of the aorta. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Mutations in a gene encoding the. cap alpha. subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling

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

Jahng, K.Y.; Ferguson, J.; Reed, S.I.

1988-06-01

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identically to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to gene encoding the ..cap alpha.. subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpal mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G/sub 1/, deposition of mating-specificmore » cell surface aggultinins, and induction of pheromone-specific mRNa, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.« less

Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment

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

Norton, Jeanette M.; Klotz, Martin G; Stein, Lisa Y

2008-01-01

The complete genome of the ammonia-oxidizing bacterium, Nitrosospira multiformis (ATCC 25196T), consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2827 putative proteins. Of these, 2026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and N. eutropha were the best match for 42% of the predicted genes in N. multiformis. The genome contains three nearly identical copies of amo and hao gene clusters as large repeats. Distinguishing features compared to N. europaea include: the presencemore » of gene clusters encoding urease and hydrogenase, a RuBisCO-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced AOB genomes. Gene clusters encoding proteins associated with outer membrane and cell envelope functions including transporters, porins, exopolysaccharide synthesis, capsule formation and protein sorting/export were abundant. Numerous sensory transduction and response regulator gene systems directed towards sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate and cyanophycin storage and utilization were identified providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.« less

Molecular characterization of the rhesus rhadinovirus (RRV) ORF4 gene and the RRV complement control protein it encodes.

PubMed

Mark, Linda; Spiller, O Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A; Wong, Scott W; Damania, Blossom; Blom, Anna M; Blackbourn, David J

2007-04-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Rapid comparison of protein binding site surfaces with Property Encoded Shape Distributions (PESD)

PubMed Central

Das, Sourav; Kokardekar, Arshad

2009-01-01

Patterns in shape and property distributions on the surface of binding sites are often conserved across functional proteins without significant conservation of the underlying amino-acid residues. To explore similarities of these sites from the viewpoint of a ligand, a sequence and fold-independent method was created to rapidly and accurately compare binding sites of proteins represented by property-mapped triangulated Gauss-Connolly surfaces. Within this paradigm, signatures for each binding site surface are produced by calculating their property-encoded shape distributions (PESD), a measure of the probability that a particular property will be at a specific distance to another on the molecular surface. Similarity between the signatures can then be treated as a measure of similarity between binding sites. As postulated, the PESD method rapidly detected high levels of similarity in binding site surface characteristics even in cases where there was very low similarity at the sequence level. In a screening experiment involving each member of the PDBBind 2005 dataset as a query against the rest of the set, PESD was able to retrieve a binding site with identical E.C. (Enzyme Commission) numbers as the top match in 79.5% of cases. The ability of the method in detecting similarity in binding sites with low sequence conservations were compared with state-of-the-art binding site comparison methods. PMID:19919089

Direct Identification of On-Bead Peptides Using Surface-Enhanced Raman Spectroscopic Barcoding System for High-Throughput Bioanalysis

PubMed Central

Kang, Homan; Jeong, Sinyoung; Koh, Yul; Geun Cha, Myeong; Yang, Jin-Kyoung; Kyeong, San; Kim, Jaehi; Kwak, Seon-Yeong; Chang, Hye-Jin; Lee, Hyunmi; Jeong, Cheolhwan; Kim, Jong-Ho; Jun, Bong-Hyun; Kim, Yong-Kweon; Hong Jeong, Dae; Lee, Yoon-Sik

2015-01-01

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery. PMID:26017924

Direct identification of on-bead peptides using surface-enhanced Raman spectroscopic barcoding system for high-throughput bioanalysis.

PubMed

Kang, Homan; Jeong, Sinyoung; Koh, Yul; Geun Cha, Myeong; Yang, Jin-Kyoung; Kyeong, San; Kim, Jaehi; Kwak, Seon-Yeong; Chang, Hye-Jin; Lee, Hyunmi; Jeong, Cheolhwan; Kim, Jong-Ho; Jun, Bong-Hyun; Kim, Yong-Kweon; Hong Jeong, Dae; Lee, Yoon-Sik

2015-05-28

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.

The unique fold and lability of the [2Fe-2S] clusters of NEET proteins mediate their key functions in health and disease.

PubMed

Karmi, Ola; Marjault, Henri-Baptiste; Pesce, Luca; Carloni, Paolo; Onuchic, Jose' N; Jennings, Patricia A; Mittler, Ron; Nechushtai, Rachel

2018-02-12

NEET proteins comprise a new class of [2Fe-2S] cluster proteins. In human, three genes encode for NEET proteins: cisd1 encodes mitoNEET (mNT), cisd2 encodes the Nutrient-deprivation autophagy factor-1 (NAF-1) and cisd3 encodes MiNT (Miner2). These recently discovered proteins play key roles in many processes related to normal metabolism and disease. Indeed, NEET proteins are involved in iron, Fe-S, and reactive oxygen homeostasis in cells and play an important role in regulating apoptosis and autophagy. mNT and NAF-1 are homodimeric and reside on the outer mitochondrial membrane. NAF-1 also resides in the membranes of the ER associated mitochondrial membranes (MAM) and the ER. MiNT is a monomer with distinct asymmetry in the molecular surfaces surrounding the clusters. Unlike its paralogs mNT and NAF-1, it resides within the mitochondria. NAF-1 and mNT share similar backbone folds to the plant homodimeric NEET protein (At-NEET), while MiNT's backbone fold resembles a bacterial MiNT protein. Despite the variation of amino acid composition among these proteins, all NEET proteins retained their unique CDGSH domain harboring their unique 3Cys:1His [2Fe-2S] cluster coordination through evolution. The coordinating exposed His was shown to convey the lability to the NEET proteins' [2Fe-2S] clusters. In this minireview, we discuss the NEET fold and its structural elements. Special attention is given to the unique lability of the NEETs' [2Fe-2S] cluster and the implication of the latter to the NEET proteins' cellular and systemic function in health and disease.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

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

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46

DOE PAGES

Balachandran, Manasi; Giannone, Richard J.; Bemis, David A.; ...

2017-08-31

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins withmore » an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.« less

Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis.

PubMed

Jones, J T; Reavy, B; Smant, G; Prior, A E

2004-01-07

We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.

Functional display of family 11 endoxylanases on the surface of phage M13.

PubMed

Beliën, T; Hertveldt, K; Van den Brande, K; Robben, J; Van Campenhout, S; Volckaert, G

2005-02-09

Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.

Stable expression of the hepatitis B virus surface antigen containing pre-S2 protein in mouse cells using a bovine papillomavirus vector.

PubMed

Yoneyama, T; Akatsuka, T; Miyamura, T

1988-08-01

The large BglII fragment (2.8 kilobases) of hepatitis B virus DNA including the transcription unit for the hepatitis B surface antigen (HBsAg) was inserted into a bovine papillomavirus vector containing the neomycin resistance gene. The recombinant DNA was transfected into mouse C127 cells. A stable transformed cell line (MS128) secreting a large amount of 22 nm HBsAg particles containing pre-S2 protein was established. The secreted HBsAg particles had the receptor for polymerized human serum albumin. Immunoprecipitation and Western blot analyses showed that HBsAg particles consisted of two major proteins of 22K and 26K encoded by the S gene and a minor protein of 35K encoded by the pre-S2 and S genes. Southern blot analysis revealed that the transfected plasmid was integrated into the host chromosomal DNA and that most of the plasmid sequences were present. These results suggest that the stable expression of the HBsAg in MS128 cells is related to the integrated state of the recombinant DNA.

[C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein].

PubMed

Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

2017-01-01

The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.

mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

PubMed Central

Kaltimbacher, Valérie; Bonnet, Crystel; Lecoeuvre, Gaëlle; Forster, Valérie; Sahel, José-Alain; Corral-Debrinski, Marisol

2006-01-01

As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface—the region coding for the mitochondrial targeting sequence and the 3′UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3′UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3′UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity. PMID:16751614

Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi

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

Whittingham, Jean L.; Blagova, Elena V.; Finn, Ciaran E.

2014-08-01

VapD is one of a set of highly homologous virulence-associated proteins from the multi-host pathogen Rhodococcus equi. The crystal structure reveals an eight-stranded β-barrel with a novel fold and a glycine rich ‘bald’ surface. Rhodococcus equi is a multi-host pathogen that infects a range of animals as well as immune-compromised humans. Equine and porcine isolates harbour a virulence plasmid encoding a homologous family of virulence-associated proteins associated with the capacity of R. equi to divert the normal processes of endosomal maturation, enabling bacterial survival and proliferation in alveolar macrophages. To provide a basis for probing the function of the Vapmore » proteins in virulence, the crystal structure of VapD was determined. VapD is a monomer as determined by multi-angle laser light scattering. The structure reveals an elliptical, compact eight-stranded β-barrel with a novel strand topology and pseudo-twofold symmetry, suggesting evolution from an ancestral dimer. Surface-associated octyl-β-d-glucoside molecules may provide clues to function. Circular-dichroism spectroscopic analysis suggests that the β-barrel structure is preceded by a natively disordered region at the N-terminus. Sequence comparisons indicate that the core folds of the other plasmid-encoded virulence-associated proteins from R. equi strains are similar to that of VapD. It is further shown that sequences encoding putative R. equi Vap-like proteins occur in diverse bacterial species. Finally, the functional implications of the structure are discussed in the light of the unique structural features of VapD and its partial structural similarity to other β-barrel proteins.« less

Binding Affinity prediction with Property Encoded Shape Distribution signatures

PubMed Central

Das, Sourav; Krein, Michael P.

2010-01-01

We report the use of the molecular signatures known as “Property-Encoded Shape Distributions” (PESD) together with standard Support Vector Machine (SVM) techniques to produce validated models that can predict the binding affinity of a large number of protein ligand complexes. This “PESD-SVM” method uses PESD signatures that encode molecular shapes and property distributions on protein and ligand surfaces as features to build SVM models that require no subjective feature selection. A simple protocol was employed for tuning the SVM models during their development, and the results were compared to SFCscore – a regression-based method that was previously shown to perform better than 14 other scoring functions. Although the PESD-SVM method is based on only two surface property maps, the overall results were comparable. For most complexes with a dominant enthalpic contribution to binding (ΔH/-TΔS > 3), a good correlation between true and predicted affinities was observed. Entropy and solvent were not considered in the present approach and further improvement in accuracy would require accounting for these components rigorously. PMID:20095526

Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement.

PubMed

Arambula, Diego; Wong, Wenge; Medhekar, Bob A; Guo, Huatao; Gingery, Mari; Czornyj, Elizabeth; Liu, Minghsun; Dey, Sanghamitra; Ghosh, Partho; Miller, Jeff F

2013-05-14

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila, an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 10(26) unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 10(19) distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3'end of ldtA. ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.

The Sus operon: a model system for starch uptake by the human gut Bacteroidetes

PubMed Central

Foley, Matthew H.; Cockburn, Darrell W.; Koropatkin, Nicole M.

2016-01-01

Resident bacteria in the densely populated human intestinal tract must efficiently compete for carbohydrate nutrition. The Bacteroidetes, a dominant bacterial phylum in the mammalian gut, encode a plethora of discrete polysaccharide utilization loci (PULs) that are selectively activated to facilitate glycan capture at the cell surface. The most well-studied PUL-encoded glycan-up-take system is the starch utilization system (Sus) of Bacteroides thetaiotaomicron. The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. In this review, we discuss what is known about the eight Sus proteins of B. thetaiotaomicron that define the Sus-like paradigm of nutrient acquisition that is exclusive to the Gram-negative Bacteroidetes. We emphasize the well-characterized outer membrane proteins SusDEF and the α-amylase SusG, each of which have unique structural features that allow them to interact with starch on the cell surface. Despite the apparent redundancy in starch-binding sites among these proteins, each has a distinct role during starch catabolism. Additionally, we consider what is known about how these proteins dynamically interact and cooperate in the membrane and propose a model for the formation of the Sus outer membrane complex. PMID:27137179

Type 3 fimbriae and biofilm formation are regulated by the transcriptional regulators MrkHI in Klebsiella pneumoniae.

PubMed

Johnson, Jeremiah G; Murphy, Caitlin N; Sippy, Jean; Johnson, Tylor J; Clegg, Steven

2011-07-01

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression.

Type 3 Fimbriae and Biofilm Formation Are Regulated by the Transcriptional Regulators MrkHI in Klebsiella pneumoniae▿

PubMed Central

Johnson, Jeremiah G.; Murphy, Caitlin N.; Sippy, Jean; Johnson, Tylor J.; Clegg, Steven

2011-01-01

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression. PMID:21571997

Phage as a template to grow bone mineral nanocrystals.

PubMed

Cao, Binrui; Xu, Hong; Mao, Chuanbin

2014-01-01

Phage display is a biotechnique that fuses functional peptides on the outer surface of filamentous phage by inserting DNA encoding the peptides into the genes of its coat proteins. The resultant peptide-displayed phage particles have been widely used as biotemplates for the synthesis of functional hybrid nanomaterials. Here, we describe the bioengineering of M13 filamentous phage to surface-display bone mineral (hydroxyapatite (HAP))-nucleating peptides derived from dentin matrix protein-1 and using the engineered phage as a biotemplate to grow HAP nanocrystals.

Druggable protein interaction sites are more predisposed to surface pocket formation than the rest of the protein surface.

PubMed

Johnson, David K; Karanicolas, John

2013-01-01

Despite intense interest and considerable effort via high-throughput screening, there are few examples of small molecules that directly inhibit protein-protein interactions. This suggests that many protein interaction surfaces may not be intrinsically "druggable" by small molecules, and elevates in importance the few successful examples as model systems for improving our fundamental understanding of druggability. Here we describe an approach for exploring protein fluctuations enriched in conformations containing surface pockets suitable for small molecule binding. Starting from a set of seven unbound protein structures, we find that the presence of low-energy pocket-containing conformations is indeed a signature of druggable protein interaction sites and that analogous surface pockets are not formed elsewhere on the protein. We further find that ensembles of conformations generated with this biased approach structurally resemble known inhibitor-bound structures more closely than equivalent ensembles of unbiased conformations. Collectively these results suggest that "druggability" is a property encoded on a protein surface through its propensity to form pockets, and inspire a model in which the crude features of the predisposed pocket(s) restrict the range of complementary ligands; additional smaller conformational changes then respond to details of a particular ligand. We anticipate that the insights described here will prove useful in selecting protein targets for therapeutic intervention.

Identification of two bvg-repressed surface proteins of Bordetella pertussis.

PubMed Central

Stenson, T H; Peppler, M S

1995-01-01

Bordetella pertussis, the etiological agent of whooping cough, has the ability to modulate its phenotype in response to environmental conditions by using the BvgAS sensory transduction system which is encoded by the vir locus (now known as bvg). The BvgAS system is part of a large family of two-component sensory transduction systems which are common to a number of pathogenic bacteria. Although much is known about the proteins which exist in the B. pertussis virulent (X-mode or phase I) phenotype, relatively little is known about the proteins produced in the avirulent (C-mode or phase III) phenotype. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing techniques to demonstrate the existence of at least 22 vir-repressed molecules which are increased in the avirulent phenotype. In addition, a series of monoclonal antibodies which are specific for the surface of avirulent B. pertussis were developed. Using immunological and protein techniques, we characterized two of these antigens as surface-exposed proteins. One of these antigens is expressed only in B. pertussis but not in the related species B. parapertussis and B. bronchiseptica. The other antigen is also present in B. parapertussis and B. bronchiseptica but is expressed at lower levels which are not regulated by bvg. The identification and characterization of vir-repressed proteins (and the genes which encode and regulate them) may help elucidate a physiological role for modulation of this obligate human pathogen. PMID:7558280

A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure

PubMed Central

Hauser, Christina T.; Tsien, Roger Y.

2007-01-01

Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (>220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (<15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn2+ ions binds tightly but reversibly to hexahistidine (His6) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca2+ from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His6 tags are accessible to the dye or antibodies, demonstrating externalization. PMID:17360414

Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

PubMed

Barz, W P; Walter, P

1999-04-01

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

PubMed Central

Barz, Wolfgang P.; Walter, Peter

1999-01-01

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δ cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins. PMID:10198056

ngs (notochord granular surface) gene encodes a novel type of intermediate filament family protein essential for notochord maintenance in zebrafish.

PubMed

Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

2013-01-25

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins.

ngs (Notochord Granular Surface) Gene Encodes a Novel Type of Intermediate Filament Family Protein Essential for Notochord Maintenance in Zebrafish*

PubMed Central

Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

2013-01-01

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins. PMID:23132861

Five Genes Encoding Surface-Exposed LPXTG Proteins Are Enriched in Hospital-Adapted Enterococcus faecium Clonal Complex 17 Isolates▿

PubMed Central

Hendrickx, Antoni P. A.; van Wamel, Willem J. B.; Posthuma, George; Bonten, Marc J. M.; Willems, Rob J. L.

2007-01-01

Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation. PMID:17873043

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, John J.; Barbour, Alan G.

1996-11-05

A method is provided herein for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.

Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

PubMed

Longo, Francesca; Motta, Sara; Mauri, Pierluigi; Landini, Paolo; Rossi, Elio

2016-11-25

In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

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

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface proteinmore » A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.« less

Druggable Protein Interaction Sites Are More Predisposed to Surface Pocket Formation than the Rest of the Protein Surface

PubMed Central

Johnson, David K.; Karanicolas, John

2013-01-01

Despite intense interest and considerable effort via high-throughput screening, there are few examples of small molecules that directly inhibit protein-protein interactions. This suggests that many protein interaction surfaces may not be intrinsically “druggable” by small molecules, and elevates in importance the few successful examples as model systems for improving our fundamental understanding of druggability. Here we describe an approach for exploring protein fluctuations enriched in conformations containing surface pockets suitable for small molecule binding. Starting from a set of seven unbound protein structures, we find that the presence of low-energy pocket-containing conformations is indeed a signature of druggable protein interaction sites and that analogous surface pockets are not formed elsewhere on the protein. We further find that ensembles of conformations generated with this biased approach structurally resemble known inhibitor-bound structures more closely than equivalent ensembles of unbiased conformations. Collectively these results suggest that “druggability” is a property encoded on a protein surface through its propensity to form pockets, and inspire a model in which the crude features of the predisposed pocket(s) restrict the range of complementary ligands; additional smaller conformational changes then respond to details of a particular ligand. We anticipate that the insights described here will prove useful in selecting protein targets for therapeutic intervention. PMID:23505360

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

PubMed

Dhalia, Rafael; Maciel, Milton; Cruz, Fábia S P; Viana, Isabelle F T; Palma, Mariana L; August, Thomas; Marques, Ernesto T A

2009-12-01

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

Functional Analysis of the p40 and p75 Proteins from Lactobacillus casei BL23

PubMed Central

Bäuerl, Christine; Pérez-Martínez, Gaspar; Yan, Fang; Polk, D. Brent; Monedero, Vicente

2011-01-01

The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria PMID:21178363

Solar to Liquid Fuels Production: Light-Driven Reduction of Carbon Dioxide to Formic Acid

DTIC Science & Technology

2014-03-29

molecular wire. The X-ray crystal structure for the E . coli FDH enzyme shows that a [4Fe-4S] cluster is located near the surface of the protein. The...CO2 to formic acid. E . coli FDH, encoded by fdhF, was chosen for this work because it is a single-subunit enzyme that has been studied in detail, and...mutagenesis was employed to change surface-located Cys11 to Gly to open a coordination site. The proteins were overproduced in E . coli and purified

Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes▿

PubMed Central

Mark, Linda; Spiller, O. Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A.; Wong, Scott W.; Damania, Blossom; Blom, Anna M.; Blackbourn, David J.

2007-01-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model. PMID:17287274

Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants.

PubMed

Lou, Xiao-Ming; Yao, Quan-Hong; Zhang, Zhen; Peng, Ri-He; Xiong, Ai-Sheng; Wang, Hua-Kun

2007-04-01

The original hepatitis B virus (HBV) large surface antigen gene was synthesized. In order to optimize the expression of this gene in tomato plants, the tobacco pathogenesis-related protein S signal peptide was fused to the 5' end of the modified gene and the sequence encoding amino acids S, E, K, D, E, and L was placed at the 3' end. The gene encoding the modified HBV large surface antigen under the control of a fruit-specific promoter was constructed and expressed in transgenic tomato plants. The expression of the antigen from transgenic plants was confirmed by PCR and reverse transcriptase PCR. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed that the maximal level of HBsAg was about 0.02% of the soluble protein in transgenic tomato fruit. The amount of HBsAg in mature fruits was found to be 65- to 171-fold larger than in small or medium fruits and leaf tissues. Examination of transgenic plant samples by transmission electron microscopy proved that HBsAg had been expressed and had accumulated. The HBsAg protein was capable of assembling into capsomers and virus-like particles. To our knowledge, this is the first time the HBV large surface antigen has been expressed in plants. This work suggests the possibility of producing a new alternative vaccine for human HBV.

Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

PubMed

Wilson, Marlena M; Anderson, D Eric; Bernstein, Harris D

2015-01-01

Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM) and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

Characterization of Plasmids in a Human Clinical Strain of Lactococcus garvieae

PubMed Central

Blanco, M. Mar; López-Campos, Guillermo H.; Cutuli, M. Teresa; Fernández-Garayzábal, José F.

2012-01-01

The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25) encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen. PMID:22768237

A newly identified protein of Leptospira interrogans mediates binding to laminin.

PubMed

Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2009-10-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

PubMed

Knodel, Markus M; Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; Targett-Adams, Paul; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

PubMed Central

Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-01

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles. PMID:29316722

Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7

PubMed Central

Kolling, Glynis L.; Matthews, Karl R.

1999-01-01

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms. PMID:10223967

Cell-free protein synthesis and assembly on a biochip

NASA Astrophysics Data System (ADS)

Heyman, Yael; Buxboim, Amnon; Wolf, Sharon G.; Daube, Shirley S.; Bar-Ziv, Roy H.

2012-06-01

Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.

Built-in adjuvanticity of genetically and protein-engineered chimeric molecules for targeting of influenza A peptide epitopes.

PubMed

Kerekov, Nikola S; Ivanova, Iva I; Mihaylova, Nikolina M; Nikolova, Maria; Prechl, Jozsef; Tchorbanov, Andrey I

2014-10-01

Highly purified, subunit, or synthetic viral antigens are known to be weakly immunogenic and potentate only the antibody, rather than cell-mediated immune responses. An alternative approach for inducing protective immunity with small viral peptides would be the direct targeting of viral epitopes to the immunocompetent cells by DNA vaccines encoding antibody fragments specific to activating cell surface co-receptor molecules. Here, we are exploring as a new genetic vaccine, a DNA chimeric molecule encoding a T and B cell epitope-containing influenza A virus hemagglutinin peptide joined to sequences encoding a single-chain variable fragment antibody fragment specific for the costimulatory B cell complement receptors 1 and 2. This recombinant DNA molecule was inserted into eukaryotic expression vector and used as a naked DNA vaccine in WT and CR1/2 KO mice. The intramuscular administration of the DNA construct resulted in the in vivo expression of an immunogenic chimeric protein, which cross-links cell surface receptors on influenza-specific B cells. The DNA vaccination was followed by prime-boosting with the protein-engineered replica of the DNA construct, thus delivering an activation intracellular signal. Immunization with an expression vector containing the described construct and boosting with the protein chimera induced a strong anti-influenza cytotoxic response, modulation of cytokine profile, and a weak antibody response in Balb/c mice. The same immunization scheme did not result in generation of influenza-specific response in mice lacking the target receptor, underlining the molecular adjuvant effect of receptor targeting.

Profiling charge complementarity and selectivity for binding at the protein surface.

PubMed

Sulea, Traian; Purisima, Enrico O

2003-05-01

A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins.

Functional Analysis of the Lactobacillus casei BL23 Sortases

PubMed Central

Muñoz-Provencio, Diego; Rodríguez-Díaz, Jesús; Collado, María Carmen; Langella, Philippe; Bermúdez-Humarán, Luis G.

2012-01-01

Sortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria. Lactobacillus casei BL23 harbors four sortase genes, two belonging to class A (srtA1 and srtA2) and two belonging to class C (srtC1 and srtC2). Class C sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili in Lactobacillus rhamnosus GG. Twenty-three genes encoding putative sortase substrates were identified in the L. casei BL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted in srtA1, srtA2, srtC1, and srtC2 and an srtA1 srtA2 double mutant were constructed. The transcription of all four sortase encoding genes was detected, but only the mutation of srtA1 resulted in a decrease in bacterial surface hydrophobicity. The β-N-acetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in the srtA1 mutant and, to a greater extent, in the srtA1 srtA2 double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in the srtA mutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for the srtA1 srtA2 strain. Mutations in srtC1 or srtC2 result in an undetectable phenotype. Together, these results suggest that SrtA1 is the housekeeping sortase in L. casei BL23 and SrtA2 would carry out redundant or complementary functions that become evident when SrtA1 activity is absent. PMID:23042174

Frequency of ace, epa and elrA Genes in Clinical and Environmental Strains of Enterococcus faecalis.

PubMed

Lysakowska, Monika Eliza; Denys, Andrzej; Sienkiewicz, Monika

2012-12-01

Surface proteins play an important role in the pathogenesis of enterococcal infections. Some of them are candidates for a vaccine, e.g., the frequency of endocarditis in rats vaccinated with Ace protein was 75 % as 12 opposed to 100 % in those who weren't. However, there are other components of enterococcal cells, such as Epa antigens or internalin-like proteins, which may be used in the prophylaxis of infections caused by them. However, also other virulence factors and resistance to antibiotics are important during enterococcal infection. Therefore, the relevance of ace, epa, elrA, other virulence genes, as well as resistance to antibiotics was investigated. 161 Enterococcus faecalis strains isolated from teaching hospitals in Lodz, cultured according to standard microbiological methods, were investigated for the presence of genes encoding surface proteins by PCR. Results were analyzed with χ(2) test. The elrA gene was found in all clinical and environmental strains, the ace gene was also widespread among E. faecalis (96.9 %). Both tested epa genes were found in the majority of isolates (83.25 %). There was correlation between the presence of esp and ace genes (p = 0.046) as well as between epa and agg genes (p = 0.0094; χ(2) test). The presence of the genes encoding surface proteins investigated in our study in the great majority of isolates implies that they would appear to be required during E. faecalis infection. Therefore, they could be excellent targets in therapy of enterococcal infections or, as some studies show, candidates for vaccines.

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

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

Zhao Haiyan, E-mail: zhaohy@ku.ed; Sequeira, Reuben D., E-mail: sequen@ku.ed; Galeva, Nadezhda A., E-mail: galeva@ku.ed

2011-01-20

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus maymore » be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.« less

Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function[OPEN

PubMed Central

2018-01-01

Plants have many, highly variable resistance (R) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize (Zea mays) Hm1, was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. PMID:29382771

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells

PubMed Central

Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2015-01-01

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

PubMed

Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2015-09-15

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex*

PubMed Central

Vincent, Maxence S.; Canestrari, Mickaël J.; Leone, Philippe; Stathopulos, Julien; Ize, Bérengère; Zoued, Abdelrahim; Cambillau, Christian; Kellenberger, Christine; Roussel, Alain

2017-01-01

The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts. PMID:28057754

A Liquid Array Platform For the Multiplexed Analysis of Synthetic Molecule-Protein Interactions

PubMed Central

Doran, Todd M.; Kodadek, Thomas

2014-01-01

Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule- and peptide-protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative comprised of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships and for certain types of serological analyses. PMID:24245981

Biomimetic surface coatings from modular amphiphilic proteins

NASA Astrophysics Data System (ADS)

Harden, James; Wan, Fan; Fischer, Stephen; Dick, Scott

2010-03-01

Recombinant DNA methods have been used to develop a library of diblock protein polymers for creating designer biofunctional interfaces. These proteins are composed of a surface-active, amphiphilic block joined to a disordered, water soluble block with an end terminal bioactive domain. The amphiphilic block has a strong affinity for many synthetic polymer surfaces, providing a facile means of imparting biological functionality to otherwise bio-neutral materials through physical self-assembly. We have incorporated a series of bioactive end domains into this diblock motif, including sequences that encode specific cell binding and signaling functions of extracellular matrix constituents (e.g. RGD and YIGSR). In this talk, we show that these diblock constructs self-assemble into biofunctional surface coatings on several model synthetic polymer materials. We demonstrate that surface adsorption of the proteins has minimal impacts on the presentation of the bioactive domains in the soluble block, and through the use of microscopic and cell proliferation assays, we show that the resulting biofunctional interfaces are capable of inducing appropriate cellular responses in a variety of human cell types.

Overproduction, purification and crystallization of a chondroitin sulfate A-binding DBL domain from a Plasmodium falciparum var2csa-encoded PfEMP1 protein

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

Higgins, Matthew K., E-mail: mkh20@cam.ac.uk

A chondroitin sulfate A-binding DBL important in placental malaria has been overproduced, purified and crystallized. Diffraction data were collected to 1.9 Å resolution. The PfEMP1 proteins of the malaria parasite Plasmodium falciparum are inserted into the membrane of infected red blood cells, where they mediate adhesion to a variety of human receptors. The DBL domains of the var2csa-encoded PfEMP1 protein play a critical role in malaria of pregnancy, tethering infected cells to the surface of the placenta through interactions with the glycosaminoglycan carbohydrate chondroitin sulfate A (CSA). A CSA-binding DBL domain has been overproduced in a bacterial expression system, purifiedmore » and crystallized. Native data sets extending to 1.9 Å resolution have been collected and phasing is under way.« less

Construction of a filamentous phage display peptide library.

PubMed

Fagerlund, Annette; Myrset, Astrid Hilde; Kulseth, Mari Ann

2014-01-01

The concept of phage display is based on insertion of random oligonucleotides at an appropriate location within a structural gene of a bacteriophage. The resulting phage will constitute a library of random peptides displayed on the surface of the bacteriophages, with the encoding genotype packaged within each phage particle. Using a phagemid/helper phage system, the random peptides are interspersed between wild-type coat proteins. Libraries of phage-expressed peptides may be used to search for novel peptide ligands to target proteins. The success of finding a peptide with a desired property in a given library is highly dependent on the diversity and quality of the library. The protocols in this chapter describe the construction of a high-diversity library of phagemid vector encoding fusions of the phage coat protein pVIII with random peptides, from which a phage library displaying random peptides can be prepared.

Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses

PubMed Central

Szécsi, Judit; Boson, Bertrand; Johnsson, Per; Dupeyrot-Lacas, Pia; Matrosovich, Mikhail; Klenk, Hans-Dieter; Klatzmann, David; Volchkov, Viktor; Cosset, François-Loïc

2006-01-01

There is an urgent need to develop novel approaches to vaccination against the emerging, highly pathogenic avian influenza viruses. Here, we engineered influenza viral-like particles (Flu-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two highly pathogenic influenza viruses of either H7N1 or H5N1 antigenic subtype. We demonstrate that, upon recovery of viral RNAs from a field strain, one can easily generate expression vectors that encode the HA, NA and M2 surface proteins of either virus and prepare high-titre Flu-VLPs. We characterise these Flu-VLPs incorporating the HA, NA and M2 proteins and we show that they induce high-titre neutralising antibodies in mice. PMID:16948862

Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins.

PubMed

Macaulay, Iain C; Tijssen, Marloes R; Thijssen-Timmer, Daphne C; Gusnanto, Arief; Steward, Michael; Burns, Philippa; Langford, Cordelia F; Ellis, Peter D; Dudbridge, Frank; Zwaginga, Jaap-Jan; Watkins, Nicholas A; van der Schoot, C Ellen; Ouwehand, Willem H

2007-04-15

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

Characterization of EhaJ, a New Autotransporter Protein from Enterohemorrhagic and Enteropathogenic Escherichia coli

PubMed Central

Easton, Donna M.; Totsika, Makrina; Allsopp, Luke P.; Phan, Minh-Duy; Idris, Adi; Wurpel, Daniël J.; Sherlock, Orla; Zhang, Bing; Venturini, Carola; Beatson, Scott A.; Mahony, Timothy J.; Cobbold, Rowland N.; Schembri, Mark A.

2011-01-01

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence. PMID:21687429

An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.

PubMed Central

Gems, D; Maizels, R M

1996-01-01

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes. Images Fig. 1 Fig. 4 PMID:8643687

Transgenic cells with increased plastoquinone levels and methods of use

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

Sayre, Richard T.; Subramanian, Sowmya; Cahoon, Edgar

Disclosed herein are transgenic cells expressing a heterologous nucleic acid encoding a prephenate dehydrogenase (PDH) protein, a heterologous nucleic acid encoding a homogentisate solanesyl transferase (HST) protein, a heterologous nucleic acid encoding a deoxyxylulose phosphate synthase (DXS) protein, or a combination of two or more thereof. In particular examples, the disclosed transgenic cells have increased plastoquinone levels. Also disclosed are methods of increasing cell growth rates or production of biomass by cultivating transgenic cells expressing a heterologous nucleic acid encoding a PDH protein, a heterologous nucleic acid encoding an HST protein, a heterologous nucleic acid encoding a DXS protein, ormore » a combination of two or more thereof under conditions sufficient to produce cell growth or biomass.« less

Crystal structure of the DNA polymerase III β subunit (β-clamp) from the extremophile Deinococcus radiodurans.

PubMed

Niiranen, Laila; Lian, Kjersti; Johnson, Kenneth A; Moe, Elin

2015-02-27

Deinococcus radiodurans is an extremely radiation and desiccation resistant bacterium which can tolerate radiation doses up to 5,000 Grays without losing viability. We are studying the role of DNA repair and replication proteins for this unusual phenotype by a structural biology approach. The DNA polymerase III β subunit (β-clamp) acts as a sliding clamp on DNA, promoting the binding and processivity of many DNA-acting proteins, and here we report the crystal structure of D. radiodurans β-clamp (Drβ-clamp) at 2.0 Å resolution. The sequence verification process revealed that at the time of the study the gene encoding Drβ-clamp was wrongly annotated in the genome database, encoding a protein of 393 instead of 362 amino acids. The short protein was successfully expressed, purified and used for crystallisation purposes in complex with Cy5-labeled DNA. The structure, which was obtained from blue crystals, shows a typical ring-shaped bacterial β-clamp formed of two monomers, each with three domains of identical topology, but with no visible DNA in electron density. A visualisation of the electrostatic surface potential reveals a highly negatively charged outer surface while the inner surface and the dimer forming interface have a more even charge distribution. The structure of Drβ-clamp was determined to 2.0 Å resolution and shows an evenly distributed electrostatic surface charge on the DNA interacting side. We hypothesise that this charge distribution may facilitate efficient movement on encircled DNA and help ensure efficient DNA metabolism in D. radiodurans upon exposure to high doses of ionizing irradiation or desiccation.

Comparative Transcriptome Analysis of Desulfovibrio Vulgaris Grown in Planktonic Culture and Mature Biofilm on a Steel Surface

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

Zhang, Weiwen; Culley, David E.; Nie, Lei

2007-08-01

The build-up of biofilms of sulphate -reducing bacteria (SRB) on metals surfaces may lead to severe corrosion of iron. To understand the processes at molecular level, in this study, a whole-genome oligonucleotide microarray was used to examine differential expression patterns between planktonic populations and mature biofilm of model SRB species Desulfovibrio vulgaris. Statistical analysis revealed that 472 genes were differentially expressed (1.5 fold or more with a p value less than 0.025) when comparing biofilm to planktonic cells. Among the differentially expressed genes were several that corresponded to biofilm formation genes identified in many aerobic bacterial biofilms (i.e., Pseudomonas speciesmore » and Escherichia coli), such as down-regulation of genes encoding flagellin, flagellar motor switch protein and chemotaxis proteins involved in cell motility and induction of genes encoding sugar transferase and glycogen synthase involved in exopolysaccharide biosynthesis. In addition, D. vulgaris biofilm-bound cells exhibited decreased transcription of genes involved in protein synthesis, energy metabolism and sulfate reduction, as well as genes involved in general stress responses. These findings were all consistent with early suggestion that the average physiology of biofilm cells were similar to planktonic cells of stationary phases. Most notably, up-regulation of large number of outer membrane proteins was observed in D. vulgaris biofilm. Although their function is still unknown, the higher expression of these genes in D. vulgaris biofilm could implicate important roles formation and maintenance of multi-cellular consortium on metal surface. The study provided insights into the metabolic networks associated with D. vulgaris biofilm formation and maintenance on an iron surface.« less

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

PubMed

Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

2016-04-19

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Acylation-dependent protein export in Leishmania.

PubMed

Denny, P W; Gokool, S; Russell, D G; Field, M C; Smith, D F

2000-04-14

The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.

Tick-Borne Transmission of Two Genetically Distinct Anaplasma marginale Strains following Superinfection of the Mammalian Reservoir Host

USDA-ARS?s Scientific Manuscript database

Strain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for genetically distinct strains of the tick-borne pathogen Anaplasma marginale that encode distinctly different surface protein variants. Supe...

Profiling Charge Complementarity and Selectivity for Binding at the Protein Surface

PubMed Central

Sulea, Traian; Purisima, Enrico O.

2003-01-01

A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins. PMID:12719221

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

Fast Kinetics of Calcium Signaling and Sensor Design

PubMed Central

Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J.

2015-01-01

Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change. PMID:26151819

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

PubMed Central

Seefeld, Ting H.; Halpern, Aaron R.; Corn, Robert M.

2012-01-01

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications. PMID:22793370

Four Proteins Encoded in the gspB-secY2A2 Operon of Streptococcus gordonii Mediate the Intracellular Glycosylation of the Platelet-Binding Protein GspB

PubMed Central

Takamatsu, Daisuke; Bensing, Barbara A.; Sullam, Paul M.

2004-01-01

Platelet binding by Streptococcus gordonii strain M99 is mediated predominantly by the cell surface glycoprotein GspB. This adhesin consists of a putative N-terminal signal peptide, two serine-rich regions (SRR1 and SRR2), a basic region between SRR1 and SRR2, and a C-terminal cell wall anchoring domain. The glycosylation of GspB is mediated at least in part by Gly and Nss, which are encoded in the secY2A2 locus immediately downstream of gspB. This region also encodes two proteins (Gtf and Orf4) that are required for the expression of GspB but whose functions have not been delineated. In this study, we further characterized the roles of Gly, Nss, Gtf, and Orf4 by investigating the expression and glycosylation of a series of glutathione S-transferase-GspB fusion proteins in M99 and in gly, nss, gtf, and orf4 mutants. Compared with fusion proteins expressed in the wild-type background, fusion proteins expressed in the mutant strain backgrounds showed altered electrophoretic mobility. In addition, the fusion proteins formed insoluble aggregates in protoplasts of the gtf and orf4 mutants. Glycan detection and lectin blot analysis revealed that SRR1 and SRR2 were glycosylated but that the basic region was unmodified. When the fusion protein was expressed in Escherichia coli, glycosylation of this protein was observed only in the presence of both gtf and orf4. These results demonstrate that Gly, Nss, Gtf, and Orf4 are all involved in the intracellular glycosylation of SRRs. Moreover, Gtf and Orf4 are essential for glycosylation, which in turn is important for the solubility of GspB. PMID:15489421

Generation and Surface Localization of Intact M Protein in Streptococcus pyogenes Are Dependent on sagA

PubMed Central

Biswas, Indranil; Germon, Pierre; McDade, Kathleen; Scott, June R.

2001-01-01

The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019–6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor. PMID:11598078

Toroidal surface complexes of bacteriophage {phi}12 are responsible for host-cell attachment

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

Leo-Macias, Alejandra; Katz, Garrett; Wei Hui

2011-06-05

Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage {phi}12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of {phi}12 demonstrates that the gene for the hexameric spike is located in {phi}12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimatesmore » derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity. - Research Highlights: > Subtomogram averaging reveals enhanced detail of a {phi}12 cystovirus surface protein complex. > The surface protein complex has a toroidal shape and six-fold symmetry. > It is encoded by the medium-size genome segment. > The proteins of the surface complex most likely are one copy of P3a and three copies of P3c.« less

Summaries of Research 1987

DTIC Science & Technology

1987-01-01

STRUCTURE OF THE GENE ENCODING THE CIRCUMSPOROZOITE PROTEIN OF PLASWODIUm YOELIJ. A RODENT MCDFL FOP EIAMINING ANTIMALARIAL SPOBOZOITE VACCINES. JOURNAL OF...BICLOGICAL CHEMISTRY 1987 MAR 5;262(7):2937-40 INFECTIOUS DISEASES 3M162770A870.AF.312-1 (DA301614) REPORT NO.E ANTIGENS, SURFACE PLAS!CDIU? AD A188...SURFACE CLONING, MOLECULAR PLASMCDIUM dERGHEI AD A185 330 NI!PI 87-C036 ROTd ?L CHUANG DM MULTIPLE EECHANIS!15 CF SEROTONERGIC SIGNAL TRANSDUCTION. LIFE

Protein immobilization techniques for microfluidic assays

PubMed Central

Kim, Dohyun; Herr, Amy E.

2013-01-01

Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

An internal thioester in a pathogen surface protein mediates covalent host binding

PubMed Central

Walden, Miriam; Edwards, John M; Dziewulska, Aleksandra M; Bergmann, Rene; Saalbach, Gerhard; Kan, Su-Yin; Miller, Ona K; Weckener, Miriam; Jackson, Rosemary J; Shirran, Sally L; Botting, Catherine H; Florence, Gordon J; Rohde, Manfred; Banfield, Mark J; Schwarz-Linek, Ulrich

2015-01-01

To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a ‘chemical harpoon’. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions. DOI: http://dx.doi.org/10.7554/eLife.06638.001 PMID:26032562

Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles [corrected].

PubMed

Strecker, Thomas; Eichler, Robert; Meulen, Jan ter; Weissenhorn, Winfried; Dieter Klenk, Hans; Garten, Wolfgang; Lenz, Oliver

2003-10-01

Lassa virus is an enveloped virus with glycoprotein spikes on its surface. It contains an RNA ambisense genome that encodes the glycoprotein precursor GP-C, the nucleoprotein NP, the polymerase L, and the Z protein. Here we demonstrate that the Lassa virus Z protein (i). is abundant in viral particles, (ii). is strongly membrane associated, (iii). is sufficient in the absence of all other viral proteins to release enveloped particles, and (iv). contains two late domains, PTAP and PPXY, necessary for the release of virus-like particles. Our data provide evidence that Z is the Lassa virus matrix protein that is the driving force for virus particle release.

Improved protein surface comparison and application to low-resolution protein structure data.

PubMed

Sael, Lee; Kihara, Daisuke

2010-12-14

Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

Targeted Resequencing and Functional Testing Identifies Low-Frequency Missense Variants in the Gene Encoding GARP as Significant Contributors to Atopic Dermatitis Risk.

PubMed

Manz, Judith; Rodríguez, Elke; ElSharawy, Abdou; Oesau, Eva-Maria; Petersen, Britt-Sabina; Baurecht, Hansjörg; Mayr, Gabriele; Weber, Susanne; Harder, Jürgen; Reischl, Eva; Schwarz, Agatha; Novak, Natalija; Franke, Andre; Weidinger, Stephan

2016-12-01

Gene-mapping studies have consistently identified a susceptibility locus for atopic dermatitis and other inflammatory diseases on chromosome band 11q13.5, with the strongest association observed for a common variant located in an intergenic region between the two annotated genes C11orf30 and LRRC32. Using a targeted resequencing approach we identified low-frequency and rare missense mutations within the LRRC32 gene encoding the protein GARP, a receptor on activated regulatory T cells that binds latent transforming growth factor-β. Subsequent association testing in more than 2,000 atopic dermatitis patients and 2,000 control subjects showed a significant excess of these LRRC32 variants in individuals with atopic dermatitis. Structural protein modeling and bioinformatic analysis predicted a disruption of protein transport upon these variants, and overexpression assays in CD4 + CD25 - T cells showed a significant reduction in surface expression of the mutated protein. Consistently, flow cytometric (FACS) analyses of different T-cell subtypes obtained from atopic dermatitis patients showed a significantly reduced surface expression of GARP and a reduced conversion of CD4 + CD25 - T cells into regulatory T cells, along with lower expression of latency-associated protein upon stimulation in carriers of the LRRC32 A407T variant. These results link inherited disturbances of transforming growth factor-β signaling with atopic dermatitis risk. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function.

PubMed

Kourelis, Jiorgos; van der Hoorn, Renier A L

2018-02-01

Plants have many, highly variable resistance ( R ) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize ( Zea mays ) Hm1 , was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. © 2018 American Society of Plant Biologists. All rights reserved.

Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

PubMed Central

Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

2013-01-01

Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. PMID:23233310

Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

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

Tao Yongguang; Song Xing; Deng Xiyun

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 couldmore » regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.« less

A genetically encoded ratiometric sensor to measure extracellular pH in microdomains bounded by basolateral membranes of epithelial cells.

PubMed

Urra, Javier; Sandoval, Moisés; Cornejo, Isabel; Barros, L Felipe; Sepúlveda, Francisco V; Cid, L Pablo

2008-10-01

Extracellular pH, especially in relatively inaccessible microdomains between cells, affects transport membrane protein activity and might have an intercellular signaling role. We have developed a genetically encoded extracellular pH sensor capable of detecting pH changes in basolateral spaces of epithelial cells. It consists of a chimerical membrane protein displaying concatenated enhanced variants of cyan fluorescence protein (ECFP) and yellow fluorescence protein (EYFP) at the external aspect of the cell surface. The construct, termed pHCECSensor01, was targeted to basolateral membranes of Madin-Darby canine kidney (MDCK) cells by means of a sequence derived from the aquaporin AQP4. The fusion of pH-sensitive EYFP with pH-insensitive ECFP allows ratiometric pH measurements. The titration curve of pHCECSensor01 in vivo had a pK (a) value of 6.5 +/- 0.04. Only minor effects of extracellular chloride on pHCECSensor01 were observed around the physiological concentrations of this anion. In MDCK cells, the sensor was able to detect changes in pH secondary to H(+) efflux into the basolateral spaces elicited by an ammonium prepulse or lactate load. This genetically encoded sensor has the potential to serve as a noninvasive tool for monitoring changes in extracellular pH microdomains in epithelial and other tissues in vivo.

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

PubMed

Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

2006-07-01

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

The Draft Genome of the Non-Host-Associated Methanobrevibacter arboriphilus Strain DH1 Encodes a Large Repertoire of Adhesin-Like Proteins

PubMed Central

Poehlein, Anja; Daniel, Rolf

2017-01-01

Methanobrevibacter arboriphilus strain DH1 is an autotrophic methanogen that was isolated from the wetwood of methane-emitting trees. This species has been of considerable interest for its unusual oxygen tolerance and has been studied as a model organism for more than four decades. Strain DH1 is closely related to other host-associated Methanobrevibacter species from intestinal tracts of animals and the rumen, making this strain an interesting candidate for comparative analysis to identify factors important for colonizing intestinal environments. Here, the genome sequence of M. arboriphilus strain DH1 is reported. The draft genome is composed of 2.445.031 bp with an average GC content of 25.44% and predicted to harbour 1964 protein-encoding genes. Among the predicted genes, there are also more than 50 putative genes for the so-called adhesin-like proteins (ALPs). The presence of ALP-encoding genes in the genome of this non-host-associated methanogen strongly suggests that target surfaces for ALPs other than host tissues also need to be considered as potential interaction partners. The high abundance of ALPs may also indicate that these types of proteins are more characteristic for specific phylogenetic groups of methanogens rather than being indicative for a particular environment the methanogens thrives in. PMID:28634433

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).

PubMed

He, M; Taussig, M J

2001-08-01

We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.

Characterization of three novel adhesins of Leptospira interrogans.

PubMed

Siqueira, Gabriela H; Atzingen, Marina V; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2013-12-01

We report cloning, expression, purification, and characterization of three predicted leptospiral membrane proteins (LIC11360, LIC11009, and LIC11975). In silico analysis and proteinase K accessibility data suggest that these proteins might be surface exposed. We show that proteins encoded by LIC11360, LIC11009 and LIC11975 genes interact with laminin in a dose-dependent and saturable manner. The proteins are referred to as leptospiral surface adhesions 23, 26, and 36 (Lsa23, Lsa26, and Lsa36), respectively. These proteins also bind plasminogen and generate active plasmin. Attachment of Lsa23 and Lsa36 to fibronectin occurs through the involvement of the 30-kDa and 70-kDa heparin-binding domains of the ligand. Dose-dependent, specific-binding of Lsa23 to the complement regulator C4BP and to a lesser extent, to factor H, suggests that this protein may interfere with the complement cascade pathways. Leptospira spp. may use these interactions as possible mechanisms during the establishment of infection.

Characterization of Three Novel Adhesins of Leptospira interrogans

PubMed Central

Siqueira, Gabriela H.; Atzingen, Marina V.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Nascimento, Ana L. T. O.

2013-01-01

We report cloning, expression, purification, and characterization of three predicted leptospiral membrane proteins (LIC11360, LIC11009, and LIC11975). In silico analysis and proteinase K accessibility data suggest that these proteins might be surface exposed. We show that proteins encoded by LIC11360, LIC11009 and LIC11975 genes interact with laminin in a dose-dependent and saturable manner. The proteins are referred to as leptospiral surface adhesions 23, 26, and 36 (Lsa23, Lsa26, and Lsa36), respectively. These proteins also bind plasminogen and generate active plasmin. Attachment of Lsa23 and Lsa36 to fibronectin occurs through the involvement of the 30-kDa and 70-kDa heparin-binding domains of the ligand. Dose-dependent, specific-binding of Lsa23 to the complement regulator C4BP and to a lesser extent, to factor H, suggests that this protein may interfere with the complement cascade pathways. Leptospira spp. may use these interactions as possible mechanisms during the establishment of infection. PMID:23958908

Mannose-specific interaction of Lactobacillus plantarum with porcine jejunal epithelium.

PubMed

Gross, Gabriele; van der Meulen, Jan; Snel, Johannes; van der Meer, Roelof; Kleerebezem, Michiel; Niewold, Theo A; Hulst, Marcel M; Smits, Mari A

2008-11-01

Host-microorganism interactions in the intestinal tract are complex, and little is known about specific nonpathogenic microbial factors triggering host responses in the gut. In this study, mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig Small Intestinal Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were compared with those of two corresponding mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of the wild-type strain associated with the intestinal surface could be observed after 8 h of perfusion when a mixture of wild-type and msa-mutant strain had been applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended to induce a decrease in jejunal net fluid absorption compared with control conditions. Furthermore, after 8 h of perfusion expression of the host gene encoding pancreatitis-associated protein, a protein with proposed bactericidal properties, was found to be upregulated by the wild-type strain only. These observations suggest a role of Msa in the induction of host responses in the pig intestine.

A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites.

PubMed

Paul, Gourab; Deshmukh, Arunaditya; Kaur, Inderjeet; Rathore, Sumit; Dabral, Surbhi; Panda, Ashutosh; Singh, Susheel Kumar; Mohmmed, Asif; Theisen, Michael; Malhotra, Pawan

2017-02-16

The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface. Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-1 65 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera. Immunoprecipitation of parasite-derived polypeptides, followed by LC-MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein-protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-1 65 and GLURP proteins. Together the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface.

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

PubMed

Sarmiento, José M; Añazco, Carolina C; Campos, Danae M; Prado, Gregory N; Navarro, Javier; González, Carlos B

2004-11-05

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Microsporidian polar tube proteins: highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups.

PubMed

Polonais, Valérie; Prensier, Gérard; Méténier, Guy; Vivarès, Christian P; Delbac, Frédéric

2005-09-01

The spore polar tube is a unique organelle required for cell invasion by fungi-related microsporidian parasites. Two major polar tube proteins (PTP1 and PTP2) are encoded by two tandemly arranged genes in Encephalitozoon species. A look at Antonospora (Nosema) locustae contigs (http://jbpc.mbl.edu/Nosema/Contigs/) revealed significant conservation in the order and orientation of various genes, despite high sequence divergence features, when comparing with Encephalitozoon cuniculi complete genome. This syntenic relationship between distantly related Encephalitozoon and Antonospora genera has been successfully exploited to identify ptp1 and ptp2 genes in two insect-infecting species assigned to the Antonospora clade (A. locustae and Paranosema grylli). Targeting of respective proteins to the polar tube was demonstrated through immunolocalization experiments with antibodies raised against recombinant proteins. Both PTPs were extracted from spores with 100mM dithiothreitol. Evidence for PTP1 mannosylation was obtained in studied species, supporting a key role of PTP1 in interactions with host cell surface.

Differential splicing generates a nervous system-specific form of Drosophila neuroglian.

PubMed

Hortsch, M; Bieber, A J; Patel, N H; Goodman, C S

1990-05-01

We recently described the characterization and cloning of Drosophila neuroglian, a member of the immunoglobulin superfamily. Neuroglian contains six immunoglobulin-like domains and five fibronectin type III domains and shows strong sequence homology to the mouse neural cell adhesion molecule L1. Here we show that the neuroglian gene generates at least two different protein products by tissue-specific alternative splicing. The two protein forms differ in their cytoplasmic domains. The long form is restricted to the surface of neurons in the CNS and neurons and some support cells in the PNS; in contrast, the short form is expressed on a wide range of other cells and tissues. Thus, whereas the mouse L1 gene appears to encode only one protein that functions largely as a neural cell adhesion molecule, its Drosophila homolog, the neuroglian gene, encodes at least two protein forms that may play two different roles, one as a neural cell adhesion molecule and the other as a more general cell adhesion molecule involved in other tissues and imaginal disc morphogenesis.

Detection of protein-protein interactions by ribosome display and protein in situ immobilisation.

PubMed

He, Mingyue; Liu, Hong; Turner, Martin; Taussig, Michael J

2009-12-31

We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.

Mutations in STAMBP, encoding a deubiquitinating enzyme, cause Microcephaly-Capillary Malformation syndrome

PubMed Central

McDonell, Laura M.; Mirzaa, Ghayda M.; Alcantara, Diana; Schwartzentruber, Jeremy; Carter, Melissa T.; Lee, Leo J.; Clericuzio, Carol L.; Graham, John M.; Morris-Rosendahl, Deborah J.; Polster, Tilman; Acsadi, Gyula; Townshend, Sharron; Williams, Simon; Halbert, Anne; Isidor, Bertrand; Smyser, Christopher D.; Paciorkowski, Alex R.; Willing, Marcia; Woulfe, John; Das, Soma; Beaulieu, Chandree L.; Marcadier, Janet; Geraghty, Michael T.; Frey, Brendan J.; Majewski, Jacek; Bulman, Dennis E.; Dobyns, William B.; O’Driscoll, Mark; Boycott, Kym M.

2014-01-01

Microcephaly-capillary malformation (MIC-CAP) syndrome exhibits severe microcephaly with progressive cortical atrophy, intractable epilepsy, profound developmental delay and multiple small capillary malformations on the skin. We employed whole-exome sequencing of five patients with MIC-CAP syndrome and identified novel recessive mutations in STAMBP, a gene encoding the deubiquitinating (DUB) isopeptidase STAMBP (STAM-binding protein)/AMSH (Associated Molecule with the SH3 domain of STAM), that plays a key role in cell surface receptor-mediated endocytosis and sorting. Patient cell lines showed reduced STAMBP expression associated with accumulation of ubiquitin-conjugated protein aggregates, elevated apoptosis and insensitive activation of the RAS-MAPK and PI3K-AKT-mTOR pathways. The latter cellular phenotype is significant considering the established connection between these pathways and their association with vascular and capillary malformations. Furthermore, our findings of a congenital human disorder caused by a defective DUB protein that functions in endocytosis, implicates ubiquitin-conjugate aggregation and elevated apoptosis as factors potentially influencing the progressive neuronal loss underlying MIC-CAP. PMID:23542699

Patchwork structure-function analysis of the Sendai virus matrix protein.

PubMed

Mottet-Osman, Geneviève; Miazza, Vincent; Vidalain, Pierre-Olivier; Roux, Laurent

2014-09-01

Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production. Copyright © 2014 Elsevier Inc. All rights reserved.

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

PubMed Central

Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P.

2016-01-01

Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity.

PubMed

Nouvel, Laurent X; Sirand-Pugnet, Pascal; Marenda, Marc S; Sagné, Eveline; Barbe, Valérie; Mangenot, Sophie; Schenowitz, Chantal; Jacob, Daniel; Barré, Aurélien; Claverol, Stéphane; Blanchard, Alain; Citti, Christine

2010-02-02

While the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study. The two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms. Overall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events.

Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity

PubMed Central

2010-01-01

Background While the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study. Results The two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms. Conclusion Overall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events. PMID:20122262

Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the gene encoding. alpha. -mannosidase II

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

Fukuda, M.N.; Masri, K.A.; Dell, A.

1990-10-01

Congenital dyserythropoietic anemia type II, or hereditary erythroblastic multinuclearity with a positive acidified-serum-lysis test (HEMPAS), is a genetic anemia in humans inherited by an autosomally recessive mode. The enzyme defect in most HEMPAS patients has previously been proposed as a lowered activity of N-acetylglucosaminyltransferase II, resulting in a lack of polylactosamine on proteins and leading to the accumulation of polylactosaminyl lipids. A recent HEMPAS case, G.C., has now been analyzed by cell-surface labeling, fast-atom-bombardment mass spectrometry of glycopeptides, and activity assay of glycosylation enzymes. Significantly decreased glycosylation of polylactosaminoglycan proteins and incompletely processed asparagine-linked oligosaccharides were detected in the erythrocytemore » membranes of G.C. These results suggest that G.C. cells contain a mutation in {alpha}-ManII-encoding gene that results in inefficient expression of {alpha}-ManII mRNA, either through reduced transcription or message instability. This report demonstrates that HEMPAS is caused by a defective gene encoding an enzyme necessary for the synthesis of asparagine-linked oligosaccharides.« less

Functional role of oppA encoding an oligopeptide-binding protein from Lactobacillus salivarius Ren in bile tolerance.

PubMed

Wang, Guohong; Li, Dan; Ma, Xiayin; An, Haoran; Zhai, Zhengyuan; Ren, Fazheng; Hao, Yanling

2015-08-01

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and some L. salivarius strains are considered as probiotics. Bile tolerance is a crucial property for probiotic bacteria to survive the transit through the GIT and exert their beneficial effects. In this work, the functional role of oppA encoding an oligopeptide transporter substrate-binding protein from L. salivarius Ren in bile salt tolerance was investigated. In silico analysis revealed that the oppA gene encodes a 61.7-kDa cell surface-anchored hydrophilic protein with a canonical lipoprotein signal peptide. Homologous overexpression of OppA was shown to confer 20-fold higher tolerance to 0.5 % oxgall in L. salivarius Ren. Furthermore, the recombinant strain exhibited 1.8-fold and 3.6-fold higher survival when exposed to the sublethal concentration of sodium taurocholate and sodium taurodeoxycholate, respectively, while no significant change was observed when exposed to sodium glycocholate and sodium glycodeoxycholate (GDCA). Our results indicate that OppA confers specific resistance to taurine-conjugated bile salts in L. salivarius Ren. In addition, the OppA overexpression strain also showed significant increased resistance to heat and salt stresses, suggesting the protective role of OppA against multiple stresses in L. salivarius Ren.

Cross-reactivity between the immunodominant determinant of the antigen I component of Streptococcus sobrinus SpaA protein and surface antigens from other members of the Streptococcus mutans group.

PubMed

Goldschmidt, R M; Curtiss, R

1990-07-01

Most members of the Streptococcus mutans group of microorganisms specify a major cell surface-associated protein, SpaA, that is defined by its antigenic properties. The region of the spaA gene from Streptococcus sobrinus 6715 encoding the immunodominant determinant of the major antigenic component (antigen I) of the SpaA protein has recently been characterized. This study examined whether recognition of the immunodominant determinant is independent of the immunized animal host and whether antibodies elicited by the immunodominant determinant cross-react with cell surface proteins from S. mutans of various serotypes. Mouse and rabbit antisera to the undenatured SpaA protein reacted similarly both with the immunodominant determinant and with other antigenic structures of the protein in Western immunoblots with SpaA polypeptides that were specified by spaA gene fragments expressed in recombinant Escherichia coli. This suggests that the antibody responses of inbred and outbred animals were similar. Furthermore, antibodies raised against both the S. sobrinus SpaA immunodominant determinant expressed by recombinant E. coli and the purified protein from S. sobrinus displayed similar strain specificities and protein band profiles towards cells surface proteins from S. mutans of various serotypes in immunodot and Western blot analyses, respectively. This suggests that for S. sobrinus serotype g, the immune response against the SpaA protein is governed by the immunodominant determinant of antigen I. In addition, it indicates that the SpaA protein domain containing the immunodominant determinant overlaps the domain conferring cross-reactivity to cell surface proteins of S. mutans of various serotypes.

The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

PubMed

Music, Nedzad; Gagnon, Carl A

2010-12-01

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1α, nsp1β, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 3' end of the viral genome encodes four minor and three major structural proteins. The GP(2a), GP₃ and GP₄ (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP₅ (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis.

Improved protein surface comparison and application to low-resolution protein structure data

PubMed Central

2010-01-01

Background Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. Results The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Conclusions Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy. PMID:21172052

Buried and accessible surface area control intrinsic protein flexibility.

PubMed

Marsh, Joseph A

2013-09-09

Proteins experience a wide variety of conformational dynamics that can be crucial for facilitating their diverse functions. How is the intrinsic flexibility required for these motions encoded in their three-dimensional structures? Here, the overall flexibility of a protein is demonstrated to be tightly coupled to the total amount of surface area buried within its fold. A simple proxy for this, the relative solvent-accessible surface area (Arel), therefore shows excellent agreement with independent measures of global protein flexibility derived from various experimental and computational methods. Application of Arel on a large scale demonstrates its utility by revealing unique sequence and structural properties associated with intrinsic flexibility. In particular, flexibility as measured by Arel shows little correspondence with intrinsic disorder, but instead tends to be associated with multiple domains and increased α-helical structure. Furthermore, the apparent flexibility of monomeric proteins is found to be useful for identifying quaternary-structure errors in published crystal structures. There is also a strong tendency for the crystal structures of more flexible proteins to be solved to lower resolutions. Finally, local solvent accessibility is shown to be a primary determinant of local residue flexibility. Overall, this work provides both fundamental mechanistic insight into the origin of protein flexibility and a simple, practical method for predicting flexibility from protein structures. © 2013 Elsevier Ltd. All rights reserved.

Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

PubMed Central

Quiroz, Felipe García; Chilkoti, Ashutosh

2015-01-01

Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level. PMID:26390327

The abundant extrachromosomal DNA content of the Spiroplasma citri GII3-3X genome

PubMed Central

Saillard, Colette; Carle, Patricia; Duret-Nurbel, Sybille; Henri, Raphaël; Killiny, Nabil; Carrère, Sébastien; Gouzy, Jérome; Bové, Joseph-Marie; Renaudin, Joël; Foissac, Xavier

2008-01-01

Background Spiroplama citri, the causal agent of citrus stubborn disease, is a bacterium of the class Mollicutes and is transmitted by phloem-feeding leafhopper vectors. In order to characterize candidate genes potentially involved in spiroplasma transmission and pathogenicity, the genome of S. citri strain GII3-3X is currently being deciphered. Results Assembling 20,000 sequencing reads generated seven circular contigs, none of which fit the 1.8 Mb chromosome map or carried chromosomal markers. These contigs correspond to seven plasmids: pSci1 to pSci6, with sizes ranging from 12.9 to 35.3 kbp and pSciA of 7.8 kbp. Plasmids pSci were detected as multiple copies in strain GII3-3X. Plasmid copy numbers of pSci1-6, as deduced from sequencing coverage, were estimated at 10 to 14 copies per spiroplasma cell, representing 1.6 Mb of extrachromosomal DNA. Genes encoding proteins of the TrsE-TraE, Mob, TraD-TraG, and Soj-ParA protein families were predicted in most of the pSci sequences, in addition to members of 14 protein families of unknown function. Plasmid pSci6 encodes protein P32, a marker of insect transmissibility. Plasmids pSci1-5 code for eight different S. citri adhesion-related proteins (ScARPs) that are homologous to the previously described protein P89 and the S. kunkelii SkARP1. Conserved signal peptides and C-terminal transmembrane alpha helices were predicted in all ScARPs. The predicted surface-exposed N-terminal region possesses the following elements: (i) 6 to 8 repeats of 39 to 42 amino acids each (sarpin repeats), (ii) a central conserved region of 330 amino acids followed by (iii) a more variable domain of about 110 amino acids. The C-terminus, predicted to be cytoplasmic, consists of a 27 amino acid stretch enriched in arginine and lysine (KR) and an optional 23 amino acid stretch enriched in lysine, aspartate and glutamate (KDE). Plasmids pSci mainly present a linear increase of cumulative GC skew except in regions presenting conserved hairpin structures. Conclusion The genome of S. citri GII3-3X is characterized by abundant extrachromosomal elements. The pSci plasmids could not only be vertically inherited but also horizontally transmitted, as they encode proteins usually involved in DNA element partitioning and cell to cell DNA transfer. Because plasmids pSci1-5 encode surface proteins of the ScARP family and pSci6 was recently shown to confer insect transmissibility, diversity and abundance of S. citri plasmids may essentially aid the rapid adaptation of S. citri to more efficient transmission by different insect vectors and to various plant hosts. PMID:18442384

Protein recognition using synthetic small-molecular binders toward optical protein sensing in vitro and in live cells.

PubMed

Kubota, Ryou; Hamachi, Itaru

2015-07-07

Chemical sensing of amino acids, peptides, and proteins provides fruitful information to understand their biological functions, as well as to develop the medical and technological applications. To detect amino acids, peptides, and proteins in vitro and in vivo, vast kinds of chemical sensors including small synthetic binders/sensors, genetically-encoded fluorescent proteins and protein-based semisynthetic biosensors have been intensely investigated. This review deals with concepts, strategies, and applications of protein recognition and sensing using small synthetic binders/sensors, which are now actively studied but still in the early stage of investigation. The recognition strategies for peptides and proteins can be divided into three categories: (i) recognition of protein substructures, (ii) protein surface recognition, and (iii) protein sensing through protein-ligand interaction. Here, we overview representative examples of protein recognition and sensing, and discuss biological or diagnostic applications such as potent inhibitors/modulators of protein-protein interactions.

Species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of Porphyromonas gingivalis HmuY

PubMed Central

2010-01-01

Background Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY. Results HmuY is a unique protein of P. gingivalis since only low amino-acid sequence homology has been found to proteins encoded in other species. It is exposed on the cell surface and highly abundant in the outer membrane of the cell, in outer-membrane vesicles, and is released into culture medium in a soluble form. The protein is produced constitutively at low levels in bacteria grown under high-iron/heme conditions and at higher levels in bacteria growing under the low-iron/heme conditions typical of dental plaque. HmuY is immunogenic and elicits high IgG antibody titers in rabbits. It is also engaged in homotypic biofilm formation by P. gingivalis. Anti-HmuY antibodies exhibit inhibitory activity against P. gingivalis growth and biofilm formation. Conclusions Here it is demonstrated that HmuY may play a significant role not only in heme acquisition, but also in biofilm accumulation on abiotic surfaces. The data also suggest that HmuY, as a surface-exposed protein, would be available for recognition by the immune response during chronic periodontitis and the production of anti-HmuY antibodies may inhibit biofilm formation. PMID:20438645

The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

PubMed

Root-Bernstein, Robert; Root-Bernstein, Meredith

2016-05-21

We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their own mRNAs, are encoded by the tRNA sequences themselves; (3) and the prediction that archaeal and prokaryotic (DNA-based) genomes were built around rRNA "genes" so that rRNA-related sequences will be found to make up an unexpectedly high proportion of these genomes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

PubMed

Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

2015-12-04

Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets

PubMed Central

Guzmán Prieto, Ana M.; Urbanus, Rolf T.; Zhang, Xinglin; Bierschenk, Damien; Koekman, C. Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P.; Pape, Marieke; Paganelli, Fernanda L.; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P. A.; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

2015-01-01

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets. PMID:26675410

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

PubMed

Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

2015-12-17

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses

PubMed Central

Montanuy, Imma; Alejo, Ali; Alcami, Antonio

2011-01-01

Eradication of smallpox was accomplished 30 yr ago, but poxviral infections still represent a public health concern due to the potential release of variola virus or the emergence of zoonotic poxviruses, such as monkeypox virus. A critical determinant of poxvirus virulence is the inhibition of interferons (IFNs) by the virus-encoded type I IFN-binding protein (IFNα/βBP). This immunomodulatory protein is secreted and has the unique property of interacting with the cell surface in order to prevent IFN-mediated antiviral responses. However, the mechanism of its attachment to the cell surface remains unknown. Using surface plasmon resonance and cell-binding assays, we report that the IFNα/βBP from vaccinia virus, the smallpox vaccine, interacts with cell surface glycosaminoglycans (GAGs). Analysis of the contribution of different regions of the protein to cell surface binding demonstrated that clusters of basic residues in the first immunoglobulin domain mediate GAG interactions. Furthermore, mutation of the GAG-interaction motifs does not affect its IFN-binding and -blocking capacity. Functional conservation of GAG-binding sites is demonstrated for the IFNα/βBP from variola and monkeypox viruses, extending our understanding of immune modulation by the most virulent human poxviruses. These results are relevant for the design of improved vaccines and intervention strategies.—Montanuy, I., Alejo, A., Alcami, A. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses. PMID:21372110

Targeted Quantitative Screening of Chromosome 18 Encoded Proteome in Plasma Samples of Astronaut Candidates.

PubMed

Kopylov, Artur T; Ilgisonis, Ekaterina V; Moysa, Alexander A; Tikhonova, Olga V; Zavialova, Maria G; Novikova, Svetlana E; Lisitsa, Andrey V; Ponomarenko, Elena A; Moshkovskii, Sergei A; Markin, Andrey A; Grigoriev, Anatoly I; Zgoda, Victor G; Archakov, Alexander I

2016-11-04

This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20-47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed an estimate of the levels of 84 of 276 proteins encoded by Chr 18. These proteins were quantified in whole and depleted plasma samples. Concentration of the proteins detected varied from 10 -6 M (transthyretin, P02766) to 10 -11 M (P4-ATPase, O43861). A minor part of the proteins (mostly representing intracellular proteins) was characterized by extremely high inter individual variations. The results provide a background for studies of a potential biomarker in plasma among proteins encoded by Chr 18. The SRM raw data are available in ProteomeXchange repository (PXD004374).

Comprehensive Evaluation of Streptococcus sanguinis Cell Wall-Anchored Proteins in Early Infective Endocarditis▿ †

PubMed Central

Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L.; Wu, Hui; Kitten, Todd

2009-01-01

Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified—a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (∼2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in S. sanguinis infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention. PMID:19703977

Comprehensive evaluation of Streptococcus sanguinis cell wall-anchored proteins in early infective endocarditis.

PubMed

Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L; Wu, Hui; Kitten, Todd

2009-11-01

Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified-a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (approximately 2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in S. sanguinis infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention.

Candida albicans Agglutinin-Like Sequence (Als) Family Vignettes: A Review of Als Protein Structure and Function

PubMed Central

Hoyer, Lois L.; Cota, Ernesto

2016-01-01

Approximately two decades have passed since the description of the first gene in the Candida albicans ALS (agglutinin-like sequence) family. Since that time, much has been learned about the composition of the family and the function of its encoded cell-surface glycoproteins. Solution of the structure of the Als adhesive domain provides the opportunity to evaluate the molecular basis for protein function. This review article is formatted as a series of fundamental questions and explores the diversity of the Als proteins, as well as their role in ligand binding, aggregative effects, and attachment to abiotic surfaces. Interaction of Als proteins with each other, their functional equivalence, and the effects of protein abundance on phenotypic conclusions are also examined. Structural features of Als proteins that may facilitate invasive function are considered. Conclusions that are firmly supported by the literature are presented while highlighting areas that require additional investigation to reveal basic features of the Als proteins, their relatedness to each other, and their roles in C. albicans biology. PMID:27014205

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins.

PubMed

Wolffe, E J; Gause, W C; Pelfrey, C M; Holland, S M; Steinberg, A D; August, J T

1990-01-05

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.

The Virulence Plasmid of Yersinia, an Antihost Genome

PubMed Central

Cornelis, Guy R.; Boland, Anne; Boyd, Aoife P.; Geuijen, Cecile; Iriarte, Maite; Neyt, Cécile; Sory, Marie-Paule; Stainier, Isabelle

1998-01-01

The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds. PMID:9841674

Molecular mechanisms for protein-encoded inheritance

PubMed Central

Wiltzius, Jed J. W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

2013-01-01

Strains are phenotypic variants, encoded by nucleic acid sequences in chromosomal inheritance and by protein “conformations” in prion inheritance and transmission. But how is a protein “conformation” stable enough to endure transmission between cells or organisms? Here new polymorphic crystal structures of segments of prion and other amyloid proteins offer structural mechanisms for prion strains. In packing polymorphism, prion strains are encoded by alternative packings (polymorphs) of β-sheets formed by the same segment of a protein; in a second mechanism, segmental polymorphism, prion strains are encoded by distinct β-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring “conformations,” capable of encoding strains. These molecular mechanisms for transfer of information into prion strains share features with the familiar mechanism for transfer of information by nucleic acid inheritance, including sequence specificity and recognition by non-covalent bonds. PMID:19684598

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection

PubMed Central

De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain

2016-01-01

ABSTRACT Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae. Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum. IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete’s foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae. Comparing gene expression during infection on guinea pigs with keratin degradation in vitro, which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo, encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates. PMID:27822542

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection.

PubMed

Tran, Van Du T; De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain; Pagni, Marco; Monod, Michel

2016-01-01

Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae . Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum . IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete's foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae . Comparing gene expression during infection on guinea pigs with keratin degradation in vitro , which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo , encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates.

Cytomegalovirus immune evasion by perturbation of endosomal trafficking

PubMed Central

Lučin, Pero; Mahmutefendić, Hana; Blagojević Zagorac, Gordana; Ilić Tomaš, Maja

2015-01-01

Cytomegaloviruses (CMVs), members of the herpesvirus family, have evolved a variety of mechanisms to evade the immune response to survive in infected hosts and to establish latent infection. They effectively hide infected cells from the effector mechanisms of adaptive immunity by eliminating cellular proteins (major histocompatibility Class I and Class II molecules) from the cell surface that display viral antigens to CD8 and CD4 T lymphocytes. CMVs also successfully escape recognition and elimination of infected cells by natural killer (NK) cells, effector cells of innate immunity, either by mimicking NK cell inhibitory ligands or by downregulating NK cell-activating ligands. To accomplish these immunoevasion functions, CMVs encode several proteins that function in the biosynthetic pathway by inhibiting the assembly and trafficking of cellular proteins that participate in immune recognition and thereby, block their appearance at the cell surface. However, elimination of these proteins from the cell surface can also be achieved by perturbation of their endosomal route and subsequent relocation from the cell surface into intracellular compartments. Namely, the physiological route of every cellular protein, including immune recognition molecules, is characterized by specific features that determine its residence time at the cell surface. In this review, we summarize the current understanding of endocytic trafficking of immune recognition molecules and perturbations of the endosomal system during infection with CMVs and other members of the herpesvirus family that contribute to their immune evasion mechanisms. PMID:25263490

Thionin-D4E1 chimeric protein protects plants against bacterial infections

DOEpatents

Stover, Eddie W; Gupta, Goutam; Hao, Guixia

2017-08-08

The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen of Mycobacterium bovis BCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis

PubMed Central

Lefèvre, Philippe; Denis, Olivier; De Wit, Lucas; Tanghe, Audrey; Vandenbussche, Paul; Content, Jean; Huygen, Kris

2000-01-01

Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test. PMID:10678905

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Positive selection on human gamete-recognition genes

PubMed Central

Stover, Daryn A.; Guerra, Vanessa; Mozaffari, Sahar V.; Ober, Carole; Mugal, Carina F.; Kaj, Ingemar

2018-01-01

Coevolution of genes that encode interacting proteins expressed on the surfaces of sperm and eggs can lead to variation in reproductive compatibility between mates and reproductive isolation between members of different species. Previous studies in mice and other mammals have focused in particular on evidence for positive or diversifying selection that shapes the evolution of genes that encode sperm-binding proteins expressed in the egg coat or zona pellucida (ZP). By fitting phylogenetic models of codon evolution to data from the 1000 Genomes Project, we identified candidate sites evolving under diversifying selection in the human genes ZP3 and ZP2. We also identified one candidate site under positive selection in C4BPA, which encodes a repetitive protein similar to the mouse protein ZP3R that is expressed in the sperm head and binds to the ZP at fertilization. Results from several additional analyses that applied population genetic models to the same data were consistent with the hypothesis of selection on those candidate sites leading to coevolution of sperm- and egg-expressed genes. By contrast, we found no candidate sites under selection in a fourth gene (ZP1) that encodes an egg coat structural protein not directly involved in sperm binding. Finally, we found that two of the candidate sites (in C4BPA and ZP2) were correlated with variation in family size and birth rate among Hutterite couples, and those two candidate sites were also in linkage disequilibrium in the same Hutterite study population. All of these lines of evidence are consistent with predictions from a previously proposed hypothesis of balancing selection on epistatic interactions between C4BPA and ZP3 at fertilization that lead to the evolution of co-adapted allele pairs. Such patterns also suggest specific molecular traits that may be associated with both natural reproductive variation and clinical infertility. PMID:29340252

Prevalence of Flp Pili-Encoding Plasmids in Cutibacterium acnes Isolates Obtained from Prostatic Tissue

PubMed Central

Davidsson, Sabina; Carlsson, Jessica; Mölling, Paula; Gashi, Natyra; Andrén, Ove; Andersson, Swen-Olof; Brzuszkiewicz, Elzbieta; Poehlein, Anja; Al-Zeer, Munir A.; Brinkmann, Volker; Scavenius, Carsten; Nazipi, Seven; Söderquist, Bo; Brüggemann, Holger

2017-01-01

Inflammation is one of the hallmarks of prostate cancer. The origin of inflammation is unknown, but microbial infections are suspected to play a role. In previous studies, the Gram-positive, low virulent bacterium Cutibacterium (formerly Propionibacterium) acnes was frequently isolated from prostatic tissue. It is unclear if the presence of the bacterium represents a true infection or a contamination. Here we investigated Cutibacterium acnes type II, also called subspecies defendens, which is the most prevalent type among prostatic C. acnes isolates. Genome sequencing of type II isolates identified large plasmids in several genomes. The plasmids are highly similar to previously identified linear plasmids of type I C. acnes strains associated with acne vulgaris. A PCR-based analysis revealed that 28.4% (21 out of 74) of all type II strains isolated from cancerous prostates carry a plasmid. The plasmid shows signatures for conjugative transfer. In addition, it contains a gene locus for tight adherence (tad) that is predicted to encode adhesive Flp (fimbrial low-molecular weight protein) pili. In subsequent experiments a tad locus-encoded putative pilin subunit was identified in the surface-exposed protein fraction of plasmid-positive C. acnes type II strains by mass spectrometry, indicating that the tad locus is functional. Additional plasmid-encoded proteins were detected in the secreted protein fraction, including two signal peptide-harboring proteins; the corresponding genes are specific for type II C. acnes, thus lacking from plasmid-positive type I C. acnes strains. Further support for the presence of Flp pili in C. acnes type II was provided by electron microscopy, revealing cell appendages in tad locus-positive strains. Our study provides new insight in the most prevalent prostatic subspecies of C. acnes, subsp. defendens, and indicates the existence of Flp pili in plasmid-positive strains. Such pili may support colonization and persistent infection of human prostates by C. acnes. PMID:29201018

Genome of the opportunistic pathogen Streptococcus sanguinis.

PubMed

Xu, Ping; Alves, Joao M; Kitten, Todd; Brown, Arunsri; Chen, Zhenming; Ozaki, Luiz S; Manque, Patricio; Ge, Xiuchun; Serrano, Myrna G; Puiu, Daniela; Hendricks, Stephanie; Wang, Yingping; Chaplin, Michael D; Akan, Doruk; Paik, Sehmi; Peterson, Darrell L; Macrina, Francis L; Buck, Gregory A

2007-04-01

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.

Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP▿

PubMed Central

Barbosa, Angela S.; Monaris, Denize; Silva, Ludmila B.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Cianciarullo, Aurora M.; Isaac, Lourdes; Abreu, Patricia A. E.

2010-01-01

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis. PMID:20404075

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation

PubMed Central

1994-01-01

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold- sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase- encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth. PMID:7962097

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation.

PubMed

Kim, Y J; Francisco, L; Chen, G C; Marcotte, E; Chan, C S

1994-12-01

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold-sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase-encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.

Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

PubMed Central

Greve, Bo; Jensen, Susanne; Phan, Hoa; Brügger, Kim; Zillig, Wolfram; She, Qunxin; Garrett, Roger A.

2005-01-01

Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase–primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini-chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1 is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA-binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these important crenarchaeal proteins. PMID:15876565

Prediction of Carbohydrate Binding Sites on Protein Surfaces with 3-Dimensional Probability Density Distributions of Interacting Atoms

PubMed Central

Tsai, Keng-Chang; Jian, Jhih-Wei; Yang, Ei-Wen; Hsu, Po-Chiang; Peng, Hung-Pin; Chen, Ching-Tai; Chen, Jun-Bo; Chang, Jeng-Yih; Hsu, Wen-Lian; Yang, An-Suei

2012-01-01

Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall prediction MCC was 0.49. Independent tests on anti-carbohydrate antibodies showed that the carbohydrate antigen binding sites were predicted with comparable accuracy. These results demonstrate that the predictors are among the best in carbohydrate binding site predictions to date. PMID:22848404

The rice blast resistance gene Ptr encodes an atypical protein required for broad spectrum disease resistance

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. We identified a novel, broad-spectrum rice blast R gene, Ptr, encoding a non-NLR protein with four Armadillo repeats. Ptr was originally identified by fast neutron mutagenesis as a ...

Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays

NASA Astrophysics Data System (ADS)

Nguyen, Huy Q.; Brower, Kara; Harink, Björn; Baxter, Brian; Thorn, Kurt S.; Fordyce, Polly M.

2017-02-01

Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1:1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled antimyc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

Alternative intronic promoters in development and disease.

PubMed

Vacik, Tomas; Raska, Ivan

2017-05-01

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.

Multifunctional Nature of the Arenavirus RING Finger Protein Z

PubMed Central

Fehling, Sarah Katharina; Lennartz, Frank; Strecker, Thomas

2012-01-01

Arenaviruses are a family of enveloped negative-stranded RNA viruses that can cause severe human disease ranging from encephalitis symptoms to fulminant hemorrhagic fever. The bi‑segmented RNA genome encodes four polypeptides: the nucleoprotein NP, the surface glycoprotein GP, the polymerase L, and the RING finger protein Z. Although it is the smallest arenavirus protein with a length of 90 to 99 amino acids and a molecular weight of approx. 11 kDa, the Z protein has multiple functions in the viral life cycle including (i) regulation of viral RNA synthesis, (ii) orchestration of viral assembly and budding, (iii) interaction with host cell proteins, and (iv) interferon antagonism. In this review, we summarize our current understanding of the structural and functional role of the Z protein in the arenavirus replication cycle. PMID:23202512

Early stages in the biogenesis of eukaryotic β-barrel proteins.

PubMed

Jores, Tobias; Rapaport, Doron

2017-09-01

The endosymbiotic organelles mitochondria and chloroplasts harbour, similarly to their prokaryotic progenitors, β-barrel proteins in their outer membrane. These proteins are encoded on nuclear DNA, translated on cytosolic ribosomes and imported into their target organelles by a dedicated machinery. Recent studies have provided insights into the import into the organelles and the membrane insertion of these proteins. Although the cytosolic stages of their biogenesis are less well defined, it is speculated that upon their synthesis, chaperones prevent β-barrel proteins from aggregation and keep them in an import-competent conformation. In this Review, we summarize the current knowledge about the biogenesis of β-barrel proteins, focusing on the early stages from the translation on cytosolic ribosomes to the recognition on the surface of the organelle. © 2017 Federation of European Biochemical Societies.

Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein.

PubMed

Salmon, D; Hanocq-Quertier, J; Paturiaux-Hanocq, F; Pays, A; Tebabi, P; Nolan, D P; Michel, A; Pays, E

1997-12-15

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

On the nature of cavities on protein surfaces: application to the identification of drug-binding sites.

PubMed

Nayal, Murad; Honig, Barry

2006-06-01

In this article we introduce a new method for the identification and the accurate characterization of protein surface cavities. The method is encoded in the program SCREEN (Surface Cavity REcognition and EvaluatioN). As a first test of the utility of our approach we used SCREEN to locate and analyze the surface cavities of a nonredundant set of 99 proteins cocrystallized with drugs. We find that this set of proteins has on average about 14 distinct cavities per protein. In all cases, a drug is bound at one (and sometimes more than one) of these cavities. Using cavity size alone as a criterion for predicting drug-binding sites yields a high balanced error rate of 15.7%, with only 71.7% coverage. Here we characterize each surface cavity by computing a comprehensive set of 408 physicochemical, structural, and geometric attributes. By applying modern machine learning techniques (Random Forests) we were able to develop a classifier that can identify drug-binding cavities with a balanced error rate of 7.2% and coverage of 88.9%. Only 18 of the 408 cavity attributes had a statistically significant role in the prediction. Of these 18 important attributes, almost all involved size and shape rather than physicochemical properties of the surface cavity. The implications of these results are discussed. A SCREEN Web server is available at http://interface.bioc.columbia.edu/screen. 2006 Wiley-Liss, Inc.

Quantum-dots-encoded-microbeads based molecularly imprinted polymer.

PubMed

Liu, Yixi; Liu, Le; He, Yonghong; He, Qinghua; Ma, Hui

2016-03-15

Quantum dots encoded microbeads have various advantages such as large surface area, superb optical properties and the ability of multiplexing. Molecularly imprinted polymer that can mimic the natural recognition entities has high affinity and selectivity for the specific analyte. Here, the concept of utilizing the quantum dots encoded microbeads as the supporting material and the polydopamine as the functional monomer to form the core-shell molecular imprinted polymer was proposed for the first time. The resulted imprinted polymer can provide various merits: polymerization can complete in aqueous environment; fabrication procedure is facile and universal; the obvious economic advantage; the thickness of the imprinting layer is highly controllable; polydopamine coating can improve the biocompatibility of the quantum dot encoded microbeads. The rabbit IgG binding and flow cytometer experiment result showed the distinct advantages of this strategy: cost-saving, facile and fast preparation procedure. Most importantly, the ability for the multichannel detection, which makes the imprinted polydopamine modified encoded-beads very attractive in protein pre-concentration, recognition, separation and biosensing. Copyright © 2015 Elsevier B.V. All rights reserved.

The evolution and diversity of a low complexity vaccine candidate, merozoite surface protein 9 (MSP-9), in Plasmodium vivax and closely related species.

PubMed

Chenet, Stella M; Pacheco, M Andreína; Bacon, David J; Collins, William E; Barnwell, John W; Escalante, Ananias A

2013-12-01

The merozoite surface protein-9 (MSP-9) has been considered a target for an anti-malarial vaccine since it is one of many proteins involved in the erythrocyte invasion, a critical step in the parasite life cycle. Orthologs encoding this antigen have been found in all known species of Plasmodium parasitic to primates. In order to characterize and investigate the extent and maintenance of MSP-9 genetic diversity, we analyzed DNA sequences of the following malaria parasite species: Plasmodium falciparum, Plasmodium reichenowi, Plasmodium chabaudi, Plasmodium yoelii, Plasmodium berghei, Plasmodium coatneyi, Plasmodium gonderi, Plasmodium knowlesi, Plasmodium inui, Plasmodium simiovale, Plasmodium fieldi, Plasmodium cynomolgi and Plasmodium vivax and evaluated the signature of natural selection in all MSP-9 orthologs. Our findings suggest that the gene encoding MSP-9 is under purifying selection in P. vivax and closely related species. We further explored how selection affected different regions of MSP-9 by comparing the polymorphisms in P. vivax and P. falciparum, and found contrasting patterns between these two species that suggest differences in functional constraints. This observation implies that the MSP-9 orthologs in human parasites may interact differently with the host immune response. Thus, studies carried out in one species cannot be directly translated into the other. Copyright © 2013 Elsevier B.V. All rights reserved.

Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili.

PubMed

Dougan, G; Dowd, G; Kehoe, M

1983-01-01

Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed.

Cloning, expression, and characterization of a peculiar choline-binding beta-galactosidase from Streptococcus mitis.

PubMed

Campuzano, Susana; Serra, Beatriz; Llull, Daniel; García, José L; García, Pedro

2009-09-01

A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative beta-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric beta-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40 degrees C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first beta-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.

Engineering M13 for phage display.

PubMed

Sidhu, S S

2001-09-01

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.

Sequence variation and structural conservation allows development of novel function and immune evasion in parasite surface protein families

PubMed Central

Higgins, Matthew K; Carrington, Mark

2014-01-01

Trypanosoma and Plasmodium species are unicellular, eukaryotic pathogens that have evolved the capacity to survive and proliferate within a human host, causing sleeping sickness and malaria, respectively. They have very different survival strategies. African trypanosomes divide in blood and extracellular spaces, whereas Plasmodium species invade and proliferate within host cells. Interaction with host macromolecules is central to establishment and maintenance of an infection by both parasites. Proteins that mediate these interactions are under selection pressure to bind host ligands without compromising immune avoidance strategies. In both parasites, the expansion of genes encoding a small number of protein folds has established large protein families. This has permitted both diversification to form novel ligand binding sites and variation in sequence that contributes to avoidance of immune recognition. In this review we consider two such parasite surface protein families, one from each species. In each case, known structures demonstrate how extensive sequence variation around a conserved molecular architecture provides an adaptable protein scaffold that the parasites can mobilise to mediate interactions with their hosts. PMID:24442723

Spleen-dependent regulation of antigenic variation in malaria parasites: Plasmodium knowlesi SICAvar expression profiles in splenic and asplenic hosts.

PubMed

Lapp, Stacey A; Korir-Morrison, Cindy; Jiang, Jianlin; Bai, Yaohui; Corredor, Vladimir; Galinski, Mary R

2013-01-01

Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA) variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1) antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+), and a related progeny clone, Pk1(B+)1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera. We have investigated SICAvar RNA and protein expression in Pk1(A+), Pk1(B+)1+, and SICA[-] parasites. The Pk1(A+) and Pk1(B+)1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry. SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+) to Pk1(B+)1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying antigenic variation in the context of the host environment.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-11-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed Central

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-01-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM. Images PMID:1656067

Self-assembled bionanostructures: proteins following the lead of DNA nanostructures

PubMed Central

2014-01-01

Natural polymers are able to self-assemble into versatile nanostructures based on the information encoded into their primary structure. The structural richness of biopolymer-based nanostructures depends on the information content of building blocks and the available biological machinery to assemble and decode polymers with a defined sequence. Natural polypeptides comprise 20 amino acids with very different properties in comparison to only 4 structurally similar nucleotides, building elements of nucleic acids. Nevertheless the ease of synthesizing polynucleotides with selected sequence and the ability to encode the nanostructural assembly based on the two specific nucleotide pairs underlay the development of techniques to self-assemble almost any selected three-dimensional nanostructure from polynucleotides. Despite more complex design rules, peptides were successfully used to assemble symmetric nanostructures, such as fibrils and spheres. While earlier designed protein-based nanostructures used linked natural oligomerizing domains, recent design of new oligomerizing interaction surfaces and introduction of the platform for topologically designed protein fold may enable polypeptide-based design to follow the track of DNA nanostructures. The advantages of protein-based nanostructures, such as the functional versatility and cost effective and sustainable production methods provide strong incentive for further development in this direction. PMID:24491139

Encoding of contextual fear memory requires de novo proteins in the prelimbic cortex

PubMed Central

Rizzo, Valerio; Touzani, Khalid; Raveendra, Bindu L.; Swarnkar, Supriya; Lora, Joan; Kadakkuzha, Beena M.; Liu, Xin-An; Zhang, Chao; Betel, Doron; Stackman, Robert W.; Puthanveettil, Sathyanarayanan V.

2016-01-01

Background Despite our understanding of the significance of the prefrontal cortex in the consolidation of long-term memories (LTM), its role in the encoding of LTM remains elusive. Here we investigated the role of new protein synthesis in the mouse medial prefrontal cortex (mPFC) in encoding contextual fear memory. Methods Because a change in the association of mRNAs to polyribosomes is an indicator of new protein synthesis, we assessed the changes in polyribosome-associated mRNAs in the mPFC following contextual fear conditioning (CFC) in the mouse. Differential gene expression in mPFC was identified by polyribosome profiling (n = 18). The role of new protein synthesis in mPFC was determined by focal inhibition of protein synthesis (n = 131) and by intra-prelimbic cortex manipulation (n = 56) of Homer 3, a candidate identified from polyribosome profiling. Results We identified several mRNAs that are differentially and temporally recruited to polyribosomes in the mPFC following CFC. Inhibition of protein synthesis in the prelimbic (PL), but not in the anterior cingulate cortex (ACC) region of the mPFC immediately after CFC disrupted encoding of contextual fear memory. Intriguingly, inhibition of new protein synthesis in the PL 6 hours after CFC did not impair encoding. Furthermore, expression of Homer 3, an mRNA enriched in polyribosomes following CFC, in the PL constrained encoding of contextual fear memory. Conclusions Our studies identify several molecular substrates of new protein synthesis in the mPFC and establish that encoding of contextual fear memories require new protein synthesis in PL subregion of mPFC. PMID:28503670

Interspecific and host-related gene expression patterns in nematode-trapping fungi.

PubMed

Andersson, Karl-Magnus; Kumar, Dharmendra; Bentzer, Johan; Friman, Eva; Ahrén, Dag; Tunlid, Anders

2014-11-11

Nematode-trapping fungi are soil-living fungi that capture and kill nematodes using special hyphal structures called traps. They display a large diversity of trapping mechanisms and differ in their host preferences. To provide insights into the genetic basis for this variation, we compared the transcriptome expressed by three species of nematode-trapping fungi (Arthrobotrys oligospora, Monacrosporium cionopagum and Arthrobotrys dactyloides, which use adhesive nets, adhesive branches or constricting rings, respectively, to trap nematodes) during infection of two different plant-pathogenic nematode hosts (the root knot nematode Meloidogyne hapla and the sugar beet cyst nematode Heterodera schachtii). The divergence in gene expression between the fungi was significantly larger than that related to the nematode species being infected. Transcripts predicted to encode secreted proteins and proteins with unknown function (orphans) were overrepresented among the highly expressed transcripts in all fungi. Genes that were highly expressed in all fungi encoded endopeptidases, such as subtilisins and aspartic proteases; cell-surface proteins containing the carbohydrate-binding domain WSC; stress response proteins; membrane transporters; transcription factors; and transcripts containing the Ricin-B lectin domain. Differentially expressed transcripts among the fungal species encoded various lectins, such as the fungal fruit-body lectin and the D-mannose binding lectin; transcription factors; cell-signaling components; proteins containing a WSC domain; and proteins containing a DUF3129 domain. A small set of transcripts were differentially expressed in infections of different host nematodes, including peptidases, WSC domain proteins, tyrosinases, and small secreted proteins with unknown function. This is the first study on the variation of infection-related gene expression patterns in nematode-trapping fungi infecting different host species. A better understanding of these patterns will facilitate the improvements of these fungi in biological control programs, by providing molecular markers for screening programs and candidates for genetic manipulations of virulence and host preferences.

Identification of the psaA Gene, Coding for Pneumococcal Surface Adhesin A, in Viridans Group Streptococci other than Streptococcus pneumoniae

PubMed Central

Jado, Isabel; Fenoll, Asunción; Casal, Julio; Pérez, Amalia

2001-01-01

The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of the psaA gene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment in Streptococcus mitis, Streptococcus oralis, and Streptococcus anginosus strains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reported Streptococcus pneumoniae serotype 6B strain. Our results confirm that psaA is present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed. PMID:11527799

Design of magnetic polyplexes taken up efficiently by dendritic cell for enhanced DNA vaccine delivery.

PubMed

Nawwab Al-Deen, F M; Selomulya, C; Kong, Y Y; Xiang, S D; Ma, C; Coppel, R L; Plebanski, M

2014-02-01

Dendritic cells (DC) targeting vaccines require high efficiency for uptake, followed by DC activation and maturation. We used magnetic vectors comprising polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles, with hyaluronic acid (HA) of different molecular weights (<10 and 900 kDa) to reduce cytotoxicity and to facilitate endocytosis of particles into DCs via specific surface receptors. DNA encoding Plasmodium yoelii merozoite surface protein 1-19 and a plasmid encoding yellow fluorescent gene were added to the magnetic complexes with various % charge ratios of HA: PEI. The presence of magnetic fields significantly enhanced DC transfection and maturation. Vectors containing a high-molecular-weight HA with 100% charge ratio of HA: PEI yielded a better transfection efficiency than others. This phenomenon was attributed to their longer molecular chains and higher mucoadhesive properties aiding DNA condensation and stability. Insights gained should improve the design of more effective DNA vaccine delivery systems.

Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence.

PubMed

Sugden, Scott M; Bego, Mariana G; Pham, Tram N Q; Cohen, Éric A

2016-03-03

The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.

Burkholderia Mallei tssM Encodes a Secreted Deubiquitinase that is Expressed Inside Infected RAW 264.7 Murine Macrophages

DTIC Science & Technology

2008-10-13

Furthermore, the encoded protein of this gene is only 30 kDa. A potential GTG start codon at position 625 also encodes a protein that is too small...horizontal bar and putative alternate translation initiation sites (ATG, GTG , and TTG) are indicated. The sizes and locations of the proteins encoded... gray line with rounded rectangles showing sequence features and motifs, including the Ala- and Pro-rich N-terminal region and the C-terminal Cys and

Nanoparticles-cell association predicted by protein corona fingerprints

NASA Astrophysics Data System (ADS)

Palchetti, S.; Digiacomo, L.; Pozzi, D.; Peruzzi, G.; Micarelli, E.; Mahmoudi, M.; Caracciolo, G.

2016-06-01

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells. Electronic supplementary information (ESI) available: Table S1. Cell viability (%) and cell association of the different nanoparticles used. Table S2. Total number of identified proteins on the different nanoparticles used. Tables S3-S18. Top 25 most abundant corona proteins identified in the protein corona of nanoparticles NP2-NP16 following 1 hour incubation with HP. Table S19. List of descriptors used. Table S20. Potential targets of protein corona fingerprints with its own interaction score (mentha) and the expression median value in Hela cells. Fig. S1 and S2. Effect of exposure to human plasma on size and zeta potential of NPs. Fig. S3. Predictive modeling of nanoparticle-cell association. See DOI: 10.1039/c6nr03898k

Drosophila gene tao-1 encodes proteins with and without a Ste20 kinase domain that affect cytoskeletal architecture and cell migration differently

PubMed Central

Pflanz, Ralf; Voigt, Aaron; Yakulov, Toma; Jäckle, Herbert

2015-01-01

Tao-1, the single representative of the Sterile 20 kinase subfamily in Drosophila, is best known for destabilizing microtubules at the actin-rich cortex, regulating the cytoskeletal architecture of cells. More recently, Tao-1 was shown to act in the Salvador–Warts–Hippo pathway by phosphorylating Hippo, regulating cell growth as well as cell polarity. Here, we show that tao-1 encodes two proteins, one with the Sterile 20 kinase domain (Tao-L) and one without it (Tao-S), and that they act in an antagonistic manner. Tao-L expression causes lamellipodia-like cell protrusions, whereas Tao-S expression results in filopodia-like structures that make cells stick to the surface they attach to. Ectopic Tao-1 expression in the anterior region of Drosophila embryos results in pole cell formation as normally observed at the posterior end. Tao-S expression causes primordial germ cells (PGCs) to adhere to the inner wall of the gut primordia and prevents proper transepithelial migration to the gonads. Conversely, RNAi knockdowns of Tao-1 cause disordered migration of PGCs out of the gut epithelium, their dispersal within the embryo and cell death. The results reveal a novel function of Tao-1 in cell migration, which is based on antagonistic activities of two proteins encoded by a single gene. PMID:25589578

Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.

PubMed

Hsu, Jack C-C; Reid, David W; Hoffman, Alyson M; Sarkar, Devanand; Nicchitta, Christopher V

2018-05-01

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease. © 2018 Hsu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and Kelp fly virus.

PubMed

Valles, Steven M; Bell, Susanne; Firth, Andrew E

2014-01-01

Solenopsis invicta virus 3 (SINV-3) is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF) of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV), an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.

MBL-associated serine proteases (MASPs) and infectious diseases.

PubMed

Beltrame, Marcia H; Boldt, Angelica B W; Catarino, Sandra J; Mendes, Hellen C; Boschmann, Stefanie E; Goeldner, Isabela; Messias-Reason, Iara

2015-09-01

The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

PubMed

Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

2010-05-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

AcmB Is an S-Layer-Associated β-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM

PubMed Central

Johnson, Brant R.

2016-01-01

ABSTRACT Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β-N-acetylglucosaminidase encoded by the gene lba0176 (acmB), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus. Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the ΔacmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro. These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. IMPORTANCE Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins. PMID:27422832

AcmB Is an S-Layer-Associated β-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM.

PubMed

Johnson, Brant R; Klaenhammer, Todd R

2016-09-15

Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β-N-acetylglucosaminidase encoded by the gene lba0176 (acmB), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the ΔacmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins. Copyright © 2016 Johnson and Klaenhammer.

Analysis of ER–mitochondria contacts using correlative fluorescence microscopy and soft X-ray tomography of mammalian cells

PubMed Central

Elgass, Kirstin D.; Smith, Elizabeth A.; LeGros, Mark A.; Larabell, Carolyn A.; Ryan, Michael T.

2015-01-01

ABSTRACT Mitochondrial fission is important for organelle transport, quality control and apoptosis. Changes to the fission process can result in a wide variety of neurological diseases. In mammals, mitochondrial fission is executed by the GTPase dynamin-related protein 1 (Drp1; encoded by DNM1L), which oligomerizes around mitochondria and constricts the organelle. The mitochondrial outer membrane proteins Mff, MiD49 (encoded by MIEF2) and MiD51 (encoded by MIEF1) are involved in mitochondrial fission by recruiting Drp1 from the cytosol to the organelle surface. In addition, endoplasmic reticulum (ER) tubules have been shown to wrap around and constrict mitochondria before a fission event. Up to now, the presence of MiD49 and MiD51 at ER–mitochondrial division foci has not been established. Here, we combine confocal live-cell imaging with correlative cryogenic fluorescence microscopy and soft x-ray tomography to link MiD49 and MiD51 to the involvement of the ER in mitochondrial fission. We gain further insight into this complex process and characterize the 3D structure of ER–mitochondria contact sites. PMID:26101352

CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane

PubMed Central

Gupta, Ankit; Balabaskaran-Nina, Praveen; Nguitragool, Wang; Saggu, Gagandeep S.; Schureck, Marc A.

2018-01-01

ABSTRACT Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. These proteins lack the multiple transmembrane domains typically present in channel-forming proteins, raising doubts about their precise roles. Using the virulent human Plasmodium falciparum parasite, we report that CLAG3 undergoes self-association and that this protein’s expression determines channel phenotype quantitatively. We overcame epigenetic silencing of clag3 paralogs and engineered parasites that express two CLAG3 isoforms simultaneously. Stoichiometric expression of these isoforms yielded intermediate channel phenotypes, in agreement with observed trafficking of both proteins to the host membrane. Coimmunoprecipitation and surface labeling revealed formation of CLAG3 oligomers. In vitro selections applied to these transfectant lines yielded distinct mutants with correlated changes in channel activity. These findings support involvement of the identified oligomers in PSAC formation and parasite nutrient acquisition. PMID:29739907

Targeting the latent cytomegalovirus reservoir with an antiviral fusion toxin protein

PubMed Central

Krishna, B. A.; Spiess, K.; Poole, E. L.; Lau, B.; Voigt, S.; Kledal, T. N.; Rosenkilde, M. M.; Sinclair, J. H.

2017-01-01

Reactivation of human cytomegalovirus (HCMV) in transplant recipients can cause life-threatening disease. Consequently, for transplant recipients, killing latently infected cells could have far-reaching clinical benefits. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, and one viral gene expressed by latently infected myeloid cells is US28. This viral gene encodes a cell surface G protein-coupled receptor (GPCR) that binds chemokines, triggering its endocytosis. We show that the expression of US28 on the surface of latently infected cells allows monocytes and their progenitor CD34+ cells to be targeted and killed by F49A-FTP, a highly specific fusion toxin protein that binds this viral GPCR. As expected, this specific targeting of latently infected cells by F49A-FTP also robustly reduces virus reactivation in vitro. Consequently, such specific fusion toxin proteins could form the basis of a therapeutic strategy for eliminating latently infected cells before haematopoietic stem cell transplantation. PMID:28148951

Variation in a surface-exposed region of the Mycoplasma pneumoniae P40 protein as a consequence of homologous DNA recombination between RepMP5 elements.

PubMed

Spuesens, Emiel B M; van de Kreeke, Nick; Estevão, Silvia; Hoogenboezem, Theo; Sluijter, Marcel; Hartwig, Nico G; van Rossum, Annemarie M C; Vink, Cornelis

2011-02-01

Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory tract infections. The first step in infection is adherence of the bacteria to the respiratory epithelium. This step is mediated by a specialized organelle, which contains several proteins (cytadhesins) that have an important function in adherence. Two of these cytadhesins, P40 and P90, represent the proteolytic products from a single 130 kDa protein precursor, which is encoded by the MPN142 gene. Interestingly, MPN142 contains a repetitive DNA element, termed RepMP5, of which homologues are found at seven other loci within the M. pneumoniae genome. It has been hypothesized that these RepMP5 elements, which are similar but not identical in sequence, recombine with their counterpart within MPN142 and thereby provide a source of sequence variation for this gene. As this variation may give rise to amino acid changes within P40 and P90, the recombination between RepMP5 elements may constitute the basis of antigenic variation and, possibly, immune evasion by M. pneumoniae. To investigate the sequence variation of MPN142 in relation to inter-RepMP5 recombination, we determined the sequences of all RepMP5 elements in a collection of 25 strains. The results indicate that: (i) inter-RepMP5 recombination events have occurred in seven of the strains, and (ii) putative RepMP5 recombination events involving MPN142 have induced amino acid changes in a surface-exposed part of the P40 protein in two of the strains. We conclude that recombination between RepMP5 elements is a common phenomenon that may lead to sequence variation of MPN142-encoded proteins.

Protein synthesis in sperm: dialog between mitochondria and cytoplasm.

PubMed

Gur, Yael; Breitbart, Haim

2008-01-30

Ejaculated sperm are capable of using mRNAs transcripts for protein translation during the final maturation steps before fertilization. In a capacitation-dependent process, nuclear-encoded mRNAs are translated by mitochondrial-type ribosomes while the cytoplasmic translation machinery is not involved. Our findings suggest that new proteins are synthesized to replace degraded proteins while swimming and waiting in the female reproductive tract before fertilization, or produced due to the specific needs of the capacitating spermatozoa. In addition, a growing number of articles have reported evidence for the correlation of nuclear-encoded mRNA and protein synthesis in somatic mitochondria. It is known that all of the proteins necessary for the replication, transcription and translation of the genes encoded in mtDNA are now encoded in the nuclear genome. This genetic investment is far out of proportion to the number of proteins involved, as there have been multiple movements and duplications of genes. However, the evolutionary retention (or secondary uptake) of the mitochondrial machinery for translation of nuclear-encoded mRNAs may shed light on this paradox.

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

Investigation of SnSPR1, a novel and abundant surface protein of Sarcocystis neurona merozoites.

PubMed

Zhang, Deqing; Howe, Daniel K

2008-04-15

An expressed sequence tag (EST) sequencing project has produced over 15,000 partial cDNA sequences from the equine pathogen Sarcocystis neurona. While many of the sequences are clear homologues of previously characterized genes, a significant number of the S. neurona ESTs do not exhibit similarity to anything in the extensive sequence databases that have been generated. In an effort to characterize parasite proteins that are novel to S. neurona, a seemingly unique gene was selected for further investigation based on its abundant representation in the collection of ESTs and the predicted presence of a signal peptide and glycolipid anchor addition on the encoded protein. The gene was expressed in E. coli, and monospecific polyclonal antiserum against the recombinant protein was produced by immunization of a rabbit. Characterization of the native protein in S. neurona merozoites and schizonts revealed that it is a low molecular weight surface protein that is expressed throughout intracellular development of the parasite. The protein was designated Surface Protein 1 (SPR1) to reflect its display on the outer surface of merozoites and to distinguish it from the ubiquitous SAG/SRS surface antigens of the heteroxenous Coccidia. Interestingly, infection assays in the presence of the polyclonal antiserum suggested that SnSPR1 plays some role in attachment and/or invasion of host cells by S. neurona merozoites. The work described herein represents a general template for selecting and characterizing the various unidentified gene sequences that are plentiful in the EST databases for S. neurona and other apicomplexans. Furthermore, this study illustrates the value of investigating these novel sequences since it can offer new candidates for diagnostic or vaccine development while also providing greater insight into the biology of these parasites.

The Bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation

USDA-ARS?s Scientific Manuscript database

Bean pod mottle virus (BPMV) is a bipartite, positive sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58 kilo-dalto...

Characterization of the 5’- and 3’-terminal subgenomic RNAs produced by a capillovirus: evidence for a CP subgenomic RNA

USDA-ARS?s Scientific Manuscript database

The members of Capillovirus genus encode two overlapping open reading frames (ORFs): ORF1 encodes a large polyprotein containing the domains of replication-associated proteins plus a coat protein (CP), and ORF2 encodes a movement protein, located within ORF1 in a different reading frame. Organizatio...

Identification of a maize nucleic acid-binding protein (NBP) belonging to a family of nuclear-encoded chloroplast proteins.

PubMed Central

Cook, W B; Walker, J C

1992-01-01

A cDNA encoding a nuclear-encoded chloroplast nucleic acid-binding protein (NBP) has been isolated from maize. Identified as an in vitro DNA-binding activity, NBP belongs to a family of nuclear-encoded chloroplast proteins which share a common domain structure and are thought to be involved in posttranscriptional regulation of chloroplast gene expression. NBP contains an N-terminal chloroplast transit peptide, a highly acidic domain and a pair of ribonucleoprotein consensus sequence domains. NBP is expressed in a light-dependent, organ-specific manner which is consistent with its involvement in chloroplast biogenesis. The relationship of NBP to the other members of this protein family and their possible regulatory functions are discussed. Images PMID:1346929

Activation of an AP1-Like Transcription Factor of the Maize Pathogen Cochliobolus heterostrophus in Response to Oxidative Stress and Plant Signals

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

Lev, Sophie; hadar, Ruthi; Amedeo, Paolo

Redox sensing is a ubiquitous mechanism regulating cellular activity. Fungal pathogens face reactive oxygen species produced by the host plant's oxidative burst in addition to endogenous reactive oxygen species produced during aerobic metabolism. An array of preformed and induced detoxifying enzymes, including superoxide dismutase, catalases, and peroxidases, could allow fungi to infect plants despite the oxidative burst. We isolated a gene (CHAP1) encoding a redox-regulated transcription factor in Cochliobolus heterostrophus, a fungal pathogen of maize. CHAP1 is a bZIP protein that possesses two cysteine-rich domains structurally and functionally related to Saccharomyces cerevisiae YAP1. Deletion of CHAP1 in C. heterostrophus resultedmore » in decreased resistance to oxidative stress caused by hydrogen peroxide and menadione, but the virulence of chap1 mutants was unaffected. Upon activation by oxidizing agents or plant signals, a green fluorescent protein (GFP)-CHAP1 fusion protein became localized in the nucleus. Expression of genes encoding antioxidant proteins was induced in the wild type but not in chap1 mutants. Activation of CHAP1 occurred from the earliest stage of plant infection, in conidial germ tubes on the leaf surface, and persisted during infection. Late in the course of infection, after extensive necrotic lesions were formed, GFP-CHAP1 redistributed to the cytosol in hyphae growing on the leaf surface. Localization of CHAP1 to the nucleus may, through changes in the redox state of the cell, provide a mechanism linking extracellular cues to transcriptional regulation during the plant-pathogen interaction.« less

Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

DOEpatents

Craft, David L.; Madduri, Krishna M.; Loper, John C.

2003-01-01

A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

tRNA travels from the cytoplasm to organelles

PubMed Central

Rubio, Mary Anne T.; Hopper, Anita K.

2011-01-01

Transfer RNAs (tRNAs) encoded by the nuclear genome are surprisingly dynamic. Although tRNAs function in protein synthesis occurring on cytoplasmic ribosomes, tRNAs can transit from the cytoplasm to the nucleus and then again return to the cytoplasm by a process known as the tRNA retrograde process. Subsets of the cytoplasmic tRNAs are also imported into mitochondria and function in mitochondrial protein synthesis. The numbers of tRNA species that are imported into mitchondria differ among organisms, ranging from just a few to the entire set needed to decode mitochondrially encoded mRNAs. For some tRNAs, import is dependent on the mitochondrial protein import machinery, whereas the majority of tRNA mitochondrial import is independent of this machinery. Although cytoplasmic proteins and proteins located on the mitochondrial surface participating in the tRNA import process have been described for several organisms, the identity of these proteins differ among organisms. Likewise, the tRNA determinants required for mitochondrial import differ among tRNA species and organisms. Here, we present an overview and discuss the current state of knowledge regarding the mechanisms involved in the tRNA retrograde process and continue with an overview of tRNA import into mitochondria. Finally, we highlight areas of future research to understand the function and regulation of movement of tRNAs between the cytoplasm and organelles. PMID:21976284

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus.

PubMed

Qiu, T; Lu, R H; Zhang, J; Zhu, Z Y

2001-07-01

The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mu1 of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mu1.

DNA encoding a DNA repair protein

DOEpatents

Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

2006-08-15

An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

Identification of Novel Surface-Exposed Proteins of Rickettsia rickettsii by Affinity Purification and Proteomics

PubMed Central

Gong, Wenping; Xiong, Xiaolu; Qi, Yong; Jiao, Jun; Duan, Changsong; Wen, Bohai

2014-01-01

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens. PMID:24950252

A phylogenomic analysis of the Actinomycetales mce operons.

PubMed

Casali, Nicola; Riley, Lee W

2007-02-26

The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins. The M. tuberculosis mce loci each include two yrbE and six mce genes, which have homology to ABC transporter permeases and substrate-binding proteins, respectively. Operons with an identical structure were identified in all Mycobacterium species examined, as well as in five other Actinomycetales genera. Some of the Actinomycetales mce operons include an mkl gene, which encodes an ATPase resembling those of ABC uptake transporters. The phylogenetic profile of Mkl orthologs exactly matched that of the Mce and YrbE proteins. Through topology and motif analyses of YrbE homologs, we identified a region within the penultimate cytoplasmic loop that may serve as the site of interaction with the putative cognate Mkl ATPase. Homologs of the exported proteins encoded adjacent to the M. tuberculosis mce operons were detected in a conserved chromosomal location downstream of the majority of Actinomycetales operons. Operons containing linked mkl, yrbE and mce genes, resembling the classic organization of an ABC importer, were found to be common in Gram-negative bacteria and appear to be associated with changes in properties of the cell surface. Evidence presented suggests that the mce operons of Actinomycetales species and related operons in Gram-negative bacteria encode a subfamily of ABC uptake transporters with a possible role in remodeling the cell envelope.

Exome sequencing identifies variants in two genes encoding the LIM-proteins NRAP and FHL1 in an Italian patient with BAG3 myofibrillar myopathy.

PubMed

D'Avila, Francesca; Meregalli, Mirella; Lupoli, Sara; Barcella, Matteo; Orro, Alessandro; De Santis, Francesca; Sitzia, Clementina; Farini, Andrea; D'Ursi, Pasqualina; Erratico, Silvia; Cristofani, Riccardo; Milanesi, Luciano; Braga, Daniele; Cusi, Daniele; Poletti, Angelo; Barlassina, Cristina; Torrente, Yvan

2016-06-01

Myofibrillar myopathies (MFMs) are genetically heterogeneous dystrophies characterized by the disintegration of Z-disks and myofibrils and are associated with mutations in genes encoding Z-disk or Z-disk-related proteins. The c.626 C > T (p.P209L) mutation in the BAG3 gene has been described as causative of a subtype of MFM. We report a sporadic case of a 26-year-old Italian woman, affected by MFM with axonal neuropathy, cardiomyopathy, rigid spine, who carries the c.626 C > T mutation in the BAG3 gene. The patient and her non-consanguineous healthy parents and brother were studied with whole exome sequencing (WES) to further investigate the genetic basis of this complex phenotype. In the patient, we found that the BAG3 mutation is associated with variants in the NRAP and FHL1 genes that encode muscle-specific, LIM domain containing proteins. Quantitative real time PCR, immunohistochemistry and Western blot analysis of the patient's muscular biopsy showed the absence of NRAP expression and FHL1 accumulation in aggregates in the affected skeletal muscle tissue. Molecular dynamic analysis of the mutated FHL1 domain showed a modification in its surface charge, which could affect its capability to bind its target proteins. To our knowledge this is the first study reporting, in a BAG3 MFM, the simultaneous presence of genetic variants in the BAG3 and FHL1 genes (previously described as independently associated with MFMs) and linking the NRAP gene to MFM for the first time.

An adhesin from hydrogen-utilizing rumen methanogen Methanobrevibacter ruminantium M1 binds a broad range of hydrogen-producing microorganisms.

PubMed

Ng, Filomena; Kittelmann, Sandra; Patchett, Mark L; Attwood, Graeme T; Janssen, Peter H; Rakonjac, Jasna; Gagic, Dragana

2016-09-01

Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein (Mru_1499) from Methanobrevibacter ruminantium M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera Epidinium and Entodinium). This unique adhesin also binds the cell surface of the bacterium Butyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein. © 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis

DOE PAGES

Plett, Jonathan M.; Yin, Hengfu; Mewalal, Ritesh; ...

2017-03-23

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa andmore » 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. Finally, we conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.« less

Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis

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

Plett, Jonathan M.; Yin, Hengfu; Mewalal, Ritesh

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as ‘effectors’, i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa andmore » 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. Finally, we conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.« less

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

Comparative differential gene expression analysis of nucleus-encoded proteins for Rafflesia cantleyi against Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Ng, Siuk-Mun; Lee, Xin-Wei; Wan, Kiew-Lian; Firdaus-Raih, Mohd

2015-09-01

Regulation of functional nucleus-encoded proteins targeting the plastidial functions was comparatively studied for a plant parasite, Rafflesia cantleyi versus a photosynthetic plant, Arabidopsis thaliana. This study involved two species of different feeding modes and different developmental stages. A total of 30 nucleus-encoded proteins were found to be differentially-regulated during two stages in the parasite; whereas 17 nucleus-encoded proteins were differentially-expressed during two developmental stages in Arabidopsis thaliana. One notable finding observed for the two plants was the identification of genes involved in the regulation of photosynthesis-related processes where these processes, as expected, seem to be present only in the autotroph.

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand

PubMed Central

Sawaswong, Vorthon; Simpalipan, Phumin; Siripoon, Napaporn; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2015-01-01

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines. PMID:25925176

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand.

PubMed

Sawaswong, Vorthon; Simpalipan, Phumin; Siripoon, Napaporn; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2015-04-01

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.

Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31

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

Lane, R.P.; Vielmetter, J.; Dreyer, W.J.

1996-08-01

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Igmore » domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumore suppressor candidate gene commonly deleted in certain human cancer tissues. 38 refs., 5 figs.« less

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

PubMed Central

Antos, John M.; Ingram, Jessica; Fang, Tao; Pishesha, Novalia; Truttmann, Matthias C.; Ploegh, Hidde L.

2017-01-01

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five amino acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. PMID:19365788

ORFeome Phage Display.

PubMed

Zantow, Jonas; Moreira, Gustavo Marçal Schmidt Garcia; Dübel, Stefan; Hust, Michael

2018-01-01

ORFeome phage display allows the efficient functional screening of entire proteomes or even metaproteomes to identify immunogenic proteins. For this purpose, randomly fragmented, whole genomes or metagenomes are cloned into a phage-display vector allowing positive selection for open reading frames (ORF) to improve the library quality. These libraries display all possible proteins encoded by a pathogen or a microbiome on the phage surface. Consequently, immunogenic proteins can be selected from these libraries using disease-related immunoglobulins from patient serum. ORFeome phage display in particular allows the identification of immunogenic proteins that are only expressed in the host-pathogen interaction but not in cultivation, as well as the detection of very low expressed and very small immunogens and immunogenic proteins of non-cultivable organisms. The identified immunogenic proteins are potential biomarkers for the development of diagnostic assays or vaccines. These articles will give an introduction to ORFeome phage-display technology and give detailed protocols to identify immunogenic proteins by phage display.

BcMF8, a putative arabinogalactan protein-encoding gene, contributes to pollen wall development, aperture formation and pollen tube growth in Brassica campestris

PubMed Central

Lin, Sue; Dong, Heng; Zhang, Fang; Qiu, Lin; Wang, Fangzhan; Cao, Jiashu; Huang, Li

2014-01-01

Background and Aims The arabinogalactan protein (AGP) gene family is involved in plant reproduction. However, little is known about the function of individual AGP genes in pollen development and pollen tube growth. In this study, Brassica campestris male fertility 8 (BcMF8), a putative AGP-encoding gene previously found to be pollen specific in Chinese cabbage (B. campestris ssp. chinensis), was investigated. Methods Real-time reverse transcription–PCR and in situ hybridization were used to analyse the expression pattern of BcMF8 in pistils. Prokaryotic expression and western blots were used to ensure that BcMF8 could encode a protein. Antisense RNA technology was applied to silence gene expression, and morphological and cytological approaches (e.g. scanning electron microscopy and transmission electron microscopy) were used to reveal abnormal phenotypes caused by gene silencing. Key Results The BcMF8 gene encoded a putative AGP protein that was located in the cell wall, and was expressed in pollen grains and pollen tubes. The functional interruption of BcMF8 by antisense RNA technology resulted in slipper-shaped and bilaterally sunken pollen with abnormal intine development and aperture formation. The inhibition of BcMF8 led to a decrease in the percentage of in vitro pollen germination. In pollen that did germinate, the pollen tubes were unstable, abnormally shaped and burst more frequently relative to controls, which corresponded to an in vivo arrest of pollen germination at the stigma surface and retarded pollen tube growth in the stylar transmitting tissues. Conclusions The phenotypic defects of antisense BcMF8 RNA lines (bcmf8) suggest a crucial function of BcMF8 in modulating the physical nature of the pollen wall and in helping in maintaining the integrity of the pollen tube wall matrix. PMID:24489019

The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

PubMed

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

2012-02-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

PubMed Central

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc

2012-01-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism. PMID:22156529

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

PubMed

Gäken, J; Jiang, J; Daniel, K; van Berkel, E; Hughes, C; Kuiper, M; Darling, D; Tavassoli, M; Galea-Lauri, J; Ford, K; Kemeny, M; Russell, S; Farzaneh, F

2000-12-01

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors.

PubMed

Kushwaha, Ambuj K; Apolis, Liana; Ito, Daisuke; Desai, Sanjay A

2018-05-03

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad-selectivity channel known as the plasmodial surface anion channel, increased Ca ++ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca ++ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N-hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca ++ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca ++ permeability, suggesting involvement of parasite-encoded proteins trafficked to the host membrane. A high-throughput chemical screen identified the first Ca ++ transport inhibitors active against Plasmodium-infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca ++ ] is consistent with parasite killing specifically via action on one or more Ca ++ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca ++ transport and may be starting points for new antimalarial drugs. © 2018 John Wiley & Sons Ltd.

Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

NASA Astrophysics Data System (ADS)

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

2016-01-01

We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

Isotope-encoded Carboxyl Group Footprinting for Mass Spectrometry-based Protein Conformational Studies

PubMed Central

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

2015-01-01

We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded “heavy” and “light” GEE are used to label separately the two states of the Orange Carotenoid Protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the “heavy” and “light” peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting. PMID:26384685

Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

DOE PAGES

Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; ...

2015-09-18

Here, we report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded “heavy” and “light” GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the “heavy”more » and “light” peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.« less

Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803

PubMed Central

Hauf, Waldemar; Watzer, Björn; Roos, Nora; Klotz, Alexander

2015-01-01

Cyanobacteria are photoautotrophic microorganisms which fix atmospheric carbon dioxide via the Calvin-Benson cycle to produce carbon backbones for primary metabolism. Fixed carbon can also be stored as intracellular glycogen, and in some cyanobacterial species like Synechocystis sp. strain PCC 6803, polyhydroxybutyrate (PHB) accumulates when major nutrients like phosphorus or nitrogen are absent. So far only three enzymes which participate in PHB metabolism have been identified in this organism, namely, PhaA, PhaB, and the heterodimeric PHB synthase PhaEC. In this work, we describe the cyanobacterial PHA surface-coating protein (phasin), which we term PhaP, encoded by ssl2501. Translational fusion of Ssl2501 with enhanced green fluorescent protein (eGFP) showed a clear colocalization to PHB granules. A deletion of ssl2501 reduced the number of PHB granules per cell, whereas the mean PHB granule size increased as expected for a typical phasin. Although deletion of ssl2501 had almost no effect on the amount of PHB, the biosynthetic activity of PHB synthase was negatively affected. Secondary-structure prediction and circular dichroism (CD) spectroscopy of PhaP revealed that the protein consists of two α-helices, both of them associating with PHB granules. Purified PhaP forms oligomeric structures in solution, and both α-helices of PhaP contribute to oligomerization. Together, these results support the idea that Ssl2501 encodes a cyanobacterial phasin, PhaP, which regulates the surface-to-volume ratio of PHB granules. PMID:25911471

The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

PubMed

Fujinami, Shun; Ito, Masahiro

2018-01-01

It is well known that the Na + cycle and the cell wall are essential for alkaline adaptation of Na + -dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na + concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na + concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

Identification of the gene encoding a 38-kilodalton immunogenic and protective antigen of Streptococcus suis.

PubMed

Okwumabua, Ogi; Chinnapapakkagari, Sharmila

2005-04-01

In our continued effort to search for a Streptococcus suis protein(s) that can serve as a vaccine candidate or a diagnostic reagent, we constructed and screened a gene library with a polyclonal antibody raised against the whole-cell protein of S. suis type 2. A clone that reacted with the antibody was identified and characterized. Analysis revealed that the gene encoding the protein is localized within a 2.0-kbp EcoRI DNA fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 445 amino acid residues with a calculated molecular mass of 46.4 kDa. By in vitro protein synthesis and Western blot experiments, the protein exhibited an electrophoretic mobility of approximately 38 kDa. At the amino acid level the deduced primary sequence shared homology with sequences of unknown function from Streptococcus pneumoniae (89%), Streptococcus mutans (86%), Lactococcus lactis (80%), Listeria monocytogenes (74%), and Clostridium perfringens (64%). Except for strains of serotypes 20, 26, 32, and 33, Southern hybridization analysis revealed the presence of the gene in strains of other S. suis serotypes and demonstrated restriction fragment length differences caused by a point mutation in the EcoRI recognition sequence. We confirmed expression of the 38-kDa protein in the hybridization-positive isolates using specific antiserum against the purified protein. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic and may serve as an antigen of diagnostic importance for the detection of most S. suis infections. Pigs immunized with the recombinant 38-kDa protein mounted antibody responses to the protein and were completely protected against challenge with a strain of a homologous serotype, the wild-type virulent strain of S. suis type 2, suggesting that it may be a good candidate for the development of a vaccine that can be used as protection against S. suis infection. Analysis of the cellular fractions of the bacterium by Western blotting revealed that the protein was present in the surface and cell wall extracts. The functional role of the protein with respect to pathogenesis and whether antibodies against the antigen confer protective immunity against diseases caused by strains of other pathogenic S. suis capsular types remains to be determined.

Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

PubMed

Lacroix, Benoît; Citovsky, Vitaly

2015-11-20

During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

PubMed

de Souza, Alessandra A; Takita, Marco A; Coletta-Filho, Helvécio D; Caldana, Camila; Yanai, Giane M; Muto, Nair H; de Oliveira, Regina C; Nunes, Luiz R; Machado, Marcos A

2004-08-15

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

The PE/PPE multigene family codes for virulence factors and is a possible source of mycobacterial antigenic variation: perhaps more?

PubMed

Akhter, Yusuf; Ehebauer, Matthias T; Mukhopadhyay, Sangita; Hasnain, Seyed E

2012-01-01

The PE/PPE multigene family codes for approximately 10% of the Mycobacterium tuberculosis proteome and is encoded by 176 open reading frames. These proteins possess, and have been named after, the conserved proline-glutamate (PE) or proline-proline-glutamate (PPE) motifs at their N-terminus. Their genes have a conserved structure and repeat motifs that could be a potential source of antigenic variation in M. tuberculosis. PE/PPE genes are scattered throughout the genome and PE/PPE pairs are usually encoded in bicistronic operons although this is not universally so. This gene family has evolved by specific gene duplication events. PE/PPE proteins are either secreted or localized to the cell surface. Several are thought to be virulence factors, which participate in evasion of the host immune response. This review summarizes the current knowledge about the gene family in order to better understand its biological function. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Cloning structural genes for Treponema pallidum immunogens and characterisation of recombinant treponemal surface protein, P2 (P2 star).

PubMed Central

Peterson, K M; Baseman, J B; Alderete, J F

1987-01-01

A genomic library consisting of partially digested 10 to 20 kilobase pair fragments of Treponema pallidum deoxyribonucleic acid (DNA) was constructed using bacteriophage lambda EMBL-3 as the vector. Positive clones expressing T pallidum antigens were detected with sera from experimentally infected rabbits. Treponemal proteins ranging in molecular weight from 37,000 daltons to 120,000 daltons were identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting of phage lysate proteins. One recombinant phage was examined further and contained an insert encoding a prominent treponemal 37,000 dalton protein. The recombinant protein was not recognised by antiserum directed against a fibronectin binding treponemal adhesion that contained the same electrophoretic mobility. Neither did antibody to the recombinant 37,000 dalton protein react with any treponemal proteins purified by fibronectin affinity chromatography. The recombinant protein in Escherichia coli lysates was labelled P2 (P2 star) to differentiate it from the comigrating adhesin protein called P2. Native P2 protein was present on T pallidum surfaces as shown by radioimmunoprecipitation assays with extrinsically labelled organisms. A cross reactive molecule like P2 was not synthesised by the avirulent spirochaete, T phagedenis biotype Reiter, which indicated that P2 is a protein specific to virulent T pallidum organisms. Finally, only sera of patients with primary syphilis possessed appreciable concentrations of antibody to recombinant P2 protein. Images PMID:3315959

Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle.

PubMed

Thompson, Joanne; Fernandez-Reyes, Delmiro; Sharling, Lisa; Moore, Sally G; Eling, Wijnand M; Kyes, Sue A; Newbold, Christopher I; Kafatos, Fotis C; Janse, Chris J; Waters, Andrew P

2007-06-01

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

The mitochondrial gene encoding ribosomal protein S12 has been translocated to the nuclear genome in Oenothera.

PubMed Central

Grohmann, L; Brennicke, A; Schuster, W

1992-01-01

The Oenothera mitochondrial genome contains only a gene fragment for ribosomal protein S12 (rps12), while other plants encode a functional gene in the mitochondrion. The complete Oenothera rps12 gene is located in the nucleus. The transit sequence necessary to target this protein to the mitochondrion is encoded by a 5'-extension of the open reading frame. Comparison of the amino acid sequence encoded by the nuclear gene with the polypeptides encoded by edited mitochondrial cDNA and genomic sequences of other plants suggests that gene transfer between mitochondrion and nucleus started from edited mitochondrial RNA molecules. Mechanisms and requirements of gene transfer and activation are discussed. Images PMID:1454526

Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture.

PubMed

Neuenfeldt, Anne; Lorber, Bernard; Ennifar, Eric; Gaudry, Agnès; Sauter, Claude; Sissler, Marie; Florentz, Catherine

2013-02-01

In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs.

Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii

PubMed Central

Wang, Sen; Zhao, Dong; Bai, Xinfeng; Zhang, Weican

2016-01-01

ABSTRACT Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii. A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN3. Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220. Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases. IMPORTANCE Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii. PMID:27742681

Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever

PubMed Central

Lin, Mingqun; Zhang, Chunbin; Gibson, Kathryn; Rikihisa, Yasuko

2009-01-01

Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. sennetsu showed that 758 (88.2%) of protein-coding genes are conserved between N. risticii and N. sennetsu. Four-way comparison of genes among N. risticii and other Anaplasmataceae showed that most genes are either shared among Anaplasmataceae (525 orthologs that generally associated with housekeeping functions), or specific to each genome (>200 genes that are mostly hypothetical proteins). Genes potentially involved in the pathogenesis of N. risticii were identified, including those encoding putative outer membrane proteins, two-component systems and a type IV secretion system (T4SS). The bipolar localization of T4SS pilus protein VirB2 on the bacterial surface was demonstrated for the first time in obligate intracellular bacteria. These data provide insights toward genomic potential of N. risticii and intracellular parasitism, and facilitate our understanding of PHF pathogenesis. PMID:19661282

Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever.

PubMed

Lin, Mingqun; Zhang, Chunbin; Gibson, Kathryn; Rikihisa, Yasuko

2009-10-01

Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. sennetsu showed that 758 (88.2%) of protein-coding genes are conserved between N. risticii and N. sennetsu. Four-way comparison of genes among N. risticii and other Anaplasmataceae showed that most genes are either shared among Anaplasmataceae (525 orthologs that generally associated with housekeeping functions), or specific to each genome (>200 genes that are mostly hypothetical proteins). Genes potentially involved in the pathogenesis of N. risticii were identified, including those encoding putative outer membrane proteins, two-component systems and a type IV secretion system (T4SS). The bipolar localization of T4SS pilus protein VirB2 on the bacterial surface was demonstrated for the first time in obligate intracellular bacteria. These data provide insights toward genomic potential of N. risticii and intracellular parasitism, and facilitate our understanding of PHF pathogenesis.

Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins▿

PubMed Central

Plant, Ewan P.; Rakauskaitė, Rasa; Taylor, Deborah R.; Dinman, Jonathan D.

2010-01-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed −1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the −1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a “golden mean” model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins. PMID:20164235

Ectromelia Virus BTB/kelch Proteins, EVM150 and EVM167, Interact with Cullin-3 Based Ubiquitin Ligases

PubMed Central

Wilton, Brianne A.; Campbell, Stephanie; Van Buuren, Nicholas; Garneau, Robyn; Furukawa, Manabu; Xiong, Yue; Barry., Michele

2008-01-01

Cellular proteins containing BTB and kelch domains have been shown to function as adapters for the recruitment of substrates to cullin-3-based ubiquitin ligases. Poxviruses are the only family of viruses known to encode multiple BTB/kelch proteins, suggesting that poxviruses may modulate the ubiquitin pathway through interaction with cullin-3. Ectromelia virus encodes four BTB/kelch proteins and one BTB-only protein. Here we demonstrate that two of the ectromelia virus encoded BTB/kelch proteins, EVM150 and EVM167, interacted with cullin-3. Similar to cellular BTB proteins, the BTB domain of EVM150 and EVM167 was necessary and sufficient for cullin-3 interaction. During infection, EVM150 and EVM167 localized to discrete cytoplasmic regions, which co-localized with cullin-3. Furthermore, EVM150 and EVM167 co-localized and interacted with conjugated ubiquitin, as demonstrated by confocal microscopy and co-immunoprecipitation. Our findings suggest that the ectromelia virus encoded BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3 potentially functioning to recruit unidentified substrates for ubiquitination. PMID:18221766

Characterization of a cDNA encoding a protein involved in formation of the skeleton during development of the sea urchin Lytechinus pictus.

PubMed

Livingston, B T; Shaw, R; Bailey, A; Wilt, F

1991-12-01

In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus pictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide, a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to proteins that form a unique beta-spiral supersecondary structure.

Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

PubMed Central

Maeland, Johan A.; Bevanger, Lars; Lyng, Randi Valsoe

2004-01-01

The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Cα) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Cα. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments. PMID:15539502

Trichoderma genes

DOEpatents

Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

2012-06-19

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus

PubMed Central

Alejo, Alí; Ruiz-Argüello, M. Begoña; Ho, Yin; Smith, Vincent P.; Saraiva, Margarida; Alcami, Antonio

2006-01-01

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis. PMID:16581912

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus.

PubMed

Alejo, Alí; Ruiz-Argüello, M Begoña; Ho, Yin; Smith, Vincent P; Saraiva, Margarida; Alcami, Antonio

2006-04-11

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.

Screening for ATM Mutations in an African-American Population to Identify a Predictor of Breast Cancer Susceptibility

DTIC Science & Technology

2006-07-01

ATM genetic variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for each mutation examined. 15. SUBJECT...women without breast cancer. An additional objective is to determine the functional impact upon the protein encoded by the ATM gene for each mutation ...each ATM variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for mutations identified. Body STATEMENT

Genetic diversity among major endemic strains of Leptospira interrogans in China

PubMed Central

He, Ping; Sheng, Yue-Ying; Shi, Yao-Zhou; Jiang, Xiu-Gao; Qin, Jin-Hong; Zhang, Zhi-Ming; Zhao, Guo-Ping; Guo, Xiao-Kui

2007-01-01

Background Leptospirosis is a world-widely distributed zoonosis. Humans become infected via exposure to pathogenic Leptospira spp. from contaminated water or soil. The availability of genomic sequences of Leptospira interrogans serovar Lai and serovar Copenhageni opened up opportunities to identify genetic diversity among different pathogenic strains of L. interrogans representing various kinds of serotypes (serogroups and serovars). Results Comparative genomic hybridization (CGH) analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with that of other 10 L. interrogans strains prevailed in China and one identified from Brazil using a microarray spotted with 3,528 protein coding sequences (CDSs) of strain Lai. The cutoff ratio of sample/reference (S/R) hybridization for detecting the absence of genes from one tested strain was set by comparing the ratio of S/R hybridization and the in silico sequence similarities of strain Lai and serovar Copenhageni strain Fiocruz L1-130. Among the 11 strains tested, 275 CDSs were found absent from at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,917 CDSs. The genes encoding fundamental cellular functions such as translation, energy production and conversion were conserved. While strain-specific genes include those that encode proteins related to either cell surface structures or carbohydrate transport and metabolism. We also found two genomic islands (GIs) in strain Lai containing genes divergently absent in other strains. Because genes encoding proteins with potential pathogenic functions are located within GIs, these elements might contribute to the variations in disease manifestation. Differences in genes involved in O-antigen biosynthesis were also identified for strains belonging to different serogroups, which offers an opportunity for future development of genomic typing tools for serological classification. Conclusion CGH analyses for pathogenic leptospiral strains prevailed in China against the L. interrogans serovar Lai strain Lai CDS-spotted microarrays revealed 2,917 common backbone CDSs and strain specific genes encoding proteins mainly related to cell surface structures and carbohydrated transport/metabolism. Of the 275 CDSs considered absent from at least one of the L. interrogans strains tested, most of them were clustered in the rfb gene cluster and two putative genomic islands (GI A and B) in strain Lai. The strain-specific genes detected via this work will provide a knowledge base for further investigating the pathogenesis of L interrogans and/or for the development of effective vaccines and/or diagnostic tools. PMID:17603913

An unusual MAP kinase is required for efficient penetration of the plant surface by Ustilago maydis

PubMed Central

Brachmann, Andreas; Schirawski, Jan; Müller, Philip; Kahmann, Regine

2003-01-01

In Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bW and bE. We have identified by RNA fingerprinting a b-regulated gene, kpp6, which encodes an unusual MAP kinase. Kpp6 is similar to a number of other fungal MAP kinases involved in mating and pathogenicity, but contains an additional N-terminal domain unrelated to other proteins. Transcription of the kpp6 gene yields two transcripts differing in length, but encoding proteins of identical mass. One transcript is upregulated by the bW/bE heterodimer, while the other is induced after pheromone stimulation. kpp6 deletion mutants are attenuated in pathogenicity. kpp6T355A,Y357F mutants carrying a non-activatable allele of kpp6 are more severely compromised in pathogenesis. These strains can still form appressoria, but are defective in the subsequent penetration of the plant cuticle. Kpp6 is expressed during all stages of the sexual life cycle except mature spores. We speculate that Kpp6 may respond to a plant signal and regulate the genes necessary for efficient penetration of plant tissue. PMID:12727886

Biofilm density and detection of biofilm-producing genes in methicillin-resistant Staphylococcus aureus strains.

PubMed

Szczuka, Ewa; Urbańska, Katarzyna; Pietryka, Marta; Kaznowski, Adam

2013-01-01

Many serious diseases caused by Staphylococcus aureus appear to be associated with biofilms. Therefore, we investigated the biofilm-forming ability of the methicillin-resistant S. aureus (MRSA) isolates collected from hospitalized patients. As many as 96 % strains had the ability to form biofilm in vitro. The majority of S. aureus strains formed biofilm in ica-dependent mechanism. However, 23 % of MRSA isolates formed biofilm in ica-independent mechanism. Half of these strains carried fnbB genes encoding surface proteins fibronectin-binding protein B involved in intercellular accumulation and biofilm development in S. aureus strains. The biofilm structures were examined via confocal laser scanning microscopy (CLSM) and three-dimensional structures were reconstructed. The images obtained in CLSM revealed that the biofilm created by ica-positive strains was different from biofilm formed by ica-negative strains. The MRSA population showed a large genetic diversity and we did not find a single clone that occurred preferentially in hospital environment. Our results demonstrated the variation in genes encoding adhesins for the host matrix proteins (elastin, laminin, collagen, fibronectin, and fibrinogen) and in the gene involved in biofilm formation (icaA) within the majority of S. aureus clones.

In Silico Identification of Epitopes in Mycobacterium avium subsp. paratuberculosis Proteins That Were Upregulated under Stress Conditions

PubMed Central

Gurung, Ratna B.; Purdie, Auriol C.; Begg, Douglas J.

2012-01-01

Johne's disease in ruminants is caused by Mycobacterium avium subsp. paratuberculosis. Diagnosis of M. avium subsp. paratuberculosis infection is difficult, especially in the early stages. To date, ideal antigen candidates are not available for efficient immunization or immunodiagnosis. This study reports the in silico selection and subsequent analysis of epitopes of M. avium subsp. paratuberculosis proteins that were found to be upregulated under stress conditions as a means to identify immunogenic candidate proteins. Previous studies have reported differential regulation of proteins when M. avium subsp. paratuberculosis is exposed to stressors which induce a response similar to dormancy. Dormancy may be involved in evading host defense mechanisms, and the host may also mount an immune response against these proteins. Twenty-five M. avium subsp. paratuberculosis proteins that were previously identified as being upregulated under in vitro stress conditions were analyzed for B and T cell epitopes by use of the prediction tools at the Immune Epitope Database and Analysis Resource. Major histocompatibility complex class I T cell epitopes were predicted using an artificial neural network method, and class II T cell epitopes were predicted using the consensus method. Conformational B cell epitopes were predicted from the relevant three-dimensional structure template for each protein. Based on the greatest number of predicted epitopes, eight proteins (MAP2698c [encoded by desA2], MAP2312c [encoded by fadE19], MAP3651c [encoded by fadE3_2], MAP2872c [encoded by fabG5_2], MAP3523c [encoded by oxcA], MAP0187c [encoded by sodA], and the hypothetical proteins MAP3567 and MAP1168c) were identified as potential candidates for study of antibody- and cell-mediated immune responses within infected hosts. PMID:22496492

The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein

PubMed Central

Miranda-Ozuna, Jesús F. T.; Hernández-García, Mar S.; Brieba, Luis G.; Benítez-Cardoza, Claudia G.; Ortega-López, Jaime; González-Robles, Arturo

2016-01-01

Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis. Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis. PMID:27481251

The role played by alternative splicing in antigenic variability in human endo-parasites.

PubMed

Hull, Rodney; Dlamini, Zodwa

2014-01-28

Endo-parasites that affect humans include Plasmodium, the causative agent of malaria, which remains one of the leading causes of death in human beings. Despite decades of research, vaccines to this and other endo-parasites remain elusive. This is in part due to the hyper-variability of the parasites surface proteins. Generally these surface proteins are encoded by a large family of genes, with only one being dominantly expressed at certain life stages. Another layer of complexity can be introduced through the alternative splicing of these surface proteins. The resulting isoforms may differ from each other with regard to cell localisation, substrate affinities and functions. They may even differ in structure to the extent that they are no longer recognised by the host's immune system. In many cases this leads to changes in the N terminus of these proteins. The geographical localisation of endo-parasitic infections around the tropics and the highest incidences of HIV-1 infection in the same areas, adds a further layer of complexity as parasitic infections affect the host immune system resulting in higher HIV infection rates, faster disease progression, and an increase in the severity of infections and complications in HIV diagnosis. This review discusses some examples of parasite surface proteins that are alternatively spliced in trypanosomes, Plasmodium and the parasitic worm Schistosoma as well as what role alternate splicing may play in the interaction between HIV and these endo-parasites.

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris

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

Zhang, Weiwen; Culley, David E.; Gritsenko, Marina A.

2006-11-03

ABSTRACT In the previous study, the whole-genome gene expression profiles of D. vulgaris in response to oxidative stress and heat shock were determined. The results showed 24-28% of the responsive genes were hypothetical proteins that have not been experimentally characterized or whose function can not be deduced by simple sequence comparison. To further explore the protecting mechanisms employed in D. vulgaris against the oxidative stress and heat shock, attempt was made in this study to infer functions of these hypothetical proteins by phylogenomic profiling along with detailed sequence comparison against various publicly available databases. By this approach we were abletomore » assign possible functions to 25 responsive hypothetical proteins. The findings included that DVU0725, induced by oxidative stress, may be involved in lipopolysaccharide biosynthesis, implying that the alternation of lipopolysaccharide on cell surface might service as a mechanism against oxidative stress in D. vulgaris. In addition, two responsive proteins, DVU0024 encoding a putative transcriptional regulator and DVU1670 encoding predicted redox protein, were sharing co-evolution atterns with rubrerythrin in Archaeoglobus fulgidus and Clostridium perfringens, respectively, implying that they might be part of the stress response and protective systems in D. vulgaris. The study demonstrated that phylogenomic profiling is a useful tool in interpretation of experimental genomics data, and also provided further insight on cellular response to oxidative stress and heat shock in D. vulgaris.« less

Reconstruction of the bifidobacterial pan-secretome reveals the network of extracellular interactions between bifidobacteria and the infant gut.

PubMed

Lugli, Gabriele Andrea; Mancino, Walter; Milani, Christian; Duranti, Sabrina; Turroni, Francesca; van Sinderen, Douwe; Ventura, Marco

2018-06-08

The repertoire of secreted proteins decoded by a microorganism represents proteins released from or associated with the cell's surface. In gut commensals, such as bifidobacteria, these proteins are perceived to be functionally relevant as they regulate the interaction with the gut environment. In the current study, we have screened the predicted proteome of over 300 bifidobacterial strains amongst the currently recognized bifidobacterial species to generate a comprehensive database encompassing bifidobacterial extracellular proteins. A glycobiome analysis of this predicted bifidobacterial secretome revealed that a correlation exists between particular bifidobacterial species and their capability to hydrolyze HMOs and intestinal glyconjugates such as mucin. Furthermore, exploration of metatranscriptomic datasets of the infant gut microbiota allowed the evaluation of the expression of bifidobacterial genes encoding extracellular proteins, represented by ABC transporter substrate-binding proteins and glycoside hydrolases enzymes involved in the degradation of human milk oligosaccharides and mucin. Overall, this study provides insights into how bifidobacteria interact with their natural yet highly complex environment, the infant gut. Importance The ecological success of bifidobacteria relies on the activity of extracellular proteins that are involved in the metabolism of nutrients and the interaction with the environment. To date, information on secreted proteins encoded by bifidobacteria are incomplete and just related to few species. In this study, we reconstructed the bifidobacterial pan-secretome, revealing extracellular proteins that modulate the interaction of bifidobacteria with their natural environment. Furthermore, a survey of secretion system between bifidobacterial genomes allowed the identification of a conserved Sec-dependent secretion machinery in all the analyzed genomes and the Tat protein translocation system in the chromosomes of 23 strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium aesculapii . Copyright © 2018 American Society for Microbiology.

A phylogenomic analysis of the Actinomycetales mce operons

PubMed Central

Casali, Nicola; Riley, Lee W

2007-01-01

Background The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins. Results The M. tuberculosis mce loci each include two yrbE and six mce genes, which have homology to ABC transporter permeases and substrate-binding proteins, respectively. Operons with an identical structure were identified in all Mycobacterium species examined, as well as in five other Actinomycetales genera. Some of the Actinomycetales mce operons include an mkl gene, which encodes an ATPase resembling those of ABC uptake transporters. The phylogenetic profile of Mkl orthologs exactly matched that of the Mce and YrbE proteins. Through topology and motif analyses of YrbE homologs, we identified a region within the penultimate cytoplasmic loop that may serve as the site of interaction with the putative cognate Mkl ATPase. Homologs of the exported proteins encoded adjacent to the M. tuberculosis mce operons were detected in a conserved chromosomal location downstream of the majority of Actinomycetales operons. Operons containing linked mkl, yrbE and mce genes, resembling the classic organization of an ABC importer, were found to be common in Gram-negative bacteria and appear to be associated with changes in properties of the cell surface. Conclusion Evidence presented suggests that the mce operons of Actinomycetales species and related operons in Gram-negative bacteria encode a subfamily of ABC uptake transporters with a possible role in remodeling the cell envelope. PMID:17324287

Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

PubMed Central

Siegrist, M. Sloan; Swarts, Benjamin M.; Fox, Douglas M.; Lim, Shion An; Bertozzi, Carolyn R.

2015-01-01

The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology. PMID:25725012

Streptococcus pyogenes collagen type I-binding Cpa surface protein. Expression profile, binding characteristics, biological functions, and potential clinical impact.

PubMed

Kreikemeyer, Bernd; Nakata, Masanobu; Oehmcke, Sonja; Gschwendtner, Caroline; Normann, Jana; Podbielski, Andreas

2005-09-30

The Streptococcus pyogenes collagen type I-binding protein Cpa (collagen-binding protein of group A streptococci) expressed by 28 serotypes of group A streptococci has been extensively characterized at the gene and protein levels. Evidence for three distinct families of cpa genes was found, all of which shared a common sequence encoding a 60-amino acid domain that accounted for selective binding to type I collagen. Surface plasmon resonance-based affinity measurements and functional studies indicated that the expression of Cpa was consistent with an attachment role for bacteria to tissue containing collagen type I. A cpa mutant displayed a significantly decreased internalization rate when incubated with HEp-2 cells but had no effect on the host cell viability. By utilizing serum from patients with a positive titer for streptolysin/DNase antibody, an increased anti-Cpa antibody titer was noted for patients with a clinical history of arthritis or osteomyelitis. Taken together, these results suggest Cpa may be a relevant matrix adhesin contributing to the pathogenesis of S. pyogenes infection of bones and joints.

The distinct proteome of placental malaria parasites.

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

Fried, Michal; Hixson, Kim K.; Anderson, Lori

Malaria proteins expressed on the surface of Plasmodium falciparum infected erythrocytes (IE) mediate adhesion and are targeted by protective immune responses. During pregnancy, IE sequester in the placenta. Placental IE bind to the molecule chondroitin sulfate A (CSA) and preferentially transcribe the gene that encodes VAR2CSA, a member of the PfEMP1 variant surface antigen family. Over successive pregnancies women develop specific immunity to CSA-binding IE and antibodies to VAR2CSA. We used tandem mass spectrometry together with accurate mass and time tag technology to study IE membrane fractions of placental parasites. VAR2CSA peptides were detected in placental IE and in IEmore » from children, but the MC variant of VAR2CSA was specifically associated with placental IE. We identified six conserved hypothetical proteins with putative TM or signal peptides that were exclusively expressed by the placental IE, and 11 such proteins that were significantly more abundant in placental IE. One of these hypothetical proteins, PFI1785w, is a 42kDa molecule detected by Western blot in parasites infecting pregnant women but not those infecting children.« less

The gene for fibroblast activation protein {alpha} (FAP), a putative cell surface-bound serine protease expressed in cancer stroma and wound healing, maps to chromosome band 2q23

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

Mathew, S.; Murty, V.V.V.S.; Chaganti, R.S.K.

The human fibroblast activation protein {alpha} (FAP{alpha}) is an inducible cell surface glycoprotein of M{sub r} 95,000 recognized by a number of monoclonal antibodies (mAbs), including the prototype mAb F19. Immunohistochemical studies have shown that FAP{alpha} expression in vivo is tightly regulated, with transient expression in some fetal mesenchymal tissues but absence of expression in most normal adult tissues. Reexpression of FAP{alpha} is observed in the reactive stromal fibroblasts of several common types of epithelial cancers, including >90% of breast, colorectal, and lung carcinomas and healing wounds. Cloning and sequence analysis of an FAP{alpha}-specific cDNA has revealed that the moleculemore » is encoded by a novel gene, FAP, which shows sequence similarity to members of the serine protease family of integral membrane proteins, namely dipeptidyl peptidase IV (DPPIV, also known as lymphocyte activation antigen, CD26, or adenosine dearoinase binding protein) and DPPX, a DPPIV-related molecule of unknown function. 15 refs., 1 fig.« less

Neisseria conserved protein DMP19 is a DNA mimic protein that prevents DNA binding to a hypothetical nitrogen-response transcription factor

PubMed Central

Wang, Hao-Ching; Ko, Tzu-Ping; Wu, Mao-Lun; Ku, Shan-Chi; Wu, Hsing-Ju; Wang, Andrew H.-J.

2012-01-01

DNA mimic proteins occupy the DNA binding sites of DNA-binding proteins, and prevent these sites from being accessed by DNA. We show here that the Neisseria conserved hypothetical protein DMP19 acts as a DNA mimic. The crystal structure of DMP19 shows a dsDNA-like negative charge distribution on the surface, suggesting that this protein should be added to the short list of known DNA mimic proteins. The crystal structure of another related protein, NHTF (Neisseria hypothetical transcription factor), provides evidence that it is a member of the xenobiotic-response element (XRE) family of transcriptional factors. NHTF binds to a palindromic DNA sequence containing a 5′-TGTNAN11TNACA-3′ recognition box that controls the expression of an NHTF-related operon in which the conserved nitrogen-response protein [i.e. (Protein-PII) uridylyltransferase] is encoded. The complementary surface charges between DMP19 and NHTF suggest specific charge–charge interaction. In a DNA-binding assay, we found that DMP19 can prevent NHTF from binding to its DNA-binding sites. Finally, we used an in situ gene regulation assay to provide evidence that NHTF is a repressor of its down-stream genes and that DMP19 can neutralize this effect. We therefore conclude that the interaction of DMP19 and NHTF provides a novel gene regulation mechanism in Neisseria spps. PMID:22373915

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

PubMed

Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

2014-04-01

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.

PubMed

van Kasteren, Puck B; Bailey-Elkin, Ben A; James, Terrence W; Ninaber, Dennis K; Beugeling, Corrine; Khajehpour, Mazdak; Snijder, Eric J; Mark, Brian L; Kikkert, Marjolein

2013-02-26

Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc-dependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-β mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual-specificity proteases.

CIP1 polypeptides and their uses

DOEpatents

Foreman, Pamela [Los Altos, CA; Van Solingen, Pieter [Naaldwijk, NL; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA

2011-04-12

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

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

Hervey, IV, William Judson; Khalsa-Moyers, Gurusahai K; Lankford, Patricia K

Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed bymore » liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid-encoding strategies for bait protein expression. This approach has the potential for enabling discovery of protein-protein interactions among the growing number of sequenced microbial species without the need for development of chromosomal insertion systems.« less

Targeted next-generation sequencing helps to decipher the genetic and phenotypic heterogeneity of hypertrophic cardiomyopathy

PubMed Central

Cecconi, Massimiliano; Parodi, Maria I.; Formisano, Francesco; Spirito, Paolo; Autore, Camillo; Musumeci, Maria B.; Favale, Stefano; Forleo, Cinzia; Rapezzi, Claudio; Biagini, Elena; Davì, Sabrina; Canepa, Elisabetta; Pennese, Loredana; Castagnetta, Mauro; Degiorgio, Dario; Coviello, Domenico A.

2016-01-01

Hypertrophic cardiomyopathy (HCM) is mainly associated with myosin, heavy chain 7 (MYH7) and myosin binding protein C, cardiac (MYBPC3) mutations. In order to better explain the clinical and genetic heterogeneity in HCM patients, in this study, we implemented a target-next generation sequencing (NGS) assay. An Ion AmpliSeq™ Custom Panel for the enrichment of 19 genes, of which 9 of these did not encode thick/intermediate and thin myofilament (TTm) proteins and, among them, 3 responsible of HCM phenocopy, was created. Ninety-two DNA samples were analyzed by the Ion Personal Genome Machine: 73 DNA samples (training set), previously genotyped in some of the genes by Sanger sequencing, were used to optimize the NGS strategy, whereas 19 DNA samples (discovery set) allowed the evaluation of NGS performance. In the training set, we identified 72 out of 73 expected mutations and 15 additional mutations: the molecular diagnosis was achieved in one patient with a previously wild-type status and the pre-excitation syndrome was explained in another. In the discovery set, we identified 20 mutations, 5 of which were in genes encoding non-TTm proteins, increasing the diagnostic yield by approximately 20%: a single mutation in genes encoding non-TTm proteins was identified in 2 out of 3 borderline HCM patients, whereas co-occuring mutations in genes encoding TTm and galactosidase alpha (GLA) altered proteins were characterized in a male with HCM and multiorgan dysfunction. Our combined targeted NGS-Sanger sequencing-based strategy allowed the molecular diagnosis of HCM with greater efficiency than using the conventional (Sanger) sequencing alone. Mutant alleles encoding non-TTm proteins may aid in the complete understanding of the genetic and phenotypic heterogeneity of HCM: co-occuring mutations of genes encoding TTm and non-TTm proteins could explain the wide variability of the HCM phenotype, whereas mutations in genes encoding only the non-TTm proteins are identifiable in patients with a milder HCM status. PMID:27600940

Molecular cloning of low-temperature-inducible ribosomal proteins from soybean.

PubMed

Kim, Kee-Young; Park, Seong-Whan; Chung, Young-Soo; Chung, Chung-Han; Kim, Jung-In; Lee, Jai-Heon

2004-05-01

Three ribosomal protein genes induced by low-temperature treatment were isolated from soybean. GmRPS13 (742 bp) encodes a 17.1 kDa protein which has 95% identity with the 40S ribosomal protein S13 of Panax ginseng (AB043974). GmRPS6 (925 bp) encodes a 28.1 kDa protein which has 94% identity with the 40S ribosomal protein S6 of Asparagus officinalis (AJ277533). GmRPL37 (494 bp) encodes a 10.7 kDa protein which has 85% identity with the 60S ribosomal protein L37 of Arabidopsis thaliana (AF370216). The expression of these ribosomal protein genes started to increase 3 d after low-temperature treatment, whereas the cold-stress protein src1 was highly induced from the first day. Such late response of these ribosomal protein genes may be due to secondary signals during cold adaptation. The induction of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under low-temperature conditions.

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

PubMed Central

2011-01-01

Background Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane domains of the baculovirus. We used this functionality to display human prorenin receptor fused with GFPuv (GFPuv-hPRR) on the surface of silkworm Bombyx mori nucleopolyhedrovirus (BmNPV) and then tested whether these baculovirus particles could be used to detect protein-protein interactions. Results BmNPV displaying GFPuv-hPRR (BmNPV-GFPuv-hPRR) was purified from hemolymph by using Sephacryl S-1000 column chromatography in the presence of 0.01% Triton X-100. Its recovery was 86% and the final baculovirus particles number was 4.98 × 108 pfu. Based on the results of enzyme-linked immunosorbent assay (ELISA), 3.1% of the total proteins in BmNPV-GFPuv-hPRR were GFPuv-hPRR. This value was similar to that calculated from the result of western blot by a densitometry (2.7%). To determine whether BmNPV-GFPuv-hPRR particles were bound to human prorenin, ELISA results were compared with those from ELISAs using protease negative BmNPV displaying β1,3-N-acetylglucosaminyltransferase 2 fused with the gene encoding GFPuv (GGT2) (BmNPV-CP--GGT2) particles, which do not display hPRR on their surfaces. Conclusion The display of on the surface of the BmNPV particles will be useful for the detection of protein-protein interactions and the screening of inhibitors and drugs in their roles as nanobioparticles. PMID:21635720

Protein-Anchoring Therapy of Biglycan for Mdx Mouse Model of Duchenne Muscular Dystrophy.

PubMed

Ito, Mikako; Ehara, Yuka; Li, Jin; Inada, Kosuke; Ohno, Kinji

2017-05-01

Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by loss-of-function mutations in DMD encoding dystrophin. No rational therapy is currently available. Utrophin is a paralog of dystrophin and is highly expressed at the neuromuscular junction. In mdx mice, utrophin is naturally upregulated throughout the muscle fibers, which mitigates muscular dystrophy. Protein-anchoring therapy was previously reported, in which a recombinant extracellular matrix (ECM) protein is delivered to and anchored to a specific target using its proprietary binding domains. Being prompted by a report that intramuscular and intraperitoneal injection of an ECM protein, biglycan, upregulates expression of utrophin and ameliorates muscle pathology in mdx mice, protein-anchoring therapy was applied to mdx mice. Recombinant adeno-associated virus serotype 8 (rAAV8) carrying hBGN encoding human biglycan was intravenously injected into 5-week-old mdx mice. The rAAV8-hBGN treatment improved motor deficits and decreased plasma creatine kinase activities. In muscle sections of treated mice, the number of central myonuclei and the distribution of myofiber sizes were improved. The treated mice increased gene expressions of utrophin and β1-syntrophin, as well as protein expressions of biglycan, utrophin, γ-sarcoglycan, dystrobrevin, and α1-syntrophin. The expression of hBGN in the skeletal muscle of the treated mice was 1.34-fold higher than that of the native mouse Bgn (mBgn). The low transduction efficiency and improved motor functions suggest that biglycan expressed in a small number of muscle fibers was likely to have been secreted and anchored to the cell surface throughout the whole muscular fibers. It is proposed that the protein-anchoring strategy can be applied not only to deficiency of an ECM protein as previously reported, but also to augmentation of a naturally induced ECM protein.

An orphan viral TNF receptor superfamily member identified in lymphocystis disease virus.

PubMed

Pontejo, Sergio M; Sánchez, Carolina; Martín, Rocío; Mulero, Victoriano; Alcami, Antonio; Alejo, Alí

2013-06-07

Lymphocystis disease virus (LCDV) is a large icosahedral dsDNA-containing virus of the Lymphocystivirus genus within the Iridoviridae family that can cause disease in more than 140 marine and freshwater fish species. While several isolates have been charcaterized and classified into distinct genotypes the complete genomic sequence is currently only available from two species, the LCDV-1, isolated from flounder (Platichtys flesus) in Europe and the LCDV-C, isolated from Japanese cultured flounder (Paralichthys olivaceus) in China. Analysis of the genome of LCDV-C showed it to encode a protein named LDVICp016 with similarities to the Tumour necrosis factor receptor (TNFR) superfamily with immunomodulatory potential. We have expressed and purified the recombinant protein LDVICp016 and screened for potential interaction partners using surface plasmon resonance. Commercially available human and mouse members of the TNF superfamily (TNFSF), along with a representative set of fish-derived TNFSF were tested.We have found the LDVICp016 protein to be secreted and we have identified a second viral TNFR encoded by ORF 095 of the same virus. None of the 42 tested proteins were found to interact with LDVICp016. We show that LDVICp016 is a secreted protein belonging to the TNF receptor family that may be part of a larger gene family in Lymphocystiviruses. While the ligand of this protein remains unknown, possibly due to the species specific nature of this interaction, further investigations into the potential role of this protein in the blockade of immune responses in its fish host are required.

An orphan viral TNF receptor superfamily member identified in lymphocystis disease virus

PubMed Central

2013-01-01

Background Lymphocystis disease virus (LCDV) is a large icosahedral dsDNA-containing virus of the Lymphocystivirus genus within the Iridoviridae family that can cause disease in more than 140 marine and freshwater fish species. While several isolates have been charcaterized and classified into distinct genotypes the complete genomic sequence is currently only available from two species, the LCDV-1, isolated from flounder (Platichtys flesus) in Europe and the LCDV-C, isolated from Japanese cultured flounder (Paralichthys olivaceus) in China. Analysis of the genome of LCDV-C showed it to encode a protein named LDVICp016 with similarities to the Tumour necrosis factor receptor (TNFR) superfamily with immunomodulatory potential. Findings We have expressed and purified the recombinant protein LDVICp016 and screened for potential interaction partners using surface plasmon resonance. Commercially available human and mouse members of the TNF superfamily (TNFSF), along with a representative set of fish-derived TNFSF were tested. We have found the LDVICp016 protein to be secreted and we have identified a second viral TNFR encoded by ORF 095 of the same virus. None of the 42 tested proteins were found to interact with LDVICp016. Conclusions We show that LDVICp016 is a secreted protein belonging to the TNF receptor family that may be part of a larger gene family in Lymphocystiviruses. While the ligand of this protein remains unknown, possibly due to the species specific nature of this interaction, further investigations into the potential role of this protein in the blockade of immune responses in its fish host are required. PMID:23758704

Staphylococcus aureus innate immune evasion is lineage-specific: a bioinfomatics study.

PubMed

McCarthy, Alex J; Lindsay, Jodi A

2013-10-01

Staphylococcus aureus is a major human pathogen, and is targeted by the host innate immune system. In response, S. aureus genomes encode dozens of secreted proteins that inhibit complement, chemotaxis and neutrophil activation resulting in successful evasion of innate immune responses. These proteins include immune evasion cluster proteins (IEC; Chp, Sak, Scn), staphylococcal superantigen-like proteins (SSLs), phenol soluble modulins (PSMs) and several leukocidins. Biochemical studies have indicated that genetic variants of these proteins can have unique functions. To ascertain the scale of genetic variation in secreted immune evasion proteins, whole genome sequences of 88 S. aureus isolates, representing 25 clonal complex (CC) lineages, in the public domain were analysed across 43 genes encoding 38 secreted innate immune evasion protein complexes. Twenty-three genes were variable, with between 2 and 15 variants, and the variants had lineage-specific distributions. They include genes encoding Eap, Ecb, Efb, Flipr/Flipr-like, Hla, Hld, Hlg, Sbi, Scin-B/C and 13 SSLs. Most of these protein complexes inhibit complement, chemotaxis and neutrophil activation suggesting that isolates from each S. aureus lineage respond to the innate immune system differently. In contrast, protein complexes that lyse neutrophils (LukSF-PVL, LukMF, LukED and PSMs) were highly conserved, but can be carried on mobile genetic elements (MGEs). MGEs also encode proteins with narrow host-specificities arguing that their acquisition has important roles in host/environmental adaptation. In conclusion, this data suggests that each lineage of S. aureus evades host immune responses differently, and that isolates can adapt to new host environments by acquiring MGEs and the immune evasion protein complexes that they encode. Cocktail therapeutics that targets multiple variant proteins may be the most appropriate strategy for controlling S. aureus infections. Copyright © 2013 Elsevier B.V. All rights reserved.

Mapping the hemagglutination domain of rotaviruses.

PubMed Central

Fuentes-Pananá, E M; López, S; Gorziglia, M; Arias, C F

1995-01-01

Most strains of animal rotaviruses are able to agglutinate erythrocytes, and the surface protein VP4 is the virus hemagglutinin. To map the hemagglutination domain on VP4 while preserving the conformation of the protein, we constructed full-length chimeras between the VP4 genes of hemagglutinating (YM) and nonhemagglutinating (KU) rotavirus strains. The parental and chimeric genes were expressed in insect cells, and the recombinant VP4 proteins were evaluated for their capacity to agglutinate human type O erythrocytes. Three chimeric genes, encoding amino acids 1 to 208 (QKU), 93 to 208 (QC), and 93 to 776 (QYM) of the YM VP4 protein in a KU VP4 background, were constructed. YM VP4 and chimeras QKU and QC were shown to specifically hemagglutinate, indicating that the region between amino acids 93 and 208 of YM VP4 is sufficient to determine the hemagglutination activity of the protein. PMID:7884915

Brain iron homeostasis, the choroid plexus, and localization of iron transport proteins.

PubMed

Rouault, Tracey A; Zhang, De-Liang; Jeong, Suh Young

2009-12-01

Maintenance of appropriate iron homeostasis in the brain is important, but the mechanisms involved in brain iron uptake are incompletely understood. Here, we have analyzed where messenger RNAs that encode iron transport proteins are expressed in the brain, using the Allen Brain atlas, and we conclude that several important iron transporters are highly expressed in the choroid plexus. Based on recent estimates of the surface area of the choroid plexus and on MRI imaging studies of manganese uptake in the brain, we propose that the choroid plexus may have a much greater role than has been previously appreciated in brain iron transport.

Ureaplasma diversum Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host

PubMed Central

Marques, Lucas M.; Rezende, Izadora S.; Barbosa, Maysa S.; Guimarães, Ana M. S.; Martins, Hellen B.; Campos, Guilherme B.; do Nascimento, Naíla C.; dos Santos, Andrea P.; Amorim, Aline T.; Santos, Verena M.; Farias, Sávio T.; Barrence, Fernanda Â. C.; de Souza, Lauro M.; Buzinhani, Melissa; Arana-Chavez, Victor E.; Zenteno, Maria E.; Amarante-Mendes, Gustavo P.; Messick, Joanne B.; Timenetsky, Jorge

2016-01-01

Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)—Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis. PMID:27603136

Ureaplasma diversum Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host.

PubMed

Marques, Lucas M; Rezende, Izadora S; Barbosa, Maysa S; Guimarães, Ana M S; Martins, Hellen B; Campos, Guilherme B; do Nascimento, Naíla C; Dos Santos, Andrea P; Amorim, Aline T; Santos, Verena M; Farias, Sávio T; Barrence, Fernanda  C; de Souza, Lauro M; Buzinhani, Melissa; Arana-Chavez, Victor E; Zenteno, Maria E; Amarante-Mendes, Gustavo P; Messick, Joanne B; Timenetsky, Jorge

2016-01-01

Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)-Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis.

Impact of Lactobacillus plantarum Sortase on Target Protein Sorting, Gastrointestinal Persistence, and Host Immune Response Modulation

PubMed Central

Remus, Daniela M.; Bongers, Roger S.; Meijerink, Marjolein; Fusetti, Fabrizia; Poolman, Bert; de Vos, Paul; Wells, Jerry M.; Bron, Peter A.

2013-01-01

Sortases are transpeptidases that couple surface proteins to the peptidoglycan of Gram-positive bacteria, and several sortase-dependent proteins (SDPs) have been demonstrated to be crucial for the interactions of pathogenic and nonpathogenic bacteria with their hosts. Here, we studied the role of sortase A (SrtA) in Lactobacillus plantarum WCFS1, a model Lactobacillus for probiotic organisms. An isogenic srtA deletion derivative was constructed which did not show residual SrtA activity. DNA microarray-based transcriptome analysis revealed that the srtA deletion had only minor impact on the full-genome transcriptome of L. plantarum, while the expression of SDP-encoding genes remained completely unaffected. Mass spectrometry analysis of the bacterial cell surface proteome, which was assessed by trypsinization of intact bacterial cells and by LiCl protein extraction, revealed that SrtA is required for the appropriate subcellular location of specific SDPs and for their covalent coupling to the cell envelope, respectively. We further found that SrtA deficiency did not affect the persistence and/or survival of L. plantarum in the gastrointestinal tract of mice. In addition, an in vitro immature dendritic cell (iDC) assay revealed that the removal of surface proteins by LiCl strongly affected the proinflammatory signaling properties of the SrtA-deficient strain but not of the wild type, which suggests a role of SDPs in host immune response modulation. PMID:23175652

Recombinant constructs of Borrelia burgdorferi

DOEpatents

Dattwyler, Raymond J.; Gomes-Solecki, Maria J. C.; Luft, Benjamin J.; Dunn, John J.

2007-02-20

Novel chimeric nucleic acids, encoding chimeric Borrelia proteins comprising OspC or an antigenic fragment thereof and OspA or an antigenic fragment thereof, are disclosed. Chimeric proteins encoded by the nucleic acid sequences are also disclosed. The chimeric proteins are useful as vaccine immunogens against Lyme borreliosis, as well as for immunodiagnostic reagents.

Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein.

PubMed

Yun, D J; Zhao, Y; Pardo, J M; Narasimhan, M L; Damsz, B; Lee, H; Abad, L R; D'Urzo, M P; Hasegawa, P M; Bressan, R A

1997-06-24

Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.

Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein

PubMed Central

Yun, Dae-Jin; Zhao, Yuan; Pardo, José M.; Narasimhan, Meena L.; Damsz, Barbara; Lee, Hyeseung; Abad, Laura R.; D’Urzo, Matilde Paino; Hasegawa, Paul M.; Bressan, Ray A.

1997-01-01

Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins. PMID:9192695

Recombinant HT{sub m4} gene, protein and assays

DOEpatents

Lim, B.; Adra, C.N.; Lelias, J.M.

1996-09-03

The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

Recombinant HT.sub.m4 gene, protein and assays

DOEpatents

Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

1996-01-01

The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

A member of a new plant gene family encoding a meprin and TRAF homology (MATH) domain-containing protein is involved in restriction of long distance movement of plant viruses

PubMed Central

Cosson, Patrick; Sofer, Luc; Schurdi-Levraud, Valérie

2010-01-01

Restriction of long distance movement of several potyviruses in Arabidopsis thaliana is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2 and RTM3 and acts as a non-conventional resistance. RTM1 encodes a protein belonging to the jacalin family and RTM2 encodes a protein which has similarities to small heat shock proteins. The recent cloning of RTM3 which encodes a protein belonging to an unknown protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its N-terminal region and a coiled-coil (CC) domain at its C-terminal end is an important breakthrough for a better understanding of this resistance process. Not only the third gene involved in this resistance has been identified and has allowed revealing a new gene family in plant but the discovery that the RTM3 protein interacts directly with RTM1 strongly suggests that the RTM proteins form a multimeric complex. However, these data also highlight striking similarities of the RTM resistance with the well known R-gene mediated resistance. PMID:20930558

Identification and Characterization of an Acinetobacter baumannii Biofilm-Associated Protein▿

PubMed Central

Loehfelm, Thomas W.; Luke, Nicole R.; Campagnari, Anthony A.

2008-01-01

We have identified a homologue to the staphylococcal biofilm-associated protein (Bap) in a bloodstream isolate of Acinetobacter baumannii. The fully sequenced open reading frame is 25,863 bp and encodes a protein with a predicted molecular mass of 854 kDa. Analysis of the nucleotide sequence reveals a repetitive structure consistent with bacterial cell surface adhesins. Bap-specific monoclonal antibody (MAb) 6E3 was generated to an epitope conserved among 41% of A. baumannii strains isolated during a recent outbreak in the U.S. military health care system. Flow cytometry confirms that the MAb 6E3 epitope is surface exposed. Random transposon mutagenesis was used to generate A. baumannii bap1302::EZ-Tn5, a mutant negative for surface reactivity to MAb 6E3 in which the transposon disrupts the coding sequence of bap. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrates that this mutant is unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. This is the first identification of a specific cell surface protein directly involved in biofilm formation by A. baumannii and suggests that Bap is involved in intercellular adhesion within the mature biofilm. PMID:18024522

Labeling proteins on live mammalian cells using click chemistry.

PubMed

Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

2015-05-01

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens.

PubMed

Alvarez, A M; Fukuhara, E; Nakase, M; Adachi, T; Aoki, N; Nakamura, R; Matsuda, T

1995-07-01

Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

RNA-guided transcriptional regulation

DOEpatents

Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

2016-02-23

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity

NASA Astrophysics Data System (ADS)

van Houdt, Rob; de Boever, Patrick; Coninx, Ilse; Janssen, Ann; Benotmane, Rafi; Leys, Natalie; Mergeay, Max

The stress response of bacterium Cupriavidus metallidurans CH34 into simulated microgravity R. Van Houdt, P. De Boever, I. Coninx, A. Janssen, M.A. Benotmane, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. We have studied the response of Cupriavidus (formerly Ralstonia) metallidurans CH34 to simulated microgravity by culturing in a Rotating Wall Vessel (RWV) bioreactor. This bioreactor technology generates a unique Low-Shear Modeled Microgravity (LSMMG) environment and is exploited as analogue for in vivo medical and space environments. Cupriavidus and Ralstonia species are relevant model bacteria since they are often isolated from the floor, air and surfaces of spacecraft assembly rooms and not only contaminate the clean rooms but have also been found prior-to-flight on surfaces of space robots such as the Mars Odyssey Orbiter and even in-flight in ISS cooling water and Shuttle drinking water. In addition, C. metallidurans CH34 is also being used in fundamental space flight experiments aimed to gain a better insight in the bacterial adaptation to space. The first objective was to elucidate the stress response of C. metallidurans CH34 grown in LSMMG compared to a normal gravity control. Transcriptomic analysis revealed that a significant part of the heat shock response was induced in LSMMG. Transcription of d naK, encoding the major heat-shock protein and a prokaryotic homologue of the eukaryotic Hsp70 protein, was induced 6.4 fold in LSMMG. DnaK is assisted by partner chaperones DnaJ and GrpE for which transcription respectively were induced 2.0 and 2.6 fold. Transcription of other chaperones known to belong to the heat shock response was also induced in LSMMG: hslV and hsl U, encoding the HslVU protease, were induced respectively 5.5 and 3.4 fold; htpG, encoding a Hsp90 family chaperone, was induced 4.6 fold and clpB was induced 4.7 fold. Transcription of the Lon protease was induced 2.5 fold. It appears that C. metallidurans CH34 experiences growth in Low-Shear Modelled Microgravity as a stressful condition eliciting the need to express the heat-shock proteins which assist protein folding, assembly, transport, repair and degradation. Challenging cells grown in simulated gravity (LSMMG) to a heat-shock for 30 min at 50° C resulted indeed in a smaller reduction (1.7 log) in cultivable cells compared to the reduction observed for cells grown in normal earth gravity (Low-Shear Gravity LSG) (4.0 log). Next to genes involved in the heat shock response, 5 of the 11 copies of uspA, encoding a widely conserved protein belonging to a superfamily whose physiological function is unknown but which is induced in response to a variety of stresses, were induced from 2.7 to 8.7 fold. In addition, LSMMG resulted in the upregulation of various genes encoding site-specific tyrosine recombinases, site-specific serine recombinase and transposases possibly indicating that Low-Shear Modeled Microgravity could elicit an adaptive response by genetic rearrangements. Finally, the parA and parB genes from pMOL30, one of the two plasmids carried by CH34 and specialized in heavy metals resistance, were strongly induced in LSMMG respectively 19.6 and 7.0 fold. The overproduction of similar proteins was also detected in C. metallidurans cells, cultured in during space flight.

Molecular cloning and nucleotide sequences of the genes for two essential proteins constituting a novel enzyme system for heptaprenyl diphosphate synthesis.

PubMed

Koike-Takeshita, A; Koyama, T; Obata, S; Ogura, K

1995-08-04

The genes encoding two dissociable components essential for Bacillus stearothermophilus heptaprenyl diphosphate synthase (all-trans-hexparenyl-diphosphate:isopentenyl-diphosphate hexaprenyl-trans-transferase, EC 2.5.1.30) were cloned, and their nucleotide sequences were determined. Sequence analyses revealed the presence of three open reading frames within 2,350 base pairs, designated as ORF-1, ORF-2, and ORF-3 in order of nucleotide sequence, which encode proteins of 220, 234, and 323 amino acids, respectively. Deletion experiments have shown that expression of the enzymatic activity requires the presence of ORF-1 and ORF-3, but ORF-2 is not essential. As a result, this enzyme was proved genetically to consist of two different protein compounds with molecular masses of 25 kDa (Component I) and 36 kDa (Component II), encoded by two of the three tandem genes. The protein encoded by ORF-1 has no similarity to any protein so far registered. However, the protein encoded by ORF-3 shows a 32% similarity to the farnesyl diphosphate synthase of the same bacterium and has seven highly conserved regions that have been shown typical in prenyltransferases (Koyama, T., Obata, S., Osabe, M., Takeshita, A., Yokoyama, K., Uchida, M., Nishino, T., and Ogura, K. (1993) J. Biochem. (Tokyo) 113, 355-363).

Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from Lactobacillus fermentum BR11 and Its Use as a Peptide Fusion Partner in Lactobacillus and Lactococcus

PubMed Central

Turner, Mark S.; Hafner, Louise M.; Walsh, Terry; Giffard, Philip M.

2004-01-01

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria. PMID:15184172

"Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions"

PubMed Central

2012-01-01

Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro. PMID:22463075

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones

PubMed Central

Glass, Jennifer B.; Kretz, Cecilia B.; Ganesh, Sangita; Ranjan, Piyush; Seston, Sherry L.; Buck, Kristen N.; Landing, William M.; Morton, Peter L.; Moffett, James W.; Giovannoni, Stephen J.; Vergin, Kevin L.; Stewart, Frank J.

2015-01-01

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO3−, NO2−, Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu. PMID:26441925

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones.

PubMed

Glass, Jennifer B; Kretz, Cecilia B; Ganesh, Sangita; Ranjan, Piyush; Seston, Sherry L; Buck, Kristen N; Landing, William M; Morton, Peter L; Moffett, James W; Giovannoni, Stephen J; Vergin, Kevin L; Stewart, Frank J

2015-01-01

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO[Formula: see text], NO[Formula: see text], Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu.

Detection of hepatitis B virus X product using an open reading frame Escherichia coli expression vector.

PubMed Central

Elfassi, E; Haseltine, W A; Dienstag, J L

1986-01-01

The genome of the hepatitis B virus (HBV) contains a sequence, designated X, capable of encoding a protein of 154 amino acids. To determine whether the putative protein synthesized from this region is antigenic, we examined the sera of HBV-infected patients for the ability to react with a hybrid protein that contained 133 amino acids encoded by the X region and portions of the bacterial ompF and beta-galactosidase genes. Some HBV-positive sera tested contained antibodies that specifically recognized the hybrid protein. All sera were from patients diagnosed as suffering from chronic active hepatitis. We conclude that the X region of HBV encodes a protein and that this protein is antigenic in some patients. Images PMID:3515347

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

PubMed Central

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

In-depth comparative analysis of malaria parasite genomes reveals protein-coding genes linked to human disease in Plasmodium falciparum genome.

PubMed

Liu, Xuewu; Wang, Yuanyuan; Liang, Jiao; Wang, Luojun; Qin, Na; Zhao, Ya; Zhao, Gang

2018-05-02

Plasmodium falciparum is the most virulent malaria parasite capable of parasitizing human erythrocytes. The identification of genes related to this capability can enhance our understanding of the molecular mechanisms underlying human malaria and lead to the development of new therapeutic strategies for malaria control. With the availability of several malaria parasite genome sequences, performing computational analysis is now a practical strategy to identify genes contributing to this disease. Here, we developed and used a virtual genome method to assign 33,314 genes from three human malaria parasites, namely, P. falciparum, P. knowlesi and P. vivax, and three rodent malaria parasites, namely, P. berghei, P. chabaudi and P. yoelii, to 4605 clusters. Each cluster consisted of genes whose protein sequences were significantly similar and was considered as a virtual gene. Comparing the enriched values of all clusters in human malaria parasites with those in rodent malaria parasites revealed 115 P. falciparum genes putatively responsible for parasitizing human erythrocytes. These genes are mainly located in the chromosome internal regions and participate in many biological processes, including membrane protein trafficking and thiamine biosynthesis. Meanwhile, 289 P. berghei genes were included in the rodent parasite-enriched clusters. Most are located in subtelomeric regions and encode erythrocyte surface proteins. Comparing cluster values in P. falciparum with those in P. vivax and P. knowlesi revealed 493 candidate genes linked to virulence. Some of them encode proteins present on the erythrocyte surface and participate in cytoadhesion, virulence factor trafficking, or erythrocyte invasion, but many genes with unknown function were also identified. Cerebral malaria is characterized by accumulation of infected erythrocytes at trophozoite stage in brain microvascular. To discover cerebral malaria-related genes, fast Fourier transformation (FFT) was introduced to extract genes highly transcribed at the trophozoite stage. Finally, 55 candidate genes were identified. Considering that parasite-infected erythrocyte surface protein 2 (PIESP2) contains gap-junction-related Neuromodulin_N domain and that anti-PIESP2 might provide protection against malaria, we chose PIESP2 for further experimental study. Our analysis revealed a limited number of genes linked to human disease in P. falciparum genome. These genes could be interesting targets for further functional characterization.

Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein.

PubMed

Abdul Halim, Mohd Farid; Karch, Kelly R; Zhou, Yitian; Haft, Daniel H; Garcia, Benjamin A; Pohlschroder, Mechthild

2015-12-28

For years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that the Haloferax volcanii SLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slg(G796F,F797G)). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ΔartA strain, the growth, cellular morphology, and cell wall of the slg(G796F,F797G) strain, in which modifications of additional H. volcanii ArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved. Prokaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeon H. volcanii as a model system, we report the first in vivo studies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood aspects of archaeal biology but also have important implications for key bacterial species, including those of the human microbiome. Additionally, insights may facilitate industrial applications, given that photosynthetic cyanobacteria encode uncharacterized homologs of this evolutionarily conserved enzyme, or may spur development of unique drug delivery systems. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

[Ph-Sensor Properties of a Fluorescent Protein from Dendronephthya sp].

PubMed

Pakhomov, A A; Chertkova, R V; Martynov, V I

2015-01-01

Genetically encoded biosensors based on fluorescent proteins are now widely applicable for monitoring pH changes in live cells. Here, we have shown that a fluorescent protein from Dendronephthya sp. (DendFP) exhibits a pronounced pH-sensitivity. Unlike most of known genetically encoded pH-sensors, fluorescence of the protein is not quenched upon medium acidification, but is shifting from the red to green spectral range. Therefore, quantitative measurements of intracellular pH are feasible by ratiometric comparison of emission intensities in the red and green spectral ranges, which makes DendFP advantageous compared with other genetically encoded pH-sensors.

Molecular cloning and characterization of alpha - galactosidase gene from Glaciozyma antarctica

NASA Astrophysics Data System (ADS)

Moheer, Reyad Qaed Al; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul

2015-09-01

Psychrophilic enzymes are proteins produced by psychrophilic organisms which recently are the limelight for industrial applications. A gene encoding α-galactosidase from a psychrophilic yeast, Glaciozyma antarctica PI12 which belongs to glycoside hydrolase family 27, was isolated and analyzed using several bioinformatic tools. The cDNA of the gene with the size of 1,404-bp encodes a protein with 467 amino acid residues. Predicted molecular weight of protein was 48.59 kDa and hence we name the gene encoding α-galactosidase as GAL48. We found that the predicted protein sequences possessed signal peptide sequence and are highly conserved among other fungal α-galactosidase.

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

PubMed

Crowdus, Carolyn A; Marsh, Antoinette E; Saville, Willliam J; Lindsay, David S; Dubey, J P; Granstrom, David E; Howe, Daniel K

2008-11-25

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

The genomic structure of the human Charcot-Leyden crystal protein gene is analogous to those of the galectin genes

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

Dyer, K.D.; Handen, J.S.; Rosenberg, H.F.

The Charcot-Leyden crystal (CLC) protein, or eosinophil lysophospholipase, is a characteristic protein of human eosinophils and basophils; recent work has demonstrated that the CLC protein is both structurally and functionally related to the galectin family of {beta}-galactoside binding proteins. The galectins as a group share a number of features in common, including a linear ligand binding site encoded on a single exon. In this work, we demonstrate that the intron-exon structure of the gene encoding CLC is analogous to those encoding the galectins. The coding sequence of the CLC gene is divided into four exons, with the entire {beta}-galactoside bindingmore » site encoded by exon III. We have isolated CLC {beta}-galactoside binding sites from both orangutan (Pongo pygmaeus) and murine (Mus musculus) genomic DNAs, both encoded on single exons, and noted conservation of the amino acids shown to interact directly with the {beta}-galactoside ligand. The most likely interpretation of these results suggests the occurrence of one or more exon duplication and insertion events, resulting in the distribution of this lectin domain to CLC as well as to the multiple galectin genes. 35 refs., 3 figs.« less

Identification of the cleavage sites of the RNA2-encoded polyproteins for two members of the genus Torradovirus by N-terminal sequencing of the virion capsid proteins.

PubMed

Ferriol, I; Silva Junior, D M; Nigg, J C; Zamora-Macorra, E J; Falk, B W

2016-11-01

Torradoviruses, family Secoviridae, are emergent bipartite RNA plant viruses. RNA1 is ca. 7kb and has one open reading frame (ORF) encoding for the protease, helicase and RNA-dependent RNA polymerase (RdRp). RNA2 is ca. 5kb and has two ORFs. RNA2-ORF1 encodes for a putative protein with unknown function(s). RNA2-ORF2 encodes for a putative movement protein and three capsid proteins. Little is known about the replication and polyprotein processing strategies of torradoviruses. Here, the cleavage sites in the RNA2-ORF2-encoded polyproteins of two torradoviruses, Tomato marchitez virus isolate M (ToMarV-M) and tomato chocolate spot virus, were determined by N-terminal sequencing, revealing that the amino acid (aa) at the -1 position of the cleavage sites is a glutamine. Multiple aa sequence comparison confirmed that this glutamine is conserved among other torradoviruses. Finally, site-directed mutagenesis of conserved aas in the ToMarV-M RdRp and protease prevented substantial accumulation of viral coat proteins or RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA-encoded libraries - an efficient small molecule discovery technology for the biomedical sciences.

PubMed

Kunig, Verena; Potowski, Marco; Gohla, Anne; Brunschweiger, Andreas

2018-06-27

DNA-encoded compound libraries are a highly attractive technology for the discovery of small molecule protein ligands. These compound collections consist of small molecules covalently connected to individual DNA sequences carrying readable information about the compound structure. DNA-tagging allows for efficient synthesis, handling and interrogation of vast numbers of chemically synthesized, drug-like compounds. They are screened on proteins by an efficient, generic assay based on Darwinian principles of selection. To date, selection of DNA-encoded libraries allowed for the identification of numerous bioactive compounds. Some of these compounds uncovered hitherto unknown allosteric binding sites on target proteins; several compounds proved their value as chemical biology probes unraveling complex biology; and the first examples of clinical candidates that trace their ancestry to a DNA-encoded library were reported. Thus, DNA-encoded libraries proved their value for the biomedical sciences as a generic technology for the identification of bioactive drug-like molecules numerous times. However, large scale experiments showed that even the selection of billions of compounds failed to deliver bioactive compounds for the majority of proteins in an unbiased panel of target proteins. This raises the question of compound library design.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

PubMed Central

Hubert, Rene S.; Vivanco, Igor; Chen, Emily; Rastegar, Shiva; Leong, Kahan; Mitchell, Steve C.; Madraswala, Rashida; Zhou, Yanhong; Kuo, James; Raitano, Arthur B.; Jakobovits, Aya; Saffran, Douglas C.; Afar, Daniel E. H.

1999-01-01

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging. PMID:10588738

Multi-Scale Surface Descriptors

PubMed Central

Cipriano, Gregory; Phillips, George N.; Gleicher, Michael

2010-01-01

Local shape descriptors compactly characterize regions of a surface, and have been applied to tasks in visualization, shape matching, and analysis. Classically, curvature has be used as a shape descriptor; however, this differential property characterizes only an infinitesimal neighborhood. In this paper, we provide shape descriptors for surface meshes designed to be multi-scale, that is, capable of characterizing regions of varying size. These descriptors capture statistically the shape of a neighborhood around a central point by fitting a quadratic surface. They therefore mimic differential curvature, are efficient to compute, and encode anisotropy. We show how simple variants of mesh operations can be used to compute the descriptors without resorting to expensive parameterizations, and additionally provide a statistical approximation for reduced computational cost. We show how these descriptors apply to a number of uses in visualization, analysis, and matching of surfaces, particularly to tasks in protein surface analysis. PMID:19834190

A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea.

PubMed Central

Reith, M; Munholland, J

1993-01-01

Extensive DNA sequencing of the chloroplast genome of the red alga Porphyra purpurea has resulted in the detection of more than 125 genes. Fifty-eight (approximately 46%) of these genes are not found on the chloroplast genomes of land plants. These include genes encoding 17 photosynthetic proteins, three tRNAs, and nine ribosomal proteins. In addition, nine genes encoding proteins related to biosynthetic functions, six genes encoding proteins involved in gene expression, and at least five genes encoding miscellaneous proteins are among those not known to be located on land plant chloroplast genomes. The increased coding capacity of the P. purpurea chloroplast genome, along with other characteristics such as the absence of introns and the conservation of ancestral operons, demonstrate the primitive nature of the P. purpurea chloroplast genome. In addition, evidence for a monophyletic origin of chloroplasts is suggested by the identification of two groups of genes that are clustered in chloroplast genomes but not in cyanobacteria. PMID:12271072

Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

Treesearch

Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

2003-01-01

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans.

PubMed

Ahn, Sang-Joon; Burne, Robert A

2007-09-01

The Streptococcus mutans atlA gene encodes an autolysin required for biofilm maturation and biogenesis of a normal cell surface. We found that the capacity to form biofilms by S. mutans, one of the principal causative agents of dental caries, was dramatically impaired by growth of the organism in an aerated environment and that cells exposed to oxygen displayed marked changes in surface protein profiles. Inactivation of the atlA gene alleviated repression of biofilm formation in the presence of oxygen. Also, the formation of long chains, a characteristic of AtlA-deficient strains, was less evident in cells grown with aeration. The SMu0629 gene is immediately upstream of atlA and encodes a product that contains a C-X-X-C motif, a characteristic of thiol-disulfide oxidoreductases. Inactivation of SMu0629 significantly reduced the levels of AtlA protein and led to resistance to autolysis. The SMu0629 mutant also displayed an enhanced capacity to form biofilms in the presence of oxygen compared to that of the parental strain. The expression of SMu0629 was shown to be under the control of the VicRK two-component system, which influences oxidative stress tolerance in S. mutans. Disruption of vicK also led to inhibition of processing of AtlA, and the mutant was hyperresistant to autolysis. When grown under aerobic conditions, the vicK mutant also showed significantly increased biofilm formation compared to strain UA159. This study illustrates the central role of AtlA and VicK in orchestrating growth on surfaces and envelope biogenesis in response to redox conditions.

Genomics Analysis of Genes Expressed in Maize Endosperm Identifies Novel Seed Proteins and Clarifies Patterns of Zein Gene Expression

PubMed Central

Woo, Young-Min; Hu, David Wang-Nan; Larkins, Brian A.; Jung, Rudolf

2001-01-01

We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although α-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the γ- and δ-zeins, and members of these gene families, especially the γ-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the α-, γ-, and δ-zein gene families, which provides evidence that γ-zeins are synthesized throughout the endosperm before α- and δ-zeins. This observation is consistent with earlier studies that suggested that γ-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD γ-zein, an 18-kD α-globulin, and a legumin-related protein. Immunolocalization of the 50-kD γ-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other γ-zeins. The 18-kD α-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm. PMID:11595803

Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate.

PubMed

Horne, Irene; Sutherland, Tara D; Harcourt, Rebecca L; Russell, Robyn J; Oakeshott, John G

2002-07-01

We isolated a bacterial strain, Agrobacterium radiobacter P230, which can hydrolyze a wide range of organophosphate (OP) insecticides. A gene encoding a protein involved in OP hydrolysis was cloned from A. radiobacter P230 and sequenced. This gene (called opdA) had sequence similarity to opd, a gene previously shown to encode an OP-hydrolyzing enzyme in Flavobacterium sp. strain ATCC 27551 and Brevundimonas diminuta MG. Insertional mutation of the opdA gene produced a strain lacking the ability to hydrolyze OPs, suggesting that this is the only gene encoding an OP-hydrolyzing enzyme in A. radiobacter P230. The OPH and OpdA proteins, encoded by opd and opdA, respectively, were overexpressed and purified as maltose-binding proteins, and the maltose-binding protein moiety was cleaved and removed. Neither protein was able to hydrolyze the aliphatic OP malathion. The kinetics of the two proteins for diethyl OPs were comparable. For dimethyl OPs, OpdA had a higher k(cat) than OPH. It was also capable of hydrolyzing the dimethyl OPs phosmet and fenthion, which were not hydrolyzed at detectable levels by OPH.

Nucleic acid encoding DS-CAM proteins and products related thereto

DOEpatents

Korenberg, Julie R.

2005-11-01

In accordance with the present invention, there are provided Down Syndrome-Cell Adhesion Molecule (DS-CAM) proteins. Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention DS-CAM proteins can be employed in a variety of ways, for example, for the production of anti-DS-CAM antibodies thereto, in therapeutic compositions and methods employing such proteins and/or antibodies. DS-CAM proteins are also useful in bioassays to identify agonists and antagonists thereto.

Design and evaluation of protein expression in a recombinant plasmid encoding epitope gp 350/220 of the Epstein-Barr virus (EBV)

NASA Astrophysics Data System (ADS)

Himmah, Karimatul; Dluha, Nurul; Anyndita, Nadya V. M.; Rifa'i, Muhaimin; Widodo

2017-05-01

The Epstein - Barr virus (EBV) causes severe infections that may lead to cancers such as nasopharyngeal carcinoma. Development of effective EBV vaccines is necessary to prevent the virus spreading throughout the community. TheEBV has a surface protein gp 350/220, which serves as an antigen to help interact with host cells. Epitopes of the protein can potentially serve as bases for a vaccine. In a previous study, we have found a conserved epitope of gp 350/220 from all strains EBV through an in silico approach. The aim of this study is to design and overproduce a recombinant peptide of epitope gp 350/220 in E. coli. DNA encoding the conserved epitope was synthesized and cloned into plasmid pET-22b(+); the recombinant plasmid was transformed into E. coli strains DH5α and BL21. The transformed plasmid DNA was isolated and confirmed by restriction using XbaI and PstI enzymes followed by DNA sequencing. Protein expression was induced by isopropyl-D-thiogalactopyranoside (IPTG) with final concentrations of 0.1, 0.2, 1, and 2 mM in consecutive times. An osmotic shock method was used to isolate protein from periplasmic fraction of E. coli DH5α and BL21. The SDS-PAGE analysis was carried out to detect peptide target (3.4 kDa). Based on this result, the induction process did not work properly, and thus needs further investigation.

Enhancing T cell activation and antiviral protection by introducing the HIV-1 protein transduction domain into a DNA vaccine.

PubMed

Leifert, J A; Lindencrona, J A; Charo, J; Whitton, J L

2001-10-10

Protein transduction domains (PTD), which can transport proteins or peptides across biological membranes, have been identified in several proteins of viral, invertebrate, and vertebrate origin. Here, we evaluate the immunological and biological consequences of including PTD in synthetic peptides and in DNA vaccines that contain CD8(+) T cell epitopes from lymphocytic choriomeningitis virus (LCMV). Synthetic PTD-peptides did not induce detectable CD8(+) T cell responses. However, fusion of an open reading frame encoding a PTD to an epitope minigene caused transfected tissue culture cells to stimulate epitope-specific T cells much more effectively. Kinetic studies indicated that the epitope reached the surface of transfected cells more rapidly and that the number of transfected cells needed to stimulate T cell responses was reduced by 35- to 50-fold when compared to cells transfected with a standard minigene plasmid. The mechanism underlying the effect of PTD linkage is not clear, but transit of the PTD-attached epitope from transfected cells to nontransfected cells (cross presentation) seemed to play, at most, a minimal role. Mice immunized once with the plasmid encoding the PTD-linked epitope showed a markedly accelerated CD8(+) T cell response and, unlike mice immunized with a standard plasmid, were completely protected against a normally lethal LCMV challenge administered only 8 days post-immunization.

cDNA, deduced polypeptide structure and chromosomal assignment of human pulmonary surfactant proteolipid, SPL(pVal)

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

Glasser, S.W.; Korfhagen, T.R.; Weaver, T.E.

1988-01-05

In hyaline membrane disease of premature infants, lack of surfactant leads to pulmonary atelectasis and respiratory distress. Hydrophobic surfactant proteins of M/sub r/ = 5000-14,000 have been isolated from mammalian surfactants which enhance the rate of spreading and the surface tension lowering properties of phospholipids during dynamic compression. The authors have characterized the amino-terminal amino acid sequence of pulmonary proteolipids from ether/ethanol extracts of bovine, canine, and human surfactant. Two distinct peptides were identified and termed SPL(pVal) and SPL(Phe). An oligonucleotide probe based on the valine-rich amino-terminal amino acid sequence of SPL(pVal) was utilized to isolate cDNA and genomic DNAmore » encoding the human protein, termed surfactant proteolipid SPL(pVal) on the basis of its unique polyvaline domain. The primary structure of a precursor protein of 20,870 daltons, containing the SPL(pVal) peptide, was deduced from the nucleotide sequence of the cDNAs. Hybrid-arrested translation and immunoprecipitation of labeled translation products of human mRNA demonstrated a precursor protein, the active hydrophobic peptide being produced by proteolytic processing. Two classes of cDNAs encoding SPL(pVal) were identified. Human SPL(pVal) mRNA was more abundant in the adult than in fetal lung. The SPL(pVal) gene locus was assigned to chromosome 8.« less

Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

PubMed

Liao, Ting-Yu Angela; Lau, Alice; Joseph, Sunil; Hytönen, Vesa; Hmama, Zakaria

2015-01-01

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Improving genetic immobilization of a cellulase on yeast cell surface for bioethanol production using cellulose.

PubMed

Yang, Jinying; Dang, Hongyue; Lu, Jian Ren

2013-04-01

In this study, Saccharomyces cerevisiae was genetically engineered to harbor the capability of utilizing celluloses for bioethanol production by displaying active cellulolytic enzymes on the cell surface. An endo-1,4-β-glucanase gene egX was cloned from Bacillus pumilus C-9 and its expression products, the EGX cellulases, were displayed on the cell surface of S. cerevisiae by fusing egX with aga2 that encodes the binding subunit of the S. cerevisiae cell wall protein α-agglutinin. To achieve high gene copies and stability, multicopy integration was obtained by integrating the fusion aga2-egX gene into the rDNA region of the S. cerevisiae chromosome. To achieve high expression and surface display efficiency, the aga2-egX gene was expressed under the control of a strong promoter. The presence of the enzymatically active cellulase fusion proteins on the S. cerevisiae cell surface was verified by carboxymethyl cellulase activity assay and immunofluorescence microscopy. This work presented a promising strategy to genetically engineer yeasts to perform efficient fermentation of cellulosic materials for bioethanol production. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

PubMed Central

McPhaul, M; Berg, P

1986-01-01

The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes. Images PMID:3466162

yadBC of Yersinia pestis, a new virulence determinant for bubonic plague.

PubMed

Forman, Stanislav; Wulff, Christine R; Myers-Morales, Tanya; Cowan, Clarissa; Perry, Robert D; Straley, Susan C

2008-02-01

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.

Gene encoding the group B streptococcal protein R4, its presence in clinical reference laboratory isolates & R4 protein pepsin sensitivity.

PubMed

Smith, B L; Flores, A; Dechaine, J; Krepela, J; Bergdall, A; Ferrieri, P

2004-05-01

R proteins were first identified by Lancefield in group B Streptococcus (GBS) as resistant to trypsin at pH8 and sensitive to pepsin at pH2. The R4 protein found predominantly in type III and some type II and V invasive isolates conforms to these criteria. The Rib protein, although structurally and epidemiologically similar to R4, was reported as resistant to both proteases. We report here the gene encoding the R4 protein from a type III group B streptococcal isolate (76-043) well characterized in our laboratory. Trypsin extracted GBS proteins were assayed for protease sensitivities by double-diffusion Ouchterlony using varying conditions for the enzyme pepsin. Standard haemoglobin assay was used to examine pepsin enzymatic activity. Thirty clinical isolates of varying protein profiles identified by double-diffusion from our reference strain laboratory were screened by PCR and Southern technique. SDS-PAGE gel purified R4 amino acid sequences were determined and used to design oligonucleotide primers for screening a 76-043 genomic library. R4 was sensitive to pepsin at pH2 but appeared resistant at pH4, the reported pH used for Rib. By standard haemoglobin assay and trypsin extract studies of R4 protein, pepsin was shown to be active at pH2, yet easily inactivated; assays of GBS surface proteins are critical at pH2. Of the amino acids initially sequenced from R4, 88 per cent (61/69) showed identity to Rib; the r4 nucleotide sequence was identical to that of rib. All isolates with strong positive protein reactions for R4 were positive in both PCR and Southern technique, whereas isolates expressing alpha, beta, R1/R4, and R5 (BPS) protein profiles were not. Sequenced PCR products aligned with identity to the R4 and Rib nucleotide sequences and confirmed the identity of these proteins and their molecular sequences.

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

PubMed Central

Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

1992-01-01

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197. Images PMID:1447140

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2014-05-27

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2014-10-28

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2015-04-14

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Methods of increasing secretion of polypeptides having biological activity

DOEpatents

Merino, Sandra

2013-10-01

The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

Characterization of two new putative adhesins of Leptospira interrogans.

PubMed

Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

2017-01-01

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.

PubMed

Ernst, Antonia M; Jekat, Stephan B; Zielonka, Sascia; Müller, Boje; Neumann, Ulla; Rüping, Boris; Twyman, Richard M; Krzyzanek, Vladislav; Prüfer, Dirk; Noll, Gundula A

2012-07-10

The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.

DNA sequence analysis of a 10 624 bp fragment of the left arm of chromosome XV from Saccharomyces cerevisiae reveals a RNA binding protein, a mitochondrial protein, two ribosomal proteins and two new open reading frames.

PubMed

Lafuente, M J; Gamo, F J; Gancedo, C

1996-09-01

We have determined the sequence of a 10624 bp DNA segment located in the left arm of chromosome XV of Saccharomyces cerevisiae. The sequence contains eight open reading frames (ORFs) longer than 100 amino acids. Two of them do not present significant homology with sequences found in the databases. The product of ORF o0553 is identical to the protein encoded by the gene SMF1. Internal to it there is another ORF, o0555 that is apparently expressed. The proteins encoded by ORFs o0559 and o0565 are identical to ribosomal proteins S19.e and L18 respectively. ORF o0550 encodes a protein with an RNA binding signature including RNP motifs and stretches rich in asparagine, glutamine and arginine.

A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein.

PubMed

Stayton, M M; Black, M; Bedbrook, J; Dunsmuir, P

1986-12-22

The 16 petunia Cab genes which have been characterized are all closely related at the nucleotide sequence level and they encode Cab precursor polypeptides which are similar in sequence and length. Here we describe a novel petunia Cab gene which encodes a unique Cab precursor protein. This protein is a member of the smallest class of Cab precursor proteins for which no gene has previously been assigned in petunia or any other species. The features of this Cab precursor protein are that it is shorter by 2-3 amino acids than the formerly characterized Cab precursors, its transit peptide sequence is unrelated, and the mature polypeptide is significantly diverged at the functionally important N terminus from other petunia Cab proteins. Gene structure also discriminates this gene which is the only intron containing Cab gene in petunia genomic DNA.

The Bean Pod Mottle Virus RNA2-Encoded 58-Kilodalton Protein P58 Is Required in cis for RNA2 Accumulation

PubMed Central

Lin, Junyan; Guo, Jiangbo; Finer, John; Dorrance, Anne E.; Redinbaugh, Margaret G.

2014-01-01

ABSTRACT Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58-kDa protein (P58) encoded by RNA2 has not been resolved. P58 and the movement protein (MP) of BPMV are two largely identical proteins differing only at their N termini, with P58 extending MP upstream by 102 amino acid residues. In this report, we unveil a unique role for P58. We show that BPMV RNA2 accumulation in infected cells was abolished when the start codon of P58 was eliminated. The role of P58 does not require the region shared by MP, as RNA2 accumulation in individual cells remained robust even when most of the MP coding sequence was removed. Importantly, the function of P58 required the P58 protein, rather than its coding RNA, as compensatory mutants could be isolated that restored RNA2 accumulation by acquiring new start codons upstream of the original one. Most strikingly, loss of P58 function could not be complemented by P58 provided in trans, suggesting that P58 functions in cis to selectively promote the accumulation of RNA2 copies that encode a functional P58 protein. Finally, we found that all RNA1-encoded proteins are cis-acting relative to RNA1. Together, our results suggest that P58 probably functions by recruiting the RNA1-encoded polyprotein to RNA2 to enable RNA2 reproduction. IMPORTANCE Bean pod mottle virus (BPMV) is one of the most important pathogens of the crop plant soybean, yet its replication mechanism is not well understood, hindering the development of knowledge-based control measures. The current study examined the replication strategy of BPMV RNA2, one of the two genomic RNA segments of this virus, and established an essential role for P58, one of the RNA2-encoded proteins, in the process of RNA2 replication. Our study demonstrates for the first time that P58 functions preferentially with the very RNA from which it is translated, thus greatly advancing our understanding of the replication mechanisms of this and related viruses. Furthermore, this study is important because it provides a potential target for BPMV-specific control, and hence could help to mitigate soybean production losses caused by this virus. PMID:24390330

HIV Nef-mediated cellular phenotypes are differentially expressed as a function of intracellular Nef concentrations.

PubMed

Liu, X; Schrager, J A; Lange, G D; Marsh, J W

2001-08-31

Nef is a regulatory protein encoded by the genome of both human and simian immunodeficiency virus. Its expression in T cells leads to CD4 and major histocompatibility complex class I modulation and either enhancement or suppression of T cell activation. How this viral protein achieves multiple and at times opposing activities has been unclear. Through direct measurements of Nef and the Nef-GFP fusion protein, we find that these events are mediated by different Nef concentrations. Relative to the intracellular concentration that down-modulates surface CD4, an order of magnitude increase in Nef-GFP expression is required for a comparable modulation of major histocompatibility complex class I, and a further 3-fold increase is necessary to suppress T cell activation.

A chromosomally encoded virulence factor protects the Lyme disease pathogen against host-adaptive immunity.

PubMed

Yang, Xiuli; Coleman, Adam S; Anguita, Juan; Pal, Utpal

2009-03-01

Borrelia burgdorferi, the bacterial pathogen of Lyme borreliosis, differentially expresses select genes in vivo, likely contributing to microbial persistence and disease. Expression analysis of spirochete genes encoding potential membrane proteins showed that surface-located membrane protein 1 (lmp1) transcripts were expressed at high levels in the infected murine heart, especially during early stages of infection. Mice and humans with diagnosed Lyme borreliosis also developed antibodies against Lmp1. Deletion of lmp1 severely impaired the pathogen's ability to persist in diverse murine tissues including the heart, and to induce disease, which was restored upon chromosomal complementation of the mutant with the lmp1 gene. Lmp1 performs an immune-related rather than a metabolic function, as its deletion did not affect microbial persistence in immunodeficient mice, but significantly decreased spirochete resistance to the borreliacidal effects of anti-B. burgdorferi sera in a complement-independent manner. These data demonstrate the existence of a virulence factor that helps the pathogen evade host-acquired immune defense and establish persistent infection in mammals.

Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring.

PubMed

He, Yong-Zhi; Fan, Ke-Qiang; Jia, Cui-Juan; Wang, Zhi-Jun; Pan, Wu-Bin; Huang, Li; Yang, Ke-Qian; Dong, Zhi-Yang

2007-05-01

A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe-SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80 degrees C and retained 50% activity after heat treatment at 95 degrees C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe(2+) was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.

The atlA operon of Streptococcus mutans: role in autolysin maturation and cell surface biogenesis.

PubMed

Ahn, Sang-Joon; Burne, Robert A

2006-10-01

The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlA-deficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

PubMed

Sattler, Ursula; Khosravi, Mojtaba; Avila, Mislay; Pilo, Paola; Langedijk, Johannes P; Ader-Ebert, Nadine; Alves, Lisa A; Plattet, Philippe; Origgi, Francesco C

2014-07-01

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretin receptor superfamily with an unusual extracellular domain

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

Hamann, J.; Hamann, D.; Lier, R.A.W.

1995-08-15

CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includesmore » the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).« less

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei; Ecker, Joseph R.

2010-02-02

The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding a F-box protein, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding a F-box protein.

Antibodies specific for HT.sub.m4

DOEpatents

Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

1998-01-01

The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

Biochemical basis for the biological clock

NASA Technical Reports Server (NTRS)

Morre, D. James; Chueh, Pin-Ju; Pletcher, Jake; Tang, Xiaoyu; Wu, Lian-Ying; Morre, Dorothy M.

2002-01-01

NADH oxidases at the external surface of plant and animal cells (ECTO-NOX proteins) exhibit stable and recurring patterns of oscillations with potentially clock-related, entrainable, and temperature-compensated period lengths of 24 min. To determine if ECTO-NOX proteins might represent the ultradian time keepers (pacemakers) of the biological clock, COS cells were transfected with cDNAs encoding tNOX proteins having a period length of 22 min or with C575A or C558A cysteine to alanine replacements having period lengths of 36 or 42 min. Here we demonstrate that such transfectants exhibited 22, 36, or 40 to 42 h circadian patterns in the activity of glyceraldehyde-3-phosphate dehydrogenase, a common clock-regulated protein, in addition to the endogenous 24 h circadian period length. The fact that the expression of a single oscillatory ECTO-NOX protein determines the period length of a circadian biochemical marker (60 X the ECTO-NOX period length) provides compelling evidence that ECTO-NOX proteins are the biochemical ultradian drivers of the cellular biological clock.

Chimeric mitochondrial peptides from contiguous regular and swinger RNA.

PubMed

Seligmann, Hervé

2016-01-01

Previous mass spectrometry analyses described human mitochondrial peptides entirely translated from swinger RNAs, RNAs where polymerization systematically exchanged nucleotides. Exchanges follow one among 23 bijective transformation rules, nine symmetric exchanges (X ↔ Y, e.g. A ↔ C) and fourteen asymmetric exchanges (X → Y → Z → X, e.g. A → C → G → A), multiplying by 24 DNA's protein coding potential. Abrupt switches from regular to swinger polymerization produce chimeric RNAs. Here, human mitochondrial proteomic analyses assuming abrupt switches between regular and swinger transcriptions, detect chimeric peptides, encoded by part regular, part swinger RNA. Contiguous regular- and swinger-encoded residues within single peptides are stronger evidence for translation of swinger RNA than previously detected, entirely swinger-encoded peptides: regular parts are positive controls matched with contiguous swinger parts, increasing confidence in results. Chimeric peptides are 200 × rarer than swinger peptides (3/100,000 versus 6/1000). Among 186 peptides with > 8 residues for each regular and swinger parts, regular parts of eleven chimeric peptides correspond to six among the thirteen recognized, mitochondrial protein-coding genes. Chimeric peptides matching partly regular proteins are rarer and less expressed than chimeric peptides matching non-coding sequences, suggesting targeted degradation of misfolded proteins. Present results strengthen hypotheses that the short mitogenome encodes far more proteins than hitherto assumed. Entirely swinger-encoded proteins could exist.

Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes.

PubMed

Hanski, E; Caparon, M

1992-07-01

Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

A protein-dependent side-chain rotamer library.

PubMed

Bhuyan, Md Shariful Islam; Gao, Xin

2011-12-14

Protein side-chain packing problem has remained one of the key open problems in bioinformatics. The three main components of protein side-chain prediction methods are a rotamer library, an energy function and a search algorithm. Rotamer libraries summarize the existing knowledge of the experimentally determined structures quantitatively. Depending on how much contextual information is encoded, there are backbone-independent rotamer libraries and backbone-dependent rotamer libraries. Backbone-independent libraries only encode sequential information, whereas backbone-dependent libraries encode both sequential and locally structural information. However, side-chain conformations are determined by spatially local information, rather than sequentially local information. Since in the side-chain prediction problem, the backbone structure is given, spatially local information should ideally be encoded into the rotamer libraries. In this paper, we propose a new type of backbone-dependent rotamer library, which encodes structural information of all the spatially neighboring residues. We call it protein-dependent rotamer libraries. Given any rotamer library and a protein backbone structure, we first model the protein structure as a Markov random field. Then the marginal distributions are estimated by the inference algorithms, without doing global optimization or search. The rotamers from the given library are then re-ranked and associated with the updated probabilities. Experimental results demonstrate that the proposed protein-dependent libraries significantly outperform the widely used backbone-dependent libraries in terms of the side-chain prediction accuracy and the rotamer ranking ability. Furthermore, without global optimization/search, the side-chain prediction power of the protein-dependent library is still comparable to the global-search-based side-chain prediction methods.

The mouse neuronal cell surface protein F3: a phosphatidylinositol- anchored member of the immunoglobulin superfamily related to chicken contactin

PubMed Central

1989-01-01

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent- insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development. PMID:2474555

The Lp_3561 and Lp_3562 Enzymes Support a Functional Divergence Process in the Lipase/Esterase Toolkit from Lactobacillus plantarum

PubMed Central

Esteban-Torres, María; Reverón, Inés; Santamaría, Laura; Mancheño, José M.; de las Rivas, Blanca; Muñoz, Rosario

2016-01-01

Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions. PMID:27486450

Adaptive antioxidant methionine accumulation in respiratory chain complexes explains the use of a deviant genetic code in mitochondria.

PubMed

Bender, Aline; Hajieva, Parvana; Moosmann, Bernd

2008-10-28

Humans and most other animals use 2 different genetic codes to translate their hereditary information: the standard code for nuclear-encoded proteins and a modern variant of this code in mitochondria. Despite the pivotal role of the genetic code for cell biology, the functional significance of the deviant mitochondrial code has remained enigmatic since its first description in 1979. Here, we show that profound and functionally beneficial alterations on the encoded protein level were causative for the AUA codon reassignment from isoleucine to methionine observed in most mitochondrial lineages. We demonstrate that this codon reassignment leads to a massive accumulation of the easily oxidized amino acid methionine in the highly oxidative inner mitochondrial membrane. This apparently paradoxical outcome can yet be smoothly settled if the antioxidant surface chemistry of methionine is taken into account, and we present direct experimental evidence that intramembrane accumulation of methionine exhibits antioxidant and cytoprotective properties in living cells. Our results unveil that methionine is an evolutionarily selected antioxidant building block of respiratory chain complexes. Collective protein alterations can thus constitute the selective advantage behind codon reassignments, which authenticates the "ambiguous decoding" hypothesis of genetic code evolution. Oxidative stress has shaped the mitochondrial genetic code.

Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen.

PubMed

Domingos, Renan F; Fernandes, Luis G; Romero, Eliete C; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2015-04-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions. © 2015 The Authors.

Small molecules targeting LapB protein prevent Listeria attachment to catfish muscle

PubMed Central

Das, Bhaskar; Lawrence, Mark

2017-01-01

Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listeriosis. L. monocytogenes lapB gene encodes a cell wall surface anchor protein, and mutation of this gene causes Listeria attenuation in mice. In this work, the potential role of Listeria LapB protein in catfish fillet attachment was investigated. To achieve this, boron-based small molecules designed to interfere with the active site of the L. monocytogenes LapB protein were developed, and their ability to prevent L. monocytogenes attachment to fish fillet was tested. Results indicated that seven out of nine different small molecules were effective in reducing the Listeria attachment to catfish fillets. Of these, three small molecules (SM3, SM5, and SM7) were highly effective in blocking Listeria attachment to catfish fillets. This study suggests an alternative strategy for reduction of L. monocytogenes contamination in fresh and frozen fish products. PMID:29253892

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Designing pH induced fold switch in proteins

NASA Astrophysics Data System (ADS)

Baruah, Anupaul; Biswas, Parbati

2015-05-01

This work investigates the computational design of a pH induced protein fold switch based on a self-consistent mean-field approach by identifying the ensemble averaged characteristics of sequences that encode a fold switch. The primary challenge to balance the alternative sets of interactions present in both target structures is overcome by simultaneously optimizing two foldability criteria corresponding to two target structures. The change in pH is modeled by altering the residual charge on the amino acids. The energy landscape of the fold switch protein is found to be double funneled. The fold switch sequences stabilize the interactions of the sites with similar relative surface accessibility in both target structures. Fold switch sequences have low sequence complexity and hence lower sequence entropy. The pH induced fold switch is mediated by attractive electrostatic interactions rather than hydrophobic-hydrophobic contacts. This study may provide valuable insights to the design of fold switch proteins.

Cloning, sequencing and expression in MEL cells of a cDNA encoding the mouse ribosomal protein S5.

PubMed

Vanegas, N; Castañeda, V; Santamaría, D; Hernández, P; Schvartzman, J B; Krimer, D B

1997-06-05

We describe the isolation and characterization of a cDNA encoding the mouse S5 ribosomal protein. It was isolated from a MEL (murine erythroleukemia) cell cDNA library by differential hybridization as a down regulated sequence during HMBA-induced differentiation. Northern series analysis showed that S5 mRNA expression is reduced 5-fold throughout the differentiation process. The mouse S5 mRNA is 760 bp long and encodes for a 204 amino acid protein with 94% homology with the human and rat S5.

Hypomorphic mutations in PGAP2, encoding a GPI-anchor-remodeling protein, cause autosomal-recessive intellectual disability.

PubMed

Hansen, Lars; Tawamie, Hasan; Murakami, Yoshiko; Mang, Yuan; ur Rehman, Shoaib; Buchert, Rebecca; Schaffer, Stefanie; Muhammad, Safia; Bak, Mads; Nöthen, Markus M; Bennett, Eric P; Maeda, Yusuke; Aigner, Michael; Reis, André; Kinoshita, Taroh; Tommerup, Niels; Baig, Shahid Mahmood; Abou Jamra, Rami

2013-04-04

PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous families. Rescue experiments with the altered proteins in PGAP2-deficient Chinese hamster ovary cell lines showed less expression of cell-surface GPI-anchored proteins DAF and CD59 than of the wild-type protein, substantiating the pathogenicity of the identified alterations. Furthermore, we observed a full rescue when we used strong promoters before the mutant cDNAs, suggesting a hypomorphic effect of the mutations. We report on alterations in the Golgi-located part of the GPI-anchor-biosynthesis pathway and extend the phenotypic spectrum of the GPI-anchor deficiencies to isolated intellectual disability with elevated ALP. GPI-anchor deficiencies can be interpreted within the concept of a disease family, and we propose that the severity of the phenotype is dependent on the location of the altered protein in the biosynthesis chain. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

An OmpA family protein, a target of the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius, controls acetic acid fermentation.

PubMed

Iida, Aya; Ohnishi, Yasuo; Horinouchi, Sueharu

2008-07-01

Via N-acylhomoserine lactones, the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius NCI1051, a gram-negative acetic acid bacterium, represses acetic acid and gluconic acid fermentation. Two-dimensional polyacrylamide gel electrophoretic analysis of protein profiles of strain NCI1051 and ginI and ginR mutants identified a protein that was produced in response to the GinI/GinR regulatory system. Cloning and nucleotide sequencing of the gene encoding this protein revealed that it encoded an OmpA family protein, named GmpA. gmpA was a member of the gene cluster containing three adjacent homologous genes, gmpA to gmpC, the organization of which appeared to be unique to vinegar producers, including "Gluconacetobacter polyoxogenes." In addition, GmpA was unique among the OmpA family proteins in that its N-terminal membrane domain forming eight antiparallel transmembrane beta-strands contained an extra sequence in one of the surface-exposed loops. Transcriptional analysis showed that only gmpA of the three adjacent gmp genes was activated by the GinI/GinR quorum-sensing system. However, gmpA was not controlled directly by GinR but was controlled by an 89-amino-acid protein, GinA, a target of this quorum-sensing system. A gmpA mutant grew more rapidly in the presence of 2% (vol/vol) ethanol and accumulated acetic acid and gluconic acid in greater final yields than strain NCI1051. Thus, GmpA plays a role in repressing oxidative fermentation, including acetic acid fermentation, which is unique to acetic acid bacteria and allows ATP synthesis via ethanol oxidation. Consistent with the involvement of gmpA in oxidative fermentation, its transcription was also enhanced by ethanol and acetic acid.

Characterization of Isolates of Streptococcus agalactiae from Diseased Farmed and Wild Marine Fish from the U.S. Gulf Coast, Latin America, and Thailand.

PubMed

Soto, Esteban; Wang, Rui; Wiles, Judy; Baumgartner, Wes; Green, Christopher; Plumb, John; Hawke, John

2015-06-01

We examined Lancefield serogroup B Streptococcus isolates recovered from diseased, cultured hybrid Striped Bass (Striped Bass Morone saxatilis × White Bass M. chrysops) and wild and cultured Gulf Killifish Fundulus grandis from coastal waters of the U.S. Gulf of Mexico (Gulf coast) and compared those isolates to strains from tilapias Oreochromis spp. reared in Mississippi, Thailand, Ecuador, and Honduras and to the original Gulf coast strain identified by Plumb et al. ( 1974 ). The isolates were subjected to phylogenetic, biochemical, and antibiotic susceptibility analyses. Genetic analysis was performed using partial sequence comparison of (1) the 16S ribosomal RNA (rRNA) gene; (2) the sipA gene, which encodes a surface immunogenic protein; (3) the cspA gene, which encodes a cell surface-associated protein; and (4) the secY gene, which encodes components of a general protein secretion pathway. Phylogenies inferred from sipA, secY, and cspA gene sequence comparisons were more discriminating than that inferred from the 16S rRNA gene sequence comparison. The U.S. Gulf coast strains showed a high degree of similarity to strains from South America and Central America and belonged to a unique group that can be distinguished from other group B streptococci. In agreement with the molecular findings, biochemical and antimicrobial resistance analyses demonstrated that the isolates recovered from the U.S. Gulf coast and Latin America were more similar to each other than to isolates from Thailand. Three laboratory challenge methods for inducing streptococcosis in Gulf Killifish were evaluated-intraperitoneal (IP) injection, immersion (IMM), and immersion plus abrasion (IMMA)-using serial dilutions of S. agalactiae isolate LADL 97-151, a representative U.S. Gulf coast strain. The dose that was lethal to 50% of test fish by 14 d postchallenge was approximately 2 CFU/fish via IP injection. In contrast, the fish that were challenged via IMM or IMMA presented cumulative mortality less than 40% by 14 d postchallenge.

Appearance of the bona fide spiral tubule of ORF virus is dependent on an intact 10-kilodalton viral protein.

PubMed

Spehner, D; De Carlo, S; Drillien, R; Weiland, F; Mildner, K; Hanau, D; Rziha, H-J

2004-08-01

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.

Research Results

NASA Astrophysics Data System (ADS)

2011-12-01

Research on Global Carbon Emission and Sequestration NSFC Funded Project Made Significant Progress in Quantum Dynamics Functional Human Blood Protein Obtained from Rice How Giant Pandas Thrive on a Bamboo Diet New Evidence of Interpersonal Violence from 129,000 Years Ago Found in China Aptamer-Mediated Efficient Capture and Release of T Lymphocytes on Nanostructured Surfaces BGI Study Results on Resequencing 50 Accessions of Rice Cast New Light on Molecular Breeding BGI Reports Study Results on Frequent Mutation of Genes Encoding UMPP Components in Kidney Cancer Research on Habitat Shift Promoting Species Diversification

The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein.

PubMed

Bleiziffer, Isabelle; Eikmeier, Julian; Pohlentz, Gottfried; McAulay, Kathryn; Xia, Guoqing; Hussain, Muzaffar; Peschel, Andreas; Foster, Simon; Peters, Georg; Heilmann, Christine

2017-01-01

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff's staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl residues can stimulate biofilm formation. Thus, sugar modifications may represent promising new targets for novel therapeutic or prophylactic measures against life-threatening S. aureus infections.

Apolipoprotein A-I mutant proteins having cysteine substitutions and polynucleotides encoding same

DOEpatents

Oda, Michael N [Benicia, CA; Forte, Trudy M [Berkeley, CA

2007-05-29

Functional Apolipoprotein A-I mutant proteins, having one or more cysteine substitutions and polynucleotides encoding same, can be used to modulate paraoxonase's arylesterase activity. These ApoA-I mutant proteins can be used as therapeutic agents to combat cardiovascular disease, atherosclerosis, acute phase response and other inflammatory related diseases. The invention also includes modifications and optimizations of the ApoA-I nucleotide sequence for purposes of increasing protein expression and optimization.

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

A lanthipeptide library used to identify a protein-protein interaction inhibitor.

PubMed

Yang, Xiao; Lennard, Katherine R; He, Chang; Walker, Mark C; Ball, Andrew T; Doigneaux, Cyrielle; Tavassoli, Ali; van der Donk, Wilfred A

2018-04-01

In this article we describe the production and screening of a genetically encoded library of 10 6 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses

PubMed Central

Johnson, Stephen M.; Eltahla, Auda A.; Aloi, Maria; Aloia, Amanda L.; McDevitt, Christopher A.; Bull, Rowena A.

2017-01-01

ABSTRACT Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3′ untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as “vesicle packets” (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools. PMID:28956770

Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

PubMed Central

Tyagi, Kriti; Hossain, Mohammad Enayet; Thakur, Vandana; Aggarwal, Praveen; Malhotra, Pawan; Mohmmed, Asif; Sharma, Yagya Dutta

2016-01-01

Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle. PMID:26954579

Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

PubMed Central

Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

2013-01-01

Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

Antibodies specific for HT{sub m4}

DOEpatents

Lim, B.; Adra, C.N.; Lelias, J.M.

1998-01-06

The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

Antibody specific for a DNA repair protein

DOEpatents

Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

2006-07-11

An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

NASA Technical Reports Server (NTRS)

Chueh, Pin-Ju; Kim, Chinpal; Cho, NaMi; Morre, Dorothy M.; Morre, D. James

2002-01-01

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus

PubMed Central

Selle, Kurt; Goh, Yong J.; Johnson, Brant R.; O’Flaherty, Sarah; Andersen, Joakim M.; Barrangou, Rodolphe; Klaenhammer, Todd R.

2017-01-01

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall. PMID:28443071

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus.

PubMed

Selle, Kurt; Goh, Yong J; Johnson, Brant R; O'Flaherty, Sarah; Andersen, Joakim M; Barrangou, Rodolphe; Klaenhammer, Todd R

2017-01-01

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus . Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus , suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N-Acetyltransferase from Trypanosoma brucei ▿ ‡ §

PubMed Central

Mariño, Karina; Güther, M. Lucia Sampaio; Wernimont, Amy K.; Qiu, Wei; Hui, Raymond; Ferguson, Michael A. J.

2011-01-01

A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed. PMID:21531872

Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the beta'-subunit of RNA polymerase.

PubMed Central

Cozens, A L; Walker, J E

1986-01-01

The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome. It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex. The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes. It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E. coli RNA polymerase and to yeast RNA polymerases II and III. PMID:3530249

WHITE STRIPE LEAF4 Encodes a Novel P-Type PPR Protein Required for Chloroplast Biogenesis during Early Leaf Development

PubMed Central

Wang, Ying; Ren, Yulong; Zhou, Kunneng; Liu, Linglong; Wang, Jiulin; Xu, Yang; Zhang, Huan; Zhang, Long; Feng, Zhiming; Wang, Liwei; Ma, Weiwei; Wang, Yunlong; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Cheng, Zhijun; Wan, Jianmin

2017-01-01

Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and perform diverse functions in organellar RNA metabolism. Despite the rice genome encodes 477 PRR proteins, the regulatory effects of PRR proteins on chloroplast development remains unknown. In this study, we report the functional characterization of the rice white stripe leaf4 (wsl4) mutant. The wsl4 mutant develops white-striped leaves during early leaf development, characterized by decreased chlorophyll content and malformed chloroplasts. Positional cloning of the WSL4 gene, together with complementation and RNA-interference tests, reveal that it encodes a novel P-family PPR protein with 12 PPR motifs, and is localized to chloroplast nucleoids. Quantitative RT-PCR analyses demonstrate that WSL4 is a low temperature response gene abundantly expressed in young leaves. Further expression analyses show that many nuclear- and plastid-encoded genes in the wsl4 mutant are significantly affected at the RNA and protein levels. Notably, the wsl4 mutant causes defects in the splicing of atpF, ndhA, rpl2, and rps12. Our findings identify WSL4 as a novel P-family PPR protein essential for chloroplast RNA group II intron splicing during early leaf development in rice. PMID:28694820

Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

DOEpatents

Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

2007-03-13

The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein.

PubMed

Miranda-Ozuna, Jesús F T; Hernández-García, Mar S; Brieba, Luis G; Benítez-Cardoza, Claudia G; Ortega-López, Jaime; González-Robles, Arturo; Arroyo, Rossana

2016-10-01

Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Plasmids encoding therapeutic agents

DOEpatents

Keener, William K [Idaho Falls, ID

2007-08-07

Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

PubMed

Adewoye, L O; Worobec, E A

2000-08-08

The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

Methods to alter levels of a DNA repair protein

DOEpatents

Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

2006-10-17

An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

Nucleic Acid Encoding A Lectin-Derived Progenitor Cell Preservation Factor

DOEpatents

Colucci, M. Gabriella; Chrispeels, Maarten J.; Moore, Jeffrey G.

2001-10-30

The invention relates to an isolated nucleic acid molecule that encodes a protein that is effective to preserve progenitor cells, such as hematopoietic progenitor cells. The nucleic acid comprises a sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof. The encoded protein has an amino acid sequence that comprises a sequence defined by SEQ ID NO:2, a homolog thereof, or a fragment thereof that contains an amino acid sequence TNNVLQVT. Methods of using the encoded protein for preserving progenitor cells in vitro, ex vivo, and in vivo are also described. The invention, therefore, include methods such as myeloablation therapies for cancer treatment wherein myeloid reconstitution is facilitated by means of the specified protein. Other therapeutic utilities are also enabled through the invention, for example, expanding progenitor cell populations ex vivo to increase chances of engraftation, improving conditions for transporting and storing progenitor cells, and facilitating gene therapy to treat and cure a broad range of life-threatening hematologic diseases.

Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.

PubMed

Morozov, Sergey Y; Milyutina, Irina A; Bobrova, Vera K; Ryazantsev, Dmitry Y; Erokhina, Tatiana N; Zavriev, Sergey K; Agranovsky, Alexey A; Solovyev, Andrey G; Troitsky, Alexey V

2015-12-01

The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

A method for partitioning the information contained in a protein sequence between its structure and function.

PubMed

Possenti, Andrea; Vendruscolo, Michele; Camilloni, Carlo; Tiana, Guido

2018-05-23

Proteins employ the information stored in the genetic code and translated into their sequences to carry out well-defined functions in the cellular environment. The possibility to encode for such functions is controlled by the balance between the amount of information supplied by the sequence and that left after that the protein has folded into its structure. We study the amount of information necessary to specify the protein structure, providing an estimate that keeps into account the thermodynamic properties of protein folding. We thus show that the information remaining in the protein sequence after encoding for its structure (the 'information gap') is very close to what needed to encode for its function and interactions. Then, by predicting the information gap directly from the protein sequence, we show that it may be possible to use these insights from information theory to discriminate between ordered and disordered proteins, to identify unknown functions, and to optimize artificially-designed protein sequences. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Heterogeneous genetic diversity pattern in Plasmodium vivax genes encoding merozoite surface proteins (MSP) -7E, -7F and -7L.

PubMed

Garzón-Ospina, Diego; Forero-Rodríguez, Johanna; Patarroyo, Manuel A

2014-12-13

The msp-7 gene has become differentially expanded in the Plasmodium genus; Plasmodium vivax has the highest copy number of this gene, several of which encode antigenic proteins in merozoites. DNA sequences from thirty-six Colombian clinical isolates from P. vivax (pv) msp-7E, -7F and -7L genes were analysed for characterizing and studying the genetic diversity of these pvmsp-7 members which are expressed during the intra-erythrocyte stage; natural selection signals producing the variation pattern so observed were evaluated. The pvmsp-7E gene was highly polymorphic compared to pvmsp-7F and pvmsp-7L which were seen to have limited genetic diversity; pvmsp-7E polymorphism was seen to have been maintained by different types of positive selection. Even though these copies seemed to be species-specific duplications, a search in the Plasmodium cynomolgi genome (P. vivax sister taxon) showed that both species shared the whole msp-7 repertoire. This led to exploring the long-term effect of natural selection by comparing the orthologous sequences which led to finding signatures for lineage-specific positive selection. The results confirmed that the P. vivax msp-7 family has a heterogeneous genetic diversity pattern; some members are highly conserved whilst others are highly diverse. The results suggested that the 3'-end of these genes encode MSP-7 proteins' functional region whilst the central region of pvmsp-7E has evolved rapidly. The lineage-specific positive selection signals found suggested that mutations occurring in msp-7s genes during host switch may have succeeded in adapting the ancestral P. vivax parasite population to humans.

Epstein-Barr virus/complement fragment C3d receptor (CR2) reacts with p53, a cellular antioncogene-encoded membrane phosphoprotein: detection by polyclonal anti-idiotypic anti-CR2 antibodies.

PubMed Central

Barel, M; Fiandino, A; Lyamani, F; Frade, R

1989-01-01

Epstein-Barr virus and the C3d fragment of the third component of complement are specific extracellular ligands for complement receptor type 2 (CR2). However, intracellular proteins that react specifically with CR2 and are involved in post-membrane signals remain unknown. We recently prepared polyclonal anti-idiotypic anti-CR2 antibodies (Ab2) by using the highly purified CR2 molecule as original immunogen. We showed that Ab2 contained anti-idiotypic specificities that mimicked extracellular domains of CR2 and detected two distinct binding sites on CR2 for its specific extracellular ligands, Epstein-Barr virus and C3d. We postulated that Ab2 might also contain specificities that could mimic intracellular domains of CR2. Here we report that Ab2, which did not react with Raji B-lymphoma cell surface components, detected specifically, among all components solubilized from Raji cell membranes, a single intracellular membrane protein of apparent molecular mass of 53 kDa. This protein was identified as the p53 cellular antioncogene-encoded membrane phosphoprotein by analyzing its antigenic properties with Pab1801, a monoclonal anti-p53 antibody, and by comparing its biochemical properties with those of p53. Additionally, solubilized and purified CR2 bound to solubilized p53 immobilized on Pab1801-Sepharose. p53, like CR2, was localized only in purified plasma membranes and nuclei of Raji cells. These data suggest strongly that p53, a cellular antioncogene-encoded phosphoprotein, reacted specifically with CR2 in Raji membranes. This interaction may represent one of the important steps through which CR2 could be involved in human B-lymphocyte proliferation and transformation. Images PMID:2557614

Multiple Functional Domains of Enterococcus faecalis Aggregation Substance Asc10 Contribute to Endocarditis Virulence ▿ †

PubMed Central

Chuang, Olivia N.; Schlievert, Patrick M.; Wells, Carol L.; Manias, Dawn A.; Tripp, Timothy J.; Dunny, Gary M.

2009-01-01

Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo. PMID:18955479

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

PubMed Central

López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M. Isabel G.

2013-01-01

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

Characterization of the cellulosomal scaffolding protein CbpC from Clostridium cellulovorans 743B.

PubMed

Nakajima, Daichi; Shibata, Toshiyuki; Tanaka, Reiji; Kuroda, Kouichi; Ueda, Mitsuyoshi; Miyake, Hideo

2017-10-01

Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 μM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

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

Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

1995-04-10

Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, althoughmore » its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.« less

Tunable geometry of bacterial inclusion bodies as substrate materials for tissue engineering

NASA Astrophysics Data System (ADS)

García-Fruitós, Elena; Seras-Franzoso, Joaquín; Vazquez, Esther; Villaverde, Antonio

2010-05-01

A spectrum of materials for biomedical applications is produced in bacteria, and some of them, such as metals or polyhydroxyalkanoates, are straightforwardly obtained as particulate entities. We have explored the biofabrication process of bacterial inclusion bodies, particulate proteinaceous materials (ranging from 50 to 500 nm in diameter) recently recognized as suitable for surface topographical modification and tissue engineering. Inclusion bodies have been widely described as spherical or pseudo-spherical particles with only minor morphological variability, mostly restricted to their size. Here we have identified a cellular gene in Escherichia coli (clpP) that controls the in vivo fabrication process of inclusion bodies. In the absence of the encoded protease, the dynamics of protein deposition is perturbed, resulting in unusual tear-shaped particles with enhanced surface-volume ratios. This fact modifies the ability of inclusion bodies to promote mammalian cell attachment and differentiation upon surface decoration. The implications of the genetic control of inclusion body geometry are discussed in the context of their biological fabrication and regarding the biomedical potential of these protein clusters in regenerative medicine.

Glucose-Induced Trophic Shift in an Endosymbiont Dinoflagellate with Physiological and Molecular Consequences1[OPEN

PubMed Central

Jinkerson, Robert E.; Clowez, Sophie; Onishi, Masayuki; Cleves, Phillip A.; Pringle, John R.

2018-01-01

Interactions between the dinoflagellate endosymbiont Symbiodinium and its cnidarian hosts (e.g. corals, sea anemones) are the foundation of coral-reef ecosystems. Carbon flow between the partners is a hallmark of this mutualism, but the mechanisms governing this flow and its impact on symbiosis remain poorly understood. We showed previously that although Symbiodinium strain SSB01 can grow photoautotrophically, it can grow mixotrophically or heterotrophically when supplied with Glc, a metabolite normally transferred from the alga to its host. Here we show that Glc supplementation of SSB01 cultures causes a loss of pigmentation and photosynthetic activity, disorganization of thylakoid membranes, accumulation of lipid bodies, and alterations of cell-surface morphology. We used global transcriptome analyses to determine if these physiological changes were correlated with changes in gene expression. Glc-supplemented cells exhibited a marked reduction in levels of plastid transcripts encoding photosynthetic proteins, although most nuclear-encoded transcripts (including those for proteins involved in lipid synthesis and formation of the extracellular matrix) exhibited little change in their abundances. However, the altered carbon metabolism in Glc-supplemented cells was correlated with modest alterations (approximately 2x) in the levels of some nuclear-encoded transcripts for sugar transporters. Finally, Glc-bleached SSB01 cells appeared unable to efficiently populate anemone larvae. Together, these results suggest links between energy metabolism and cellular physiology, morphology, and symbiotic interactions. However, the results also show that in contrast to many other organisms, Symbiodinium can undergo dramatic physiological changes that are not reflected by major changes in the abundances of nuclear-encoded transcripts and thus presumably reflect posttranscriptional regulatory processes. PMID:29217594

Mitis group streptococci express variable pilus islet 2 pili.

PubMed

Zähner, Dorothea; Gandhi, Ashish R; Yi, Hong; Stephens, David S

2011-01-01

Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguinis are members of the Mitis group of streptococci and agents of oral biofilm, dental plaque and infective endocarditis, disease processes that involve bacteria-bacteria and bacteria-host interactions. Their close relative, the human pathogen S. pneumoniae uses pilus-islet 2 (PI-2)-encoded pili to facilitate adhesion to eukaryotic cells. PI-2 pilus-encoding genetic islets were identified in S. oralis, S. mitis, and S. sanguinis, but were absent from other isolates of these species. The PI-2 islets resembled the genetic organization of the PI-2 islet of S. pneumoniae, but differed in the genes encoding the structural pilus proteins PitA and PitB. Two and three variants of pitA (a pseudogene in S. pneumoniae) and pitB, respectively, were identified that showed ≈20% difference in nucleotide as well as corresponding protein sequence. Species-independent combinations of pitA and pitB variants indicated prior intra- and interspecies horizontal gene transfer events. Polyclonal antisera developed against PitA and PitB of S. oralis type strain ATCC35037 revealed that PI-2 pili in oral streptococci were composed of PitA and PitB. Electronmicrographs showed pilus structures radiating >700 nm from the bacterial surface in the wild type strain, but not in an isogenic PI-2 deletion mutant. Anti-PitB-antiserum only reacted with pili containing the same PitB variant, whereas anti-PitA antiserum was cross-reactive with the other PitA variant. Electronic multilocus sequence analysis revealed that all PI-2-encoding oral streptococci were closely-related and cluster with non-PI-2-encoding S. oralis strains. This is the first identification of PI-2 pili in Mitis group oral streptococci. The findings provide a striking example of intra- and interspecies horizontal gene transfer. The PI-2 pilus diversity provides a possible key to link strain-specific bacterial interactions and/or tissue tropisms with pathogenic traits in the Mitis group streptococci.

Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch Migrase

PubMed Central

Dresser, Ashley R.; Hardy, Pierre-Olivier; Chaconas, George

2009-01-01

Persistent infection by pathogenic organisms requires effective strategies for the defense of these organisms against the host immune response. A common strategy employed by many pathogens to escape immune recognition and clearance is to continually vary surface epitopes through recombinational shuffling of genetic information. Borrelia burgdorferi, a causative agent of Lyme borreliosis, encodes a surface-bound lipoprotein, VlsE. This protein is encoded by the vlsE locus carried at the right end of the linear plasmid lp28-1. Adjacent to the expression locus are 15 silent cassettes carrying information that is moved into the vlsE locus through segmental gene conversion events. The protein players and molecular mechanism of recombinational switching at vlsE have not been characterized. In this study, we analyzed the effect of the independent disruption of 17 genes that encode factors involved in DNA recombination, repair or replication on recombinational switching at the vlsE locus during murine infection. In Neisseria gonorrhoeae, 10 such genes have been implicated in recombinational switching at the pilE locus. Eight of these genes, including recA, are either absent from B. burgdorferi, or do not show an obvious requirement for switching at vlsE. The only genes that are required in both organisms are ruvA and ruvB, which encode subunits of a Holliday junction branch migrase. Disruption of these genes results in a dramatic decrease in vlsE recombination with a phenotype similar to that observed for lp28-1 or vls-minus spirochetes: productive infection at week 1 with clearance by day 21. In SCID mice, the persistence defect observed with ruvA and ruvB mutants was fully rescued as previously observed for vlsE-deficient B. burgdorferi. We report the requirement of the RuvAB branch migrase in recombinational switching at vlsE, the first essential factor to be identified in this process. These findings are supported by the independent work of Lin et al. in the accompanying article, who also found a requirement for the RuvAB branch migrase. Our results also indicate that the mechanism of switching at vlsE in B. burgdorferi is distinct from switching at pilE in N. gonorrhoeae, which is the only other organism analyzed genetically in detail. Finally, our findings suggest a unique mechanism for switching at vlsE and a role for currently unidentified B. burgdorferi proteins in this process. PMID:19997508

Photo-Induced Click Chemistry for DNA Surface Structuring by Direct Laser Writing.

PubMed

Kerbs, Antonina; Mueller, Patrick; Kaupp, Michael; Ahmed, Ishtiaq; Quick, Alexander S; Abt, Doris; Wegener, Martin; Niemeyer, Christof M; Barner-Kowollik, Christopher; Fruk, Ljiljana

2017-04-11

Oligonucleotides containing photo-caged dienes were prepared and shown to react quantitatively in a light-induced Diels-Alder cycloaddition with functional maleimides in aqueous solution within minutes. Due to its high yield and fast rate, the reaction was exploited for DNA surface patterning with sub-micrometer resolution employing direct laser writing (DLW). Functional DNA arrays were written by direct laser writing (DLW) in variable patterns, which were further encoded with fluorophores and proteins through DNA directed immobilization. This mild and efficient light-driven platform technology holds promise for the fabrication of complex bioarrays with sub-micron resolution. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases

USDA-ARS?s Scientific Manuscript database

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Sugar beet (Beta vulgaris L.) PGIP genes, BvPGIP1, BvPGIP2 and BvPGIP3, were isolated from two breeding lines, F1016 and F1010. Full-length cDNA sequences of the three BvPGIP genes encod...

HT.sub.m4 methods of treatment and assays, agonists and antagonists

DOEpatents

Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

1999-01-01

The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

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

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

Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong

2016-01-08

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively boundmore » the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.« less

Subversion of cytokine networks by virally encoded decoy receptors

PubMed Central

Epperson, Megan L.; Lee, Chung A.; Fremont, Daved H.

2012-01-01

Summary During the course of evolution, viruses have captured or created a diverse array of open reading frames that encode for proteins that serve to evade and sabotage the host innate and adaptive immune responses, which would otherwise lead to their elimination. These viral genomes are some of the best textbooks of immunology ever written. The established arsenal of immunomodulatory proteins encoded by viruses is large and growing and includes specificities for virtually all known inflammatory pathways and targets. The focus of this review is on herpes and poxvirus-encoded cytokine and chemokine binding proteins that serve to undermine the coordination of host immune surveillance. Structural and mechanistic studies of these decoy receptors have provided a wealth of information, not only about viral pathogenesis but also about the inner workings of cytokine signaling networks. PMID:23046131

Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications

PubMed Central

Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grażyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

2012-01-01

The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application. PMID:23305359

Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

PubMed

Ito, Mikako; Ohno, Kinji

2018-02-20

Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression of utrophin and DAPC component proteins. We propose that protein-anchoring therapy could be applied to hereditary/acquired defects in ECM and secreted proteins, as well as therapeutic overexpression of such factors. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms

PubMed Central

Rose, Sasha J.

2016-01-01

ABSTRACT Extracellular DNA (eDNA) is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM). Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae. The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate. PMID:27923918

MARS: A protein family involved in the formation of vertical skeletal elements.

PubMed

Abehsera, Shai; Peles, Shani; Tynyakov, Jenny; Bentov, Shmuel; Aflalo, Eliahu D; Li, Shihao; Li, Fuhua; Xiang, Jianhai; Sagi, Amir

2017-05-01

Vertical organizations of skeletal elements are found in various vertebrate teeth and invertebrate exoskeletons. The molecular mechanism behind the development of such structural organizations is poorly known, although it is generally held that organic matrix proteins play an essential role. While most crustacean cuticular organizations exhibit horizontal chitinous layering, a typical vertical organization is found towards the surface of the teeth in the mandibles of the crayfish Cherax quadricarinatus. Candidate genes encoding for mandible-forming structural proteins were mined in C. quadricarinatus molt-related transcriptomic libraries by using a binary patterning approach. A new protein family, termed the Mandible Alanine Rich Structural (MARS) protein family, with a modular sequence design predicted to form fibers, was found. Investigations of spatial and temporal expression of the different MARS genes suggested specific expression in the mandibular teeth-forming epithelium, particularly during the formation of the chitinous vertical organization. MARS loss-of-function RNAi experiments resulted in the collapse of the organization of the chitin fibers oriented vertically to the surface of the crayfish mandibular incisor tooth. A general search of transcriptomic libraries suggested conservation of MARS proteins across a wide array of crustaceans. Our results provide a first look into the molecular mechanism used to build the complex crustacean mandible and into the specialized vertical structural solution that has evolved in skeletal elements. Copyright © 2017 Elsevier Inc. All rights reserved.

Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage.

PubMed

Guan, Su Hua; Belsham, Graham J

2017-04-01

Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

Many Saccharomyces cerevisiae Cell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent.

PubMed

Bester, Michael C; Jacobson, Dan; Bauer, Florian F

2012-01-01

The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.

High-level expression of a synthetic gene encoding a sweet protein, monellin, in Escherichia coli.

PubMed

Chen, Zhongjun; Cai, Heng; Lu, Fuping; Du, Lianxiang

2005-11-01

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE.

Whole Genome Sequences of Three Treponema pallidum ssp. pertenue Strains: Yaws and Syphilis Treponemes Differ in Less than 0.2% of the Genome Sequence

PubMed Central

Chen, Lei; Pospíšilová, Petra; Strouhal, Michal; Qin, Xiang; Mikalová, Lenka; Norris, Steven J.; Muzny, Donna M.; Gibbs, Richard A.; Fulton, Lucinda L.; Sodergren, Erica; Weinstock, George M.; Šmajs, David

2012-01-01

Background The yaws treponemes, Treponema pallidum ssp. pertenue (TPE) strains, are closely related to syphilis causing strains of Treponema pallidum ssp. pallidum (TPA). Both yaws and syphilis are distinguished on the basis of epidemiological characteristics, clinical symptoms, and several genetic signatures of the corresponding causative agents. Methodology/Principal Findings To precisely define genetic differences between TPA and TPE, high-quality whole genome sequences of three TPE strains (Samoa D, CDC-2, Gauthier) were determined using next-generation sequencing techniques. TPE genome sequences were compared to four genomes of TPA strains (Nichols, DAL-1, SS14, Chicago). The genome structure was identical in all three TPE strains with similar length ranging between 1,139,330 bp and 1,139,744 bp. No major genome rearrangements were found when compared to the four TPA genomes. The whole genome nucleotide divergence (dA) between TPA and TPE subspecies was 4.7 and 4.8 times higher than the observed nucleotide diversity (π) among TPA and TPE strains, respectively, corresponding to 99.8% identity between TPA and TPE genomes. A set of 97 (9.9%) TPE genes encoded proteins containing two or more amino acid replacements or other major sequence changes. The TPE divergent genes were mostly from the group encoding potential virulence factors and genes encoding proteins with unknown function. Conclusions/Significance Hypothetical genes, with genetic differences, consistently found between TPE and TPA strains are candidates for syphilitic treponemes virulence factors. Seventeen TPE genes were predicted under positive selection, and eleven of them coded either for predicted exported proteins or membrane proteins suggesting their possible association with the cell surface. Sequence changes between TPE and TPA strains and changes specific to individual strains represent suitable targets for subspecies- and strain-specific molecular diagnostics. PMID:22292095

Comparative genome analysis reveals genetic adaptation to versatile environmental conditions and importance of biofilm lifestyle in Comamonas testosteroni.

PubMed

Wu, Yichao; Arumugam, Krithika; Tay, Martin Qi Xiang; Seshan, Hari; Mohanty, Anee; Cao, Bin

2015-04-01

Comamonas testosteroni is an important environmental bacterium capable of degrading a variety of toxic aromatic pollutants and has been demonstrated to be a promising biocatalyst for environmental decontamination. This organism is often found to be among the primary surface colonizers in various natural and engineered ecosystems, suggesting an extraordinary capability of this organism in environmental adaptation and biofilm formation. The goal of this study was to gain genetic insights into the adaption of C. testosteroni to versatile environments and the importance of a biofilm lifestyle. Specifically, a draft genome of C. testosteroni I2 was obtained. The draft genome is 5,778,710 bp in length and comprises 110 contigs. The average G+C content was 61.88 %. A total of 5365 genes with 5263 protein-coding genes were predicted, whereas 4324 (80.60 % of total genes) protein-encoding genes were associated with predicted functions. The catabolic genes responsible for biodegradation of steroid and other aromatic compounds on draft genome were identified. Plasmid pI2 was found to encode a complete pathway for aniline degradation and a partial catabolic pathway for chloroaniline. This organism was found to be equipped with a sophisticated signaling system which helps it find ideal niches and switch between planktonic and biofilm lifestyles. A large number of putative multi-drug-resistant genes coding for abundant outer membrane transporters, chaperones, and heat shock proteins for the protection of cellular function were identified in the genome of strain I2. In addition, the genome of strain I2 was predicted to encode several proteins involved in producing, secreting, and uptaking siderophores under iron-limiting conditions. The genome of strain I2 contains a number of genes responsible for the synthesis and secretion of exopolysaccharides, an extracellular component essential for biofilm formation. Overall, our results reveal the genomic features underlying the adaption of C. testosteroni to versatile environments and highlighting the importance of its biofilm lifestyle.

Virus-encoded chemokine receptors--putative novel antiviral drug targets.

PubMed

Rosenkilde, Mette M

2005-01-01

Large DNA viruses, in particular herpes- and poxviruses, have evolved proteins that serve as mimics or decoys for endogenous proteins in the host. The chemokines and their receptors serve key functions in both innate and adaptive immunity through control of leukocyte trafficking, and have as such a paramount role in the antiviral immune responses. It is therefore not surprising that viruses have found ways to exploit and subvert the chemokine system by means of molecular mimicry. By ancient acts of molecular piracy and by induction and suppression of endogenous genes, viruses have utilized chemokines and their receptors to serve a variety of roles in viral life-cycle. This review focuses on the pharmacology of virus-encoded chemokine receptors, yet also the family of virus-encoded chemokines and chemokine-binding proteins will be touched upon. Key properties of the virus-encoded receptors, compared to their closest endogenous homologs, are interactions with a wider range of chemokines, which can act as agonists, antagonists and inverse agonists, and the exploitation of many signal transduction pathways. High constitutive activity is another key property of some--but not all--of these receptors. The chemokine receptors belong to the superfamily of G-protein coupled 7TM receptors that per se are excellent drug targets. At present, non-peptide antagonists have been developed against many chemokine receptors. The potentials of the virus-encoded chemokine receptors as drug targets--ie. as novel antiviral strategies--will be highlighted here together with the potentials of the virus-encoded chemokines and chemokine-binding proteins as novel anti-inflammatory biopharmaceutical strategies.

[The importance of C-terminal aspartic acid residue (D141) to the antirestriction activity of the ArdB (R64) protein].

PubMed

Kudryavtseva, A A; Osetrova, M S; Livinyuk, V Ya; Manukhov, I V; Zavilgelsky, G B

2017-01-01

Antirestriction proteins of the ArdB/KlcA family are specific inhibitors of restriction (endonuclease) activity of type-I restriction/modification enzymes. The effect of conserved amino acid residues on the antirestriction activity of the ArdB protein encoded by the transmissible R64 (IncI1) plasmid has been investigated. An analysis of the amino acid sequences of ArdB homologues demonstrated the presence of four groups of conserved residues ((1) R16, E32, and W51; (2) Y46 and G48; (3) S81, D83 and E132, and (4) N77, L(I)140, and D141) on the surface of the protein globule. Amino acid residues of the fourth group showed a unique localization pattern with the terminal residue protruding beyond the globule surface. The replacement of two conserved amino acids (D141 and N77) located in the close vicinity of each other on the globule surface showed that the C-terminal D141 is essential for the antirestriction activity of ArdB. The deletion of this residue, as well as replacement by a hydrophobic threonine residue (D141T), completely abolished the antirestriction activity of ArdB. The synonymous replacement of D141 by a glutamic acid residue (D141E) caused an approximately 30-fold decrease of the antirestriction activity of ArdB, and the point mutation N77A caused an approximately 20-fold decrease in activity. The residues D141 and N77 located on the surface of the protein globule are presumably essential for the formation of a contact between ArdB and a currently unknown factor that modulates the activity of type-I restriction/modification enzymes.

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

NASA Technical Reports Server (NTRS)

Sedbrook, John C.; Carroll, Kathleen L.; Hung, Kai F.; Masson, Patrick H.; Somerville, Chris R.

2002-01-01

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

The Prader-Willi syndrome proteins MAGEL2 and necdin regulate leptin receptor cell surface abundance through ubiquitination pathways.

PubMed

Wijesuriya, Tishani Methsala; De Ceuninck, Leentje; Masschaele, Delphine; Sanderson, Matthea R; Carias, Karin Vanessa; Tavernier, Jan; Wevrick, Rachel

2017-11-01

In Prader-Willi syndrome (PWS), obesity is caused by the disruption of appetite-controlling pathways in the brain. Two PWS candidate genes encode MAGEL2 and necdin, related melanoma antigen proteins that assemble into ubiquitination complexes. Mice lacking Magel2 are obese and lack leptin sensitivity in hypothalamic pro-opiomelanocortin neurons, suggesting dysregulation of leptin receptor (LepR) activity. Hypothalamus from Magel2-null mice had less LepR and altered levels of ubiquitin pathway proteins that regulate LepR processing (Rnf41, Usp8, and Stam1). MAGEL2 increased the cell surface abundance of LepR and decreased their degradation. LepR interacts with necdin, which interacts with MAGEL2, which complexes with RNF41 and USP8. Mutations in the MAGE homology domain of MAGEL2 suppress RNF41 stabilization and prevent the MAGEL2-mediated increase of cell surface LepR. Thus, MAGEL2 and necdin together control LepR sorting and degradation through a dynamic ubiquitin-dependent pathway. Loss of MAGEL2 and necdin may uncouple LepR from ubiquitination pathways, providing a cellular mechanism for obesity in PWS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mutations in iron-sulfur cluster proteins that improve xylose utilization

DOEpatents

Froehlich, Allan; Henningsen, Brooks; Covalla, Sean; Zelle, Rintze M.

2018-03-20

There is provided an engineered host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism; and (b) at least one gene encoding a polypeptide having xylose isomerase activity, and methods of their use thereof.

Involvement of T6 pili in biofilm formation by serotype M6 Streptococcus pyogenes.

PubMed

Kimura, Keiji Richard; Nakata, Masanobu; Sumitomo, Tomoko; Kreikemeyer, Bernd; Podbielski, Andreas; Terao, Yutaka; Kawabata, Shigetada

2012-02-01

The group A streptococcus (GAS) Streptococcus pyogenes is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (fibronectin-binding protein, collagen-binding protein, and trypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm in vitro. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the tee6 gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the fctX and srtB genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.

A highly divergent gene cluster in honey bees encodes a novel silk family.

PubMed

Sutherland, Tara D; Campbell, Peter M; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Wanjura, Wolfgang J; Haritos, Victoria S

2006-11-01

The pupal cocoon of the domesticated silk moth Bombyx mori is the best known and most extensively studied insect silk. It is not widely known that Apis mellifera larvae also produce silk. We have used a combination of genomic and proteomic techniques to identify four honey bee fiber genes (AmelFibroin1-4) and two silk-associated genes (AmelSA1 and 2). The four fiber genes are small, comprise a single exon each, and are clustered on a short genomic region where the open reading frames are GC-rich amid low GC intergenic regions. The genes encode similar proteins that are highly helical and predicted to form unusually tight coiled coils. Despite the similarity in size, structure, and composition of the encoded proteins, the genes have low primary sequence identity. We propose that the four fiber genes have arisen from gene duplication events but have subsequently diverged significantly. The silk-associated genes encode proteins likely to act as a glue (AmelSA1) and involved in silk processing (AmelSA2). Although the silks of honey bees and silkmoths both originate in larval labial glands, the silk proteins are completely different in their primary, secondary, and tertiary structures as well as the genomic arrangement of the genes encoding them. This implies independent evolutionary origins for these functionally related proteins.

Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

DOEpatents

Laible, Philip D; Hanson, Deborah K

2013-06-04

The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1

PubMed Central

Salomon, Dor; Klimko, John A.

2014-01-01

The marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity. PMID:24987102

Variation of mucin adhesion, cell surface characteristics, and molecular mechanisms among Lactobacillus plantarum isolated from different habitats.

PubMed

Buntin, Nirunya; de Vos, Willem M; Hongpattarakere, Tipparat

2017-10-01

The adhesion ability to mucin varied greatly among 18 Lactobacillus plantarum isolates depending on their isolation habitats. Such ability remained at high level even though they were sequentially exposed to the gastrointestinal (GI) stresses. The majority of L. plantarum isolated from shrimp intestine and about half of food isolates exhibited adhesion ability (51.06-55.04%) about the same as the well-known adhesive L. plantarum 299v. Interestingly, five infant isolates of CIF17A2, CIF17A4, CIF17A5, CIF17AN2, and CIF17AN8 exhibited extremely high adhesion ranging from 62.69 to 72.06%. Such highly adhesive property correlating to distinctively high cell surface hydrophobicity was significantly weaken after pretreatment with LiCl and guanidine-HCl confirming the entailment of protein moiety. Regarding the draft genome information, all molecular structures of major cell wall-anchored proteins involved in the adhesion based on L. plantarum WCSF1, including lp_0964, lp_1643, lp_3114, lp_2486, lp_3127, and lp_3059 orthologues were detected in all isolates. Exceptionally, the gene-trait matching between yeast agglutination assay and the relevant mannose-specific adhesin (lp_1229) encoding gene confirmed the Msa absence in five infant isolates expressed distinctively high adhesion. Interestingly, the predicted flagellin encoding genes (fliC) firstly revealed in lp_1643, lp_2486, and lp_3114 orthologues may potentially contribute to such highly adhesive property of these isolates.

In silico analysis of the polygalacturonase inhibiting protein 1 from apple, Malus domestica.

PubMed

Matsaunyane, Lerato Bt; Oelofse, Dean; Dubery, Ian A

2015-03-11

The Malus domestica polygalacturonase inhibiting protein 1 (MdPGIP1) gene, encoding the M. domestica polygalacturonase inhibiting protein 1 (MdPGIP1), was isolated from the Granny Smith apple cultivar (GenBank accession no. DQ185063). The gene was used to transform tobacco and potato for enhanced resistance against fungal diseases. Analysis of the MdPGIP1 nucleotide sequence revealed that the gene comprises 993 nucleotides that encode a 330 amino acid polypeptide. In silico characterization of the MdPGIP1 polypeptide revealed domains typical of PGIP proteins, which include a 24 amino acid putative signal peptide, a potential cleavage site [Alanine-Leucine-Serine (ALS)] for the signal peptide, a 238 amino acid leucine-rich repeat (LRR) domain, a 46 amino acid N-terminal domain and a 22 amino acid C-terminal domain. The hydropathic evaluation of MdPGIP1 indicated a repetitive hydrophobic motif in the LRR domain and a hydrophilic surface area consistent with a globular protein. The typical consensus glycosylation sequence of Asn-X-Ser/Thr was identified in MdPGIP1, indicating potential N-linked glycosylation of MdPGIP1. The molecular mass of non-glycosylated MdPGIP1 was calculated as 36.615 kDa and the theoretical isoelectric point as 6.98. Furthermore, the secondary and tertiary structure of MdPGIP1 was modelled, and revealed that MdPGIP1 is a curved and elongated molecule that contains sheet B1, sheet B2 and 310-helices on its LRR domain. The overall properties of the MdPGIP1 protein is similar to that of the prototypical Phaseolus vulgaris PGIP 2 (PvPGIP2), and the detected differences supported its use in biotechnological applications as an inhibitor of targeted fungal polygalacturonases (PGs).

The Coat Protein and NIa Protease of Two Potyviridae Family Members Independently Confer Superinfection Exclusion

PubMed Central

French, Roy

2016-01-01

ABSTRACT Superinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans, and animals, its mechanisms, including involvement of specific viral determinants, are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses, Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) and Triticum mosaic virus (TriMV; genus Poacevirus, family Potyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, or NIb cistrons permitted efficient superinfection by WSMV expressing green fluorescent protein (WSMV-GFP). In contrast, wheat infected with TriMV expressing WSMV NIa-Pro or coat protein (CP) substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP rather than their encoding RNAs, as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200-amino-acid (aa) middle fragment (aa 101 to 300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE and that these two proteins trigger SIE independently of each other. IMPORTANCE Superinfection exclusion (SIE) is an antagonistic virus-virus interaction that prevents secondary invasions by identical or closely related viruses in the same host cells. Although known to occur in diverse viruses, SIE remains an enigma in terms of key molecular determinants and action mechanisms. In this study, we found that Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) encode two independently functioning cistrons that serve as effectors of SIE at the protein but not the RNA level. The coat protein and NIa-Pro encoded by these two viruses, when expressed from a heterologous virus, exerted SIE to the cognate viruses. The identification of virus-encoded effectors of SIE and their transgenic expression could potentially facilitate the development of virus-resistant crop plants. Additionally, functional conservation of SIE in diverse virus groups suggests that a better understanding of the underlying mechanisms of SIE could facilitate the development of novel antiviral therapies against viral diseases. PMID:27681136

Surface-Displayed IL-10 by Recombinant Lactobacillus plantarum Reduces Th1 Responses of RAW264.7 Cells Stimulated with Poly(I:C) or LPS.

PubMed

Cai, Ruopeng; Jiang, Yanlong; Yang, Wei; Yang, Wentao; Shi, Shaohua; Shi, Chunwei; Hu, Jingtao; Gu, Wei; Ye, Liping; Zhou, Fangyu; Gong, Qinglong; Han, Wenyu; Yang, Guilian; Wang, Chunfeng

2016-02-01

Recently, poly-γ-glutamic acid synthetase A (pgsA) has been applied to display exogenous proteins on the surface of Lactobacillus casei or Lactococcus lactis, which results in a surfacedisplayed component of bacteria. However, the ability of carrying genes encoded by plasmids and the expression efficiency of recombinant bacteria can be somewhat affected by the longer gene length of pgsA (1,143 bp); therefore, a truncated gene, pgsA, was generated based on the characteristics of pgsA by computational analysis. Using murine IL-10 as an exogenous gene, recombinant Lactobacillus plantarum was constructed and the capacity of the surface-displayed protein and functional differences between exogenous proteins expressed by these strains were evaluated. Surface expression of IL-10 on both recombinant bacteria with anchorins and the higher expression levels in L. plantarum-pgsA'-IL-10 were confirmed by western blot assay. Most importantly, up-regulation of IL-1β, IL-6, TNF-α, IFN-γ, and the nuclear transcription factor NF-κB p65 in RAW264.7 cells after stimulation with Poly(I:C) or LPS was exacerbated after co-culture with L. plantarum-pgsA. By contrast, IL-10 expressed by these recombinant strains could reduce these factors, and the expression of these factors was associated with recombinant strains that expressed anchorin (especially in L. plantarum-pgsA'-IL-10) and was significantly lower compared with the anchorin-free strains. These findings indicated that exogenous proteins could be successfully displayed on the surface of L. plantarum by pgsA or pgsA', and the expression of recombinant bacteria with pgsA' was superior compared with bacteria with pgsA.

Nucleic acids encoding plant glutamine phenylpyruvate transaminase (GPT) and uses thereof

DOEpatents

Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

2016-03-29

Glutamine phenylpyruvate transaminase (GPT) proteins, nucleic acid molecules encoding GPT proteins, and uses thereof are disclosed. Provided herein are various GPT proteins and GPT gene coding sequences isolated from a number of plant species. As disclosed herein, GPT proteins share remarkable structural similarity within plant species, and are active in catalyzing the synthesis of 2-hydroxy-5-oxoproline (2-oxoglutaramate), a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism.

Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas.

PubMed

Tapia, Lorena I; Shaw, Chad A; Aideyan, Letisha O; Jewell, Alan M; Dawson, Brian C; Haq, Taha R; Piedra, Pedro A

2014-01-01

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community.

Gene Sequence Variability of the Three Surface Proteins of Human Respiratory Syncytial Virus (HRSV) in Texas

PubMed Central

Tapia, Lorena I.; Shaw, Chad A.; Aideyan, Letisha O.; Jewell, Alan M.; Dawson, Brian C.; Haq, Taha R.; Piedra, Pedro A.

2014-01-01

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004–2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community. PMID:24625544

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

PubMed

Yassin, Atteyet F; Langenberg, Stefan; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Mukherjee, Supratim; Reddy, T B K; Daum, Chris; Shapiro, Nicole; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos C

2017-01-01

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

PubMed

Liszewska, Frantz; Gaganidze, Dali; Sirko, Agnieszka

2005-01-01

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

Isolation and in silico analysis of a novel H+-pyrophosphatase gene orthologue from the halophytic grass Leptochloa fusca

NASA Astrophysics Data System (ADS)

Rauf, Muhammad; Saeed, Nasir A.; Habib, Imran; Ahmed, Moddassir; Shahzad, Khurram; Mansoor, Shahid; Ali, Rashid

2017-02-01

Structure prediction can provide information about function and active sites of protein which helps to design new functional proteins. H+-pyrophosphatase is transmembrane protein involved in establishing proton motive force for active transport of Na+ across membrane by Na+/H+ antiporters. A full length novel H+-pyrophosphatase gene was isolated from halophytic grass Leptochloa fusca using RT-PCR and RACE method. Full length LfVP1 gene sequence of 2292 nucleotides encodes protein of 764 amino acids. DNA and protein sequences were used for characterization using bioinformatics tools. Various important potential sites were predicted by PROSITE webserver. Primary structural analysis showed LfVP1 as stable protein and Grand average hydropathy (GRAVY) indicated that LfVP1 protein has good hydrosolubility. Secondary structure analysis showed that LfVP1 protein sequence contains significant proportion of alpha helix and random coil. Protein membrane topology suggested the presence of 14 transmembrane domains and presence of catalytic domain in TM3. Three dimensional structure from LfVP1 protein sequence also indicated the presence of 14 transmembrane domains and hydrophobicity surface model showed amino acid hydrophobicity. Ramachandran plot showed that 98% amino acid residues were predicted in the favored region.

Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

PubMed

Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

2008-11-01

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

Non-essential MCM-related proteins mediate a response to DNA damage in the archaeon Methanococcus maripaludis.

PubMed

Walters, Alison D; Chong, James P J

2017-05-01

The single minichromosome maintenance (MCM) protein found in most archaea has been widely studied as a simplified model for the MCM complex that forms the catalytic core of the eukaryotic replicative helicase. Organisms of the order Methanococcales are unusual in possessing multiple MCM homologues. The Methanococcus maripaludis S2 genome encodes four MCM homologues, McmA-McmD. DNA helicase assays reveal that the unwinding activity of the three MCM-like proteins is highly variable despite sequence similarities and suggests additional motifs that influence MCM function are yet to be identified. While the gene encoding McmA could not be deleted, strains harbouring individual deletions of genes encoding each of the other MCMs display phenotypes consistent with these proteins modulating DNA damage responses. M. maripaludis S2 is the first archaeon in which MCM proteins have been shown to influence the DNA damage response.

Role of Soybean mosaic virus-encoded proteins in seed and aphid transmission in soybean

USDA-ARS?s Scientific Manuscript database

Soybean mosaic virus (SMV) is seed and aphid transmitted and can cause significant reductions in yield and seed quality in soybean, Glycine max. The roles in seed and aphid transmission of selected SMV-encoded proteins were investigated by constructing chimeric recombinants between SMV 413 (efficien...

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium

PubMed Central

Sillanpää, Jouko; Nallapareddy, Sreedhar R.; Prakash, Vittal P.; Qin, Xiang; Hook, Magnus; Weinstock, George M.; Murray, Barbara E.

2009-01-01

SUMMARY Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about pathogenic determinants of this organism. We have previously identified a cell wall anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad spectrum binding to extracellular matrix proteins. Here, we analyzed the draft genome of strain TX0016 for potential MSCRAMMs (microbial surface component recognizing adhesive matrix molecules). Genome-based bioinformatics identified 22 predicted cell wall anchored E. faeciumsurface proteins (Fms) of which 15 (including Acm) have typical characteristics of MSCRAMMs including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one (Fms10, redesignated Scm for second collagen adhesin of E. faeciu m) revealed that recombinant Scm65 (A- and B-domains) and Scm36 (A-domain) bound efficiently to collagen type V in a concentration dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism of recombinant Scm36 and of Acm37 indicated that these proteins are rich in β-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; 9 of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated as EbpCfm), detected a “ladder” pattern of high-molecular weight protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpCfm is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpAfm), fms5 (ebpBfm) and ebpCfm are co-transcribed, consistent with pilus-encoding gene clusters of other gram-positive bacteria. All 15 genes occurred frequently in 30 clinically-derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen. PMID:18832325

Molecular characterization of calreticulin from Anopheles stephensi midgut cells and functional assay of the recombinant calreticulin with Plasmodium berghei ookinetes.

PubMed

Borhani Dizaji, Nahid; Basseri, Hamid Reza; Naddaf, Saied Reza; Heidari, Mansour

2014-10-25

Transmission blocking vaccines (TBVs) that target the antigens on the midgut epithelium of Anopheles mosquitoes are among the promising tools for the elimination of the malaria parasite. Characterization and analysis of effective antigens is the first step to design TBVs. Calreticulin (CRT), a lectin-like protein, from Anopheles albimanus midgut, has shown antigenic features, suggesting a promising and novel TBV target. CRT is a highly conserved protein with similar features in vertebrates and invertebrates including anopheline. We cloned the full-length crt gene from malaria vector, Anopheles stephensi (AsCrt) and explored the interaction of recombinant AsCrt protein, expressed in a prokaryotic system (pGEX-6p-1), with surface proteins of Plasmodium berghei ookinetes by immunofluorescence assay. The cellular localization of AsCrt was determined using the baculovirus expression system. Sequence analysis of the whole cDNA of AsCrt revealed that AsCrt contains an ORF of 1221 bp. The amino acid sequence of AsCrt protein obtained in this study showed 64% homology with similar protein in human. The AsCrt shares the most common features of CRTs from other species. This gene encodes a 406 amino-acid protein with a molecular mass of 46 kDa, which contains a predicted 16 amino-acid signal peptides, conserved cysteine residues, a proline-rich region, and highly acidic C-terminal domain with endoplasmic reticulum retrieval sequence HDEL. The production of GST-AsCrt recombinant protein was confirmed by Western blot analysis using an antibody against the GST protein. The FITC-labeled GST-AsCrt exhibited a significant interaction with P. berghei ookinete surface proteins. Purified recombinant GST-AsCrt, labeled with FITC, displayed specific binding to the surface of P. berghei ookinetes in comparison with control. Moreover, the expression of AsCrt in baculovirus expression system indicated that AsCrt was localized on the surface of Sf9 cells. Our results suggest that AsCrt could be utilized as a potential target for future studies in TBV area for malaria control. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of Western Equine Encephalitis Virus Structural Proteins That Confer Protection after DNA Vaccination▿

PubMed Central

Gauci, Penelope J.; Wu, Josh Q. H.; Rayner, George A.; Barabé, Nicole D.; Nagata, Leslie P.; Proll, David F.

2010-01-01

DNA vaccines encoding different portions of the structural proteins of western equine encephalitis virus were tested for the efficacy of their protection in a 100% lethal mouse model of the virus. The 6K-E1 structural protein encoded by the DNA vaccine conferred complete protection against challenge with the homologous strain and limited protection against challenge with a heterologous strain. PMID:19923571

Repression of small toxic protein synthesis by the Sib and OhsC small RNAs.

PubMed

Fozo, Elizabeth M; Kawano, Mitsuoki; Fontaine, Fanette; Kaya, Yusuf; Mendieta, Kathy S; Jones, Kristi L; Ocampo, Alejandro; Rudd, Kenneth E; Storz, Gisela

2008-12-01

The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, although the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five-sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18- to 19-amino-acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell.

Repression of small toxic protein synthesis by the Sib and OhsC small RNAs

PubMed Central

Fozo, Elizabeth M.; Kawano, Mitsuoki; Fontaine, Fanette; Kaya, Yusuf; Mendieta, Kathy S.; Jones, Kristi L.; Ocampo, Alejandro; Rudd, Kenneth E.; Storz, Gisela

2008-01-01

Summary The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, though the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18–19 amino acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by co-expression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell. PMID:18710431

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

PubMed Central

Fang, H; Green, N

1994-01-01

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane. Images PMID:7841522

Two Crinivirus-specific proteins of Lettuce infectious yellows virus (LIYV), P26 and P9, are self-interacting.

PubMed

Stewart, Lucy R; Hwang, Min Sook; Falk, Bryce W

2009-11-01

Interactions of Lettuce infectious yellows virus (LIYV)-encoded proteins were tested by yeast-two-hybrid (Y2H) assays. LIYV-encoded P34, Hsp70h, P59, CP, CPm, and P26 were tested in all possible pairwise combinations. Interaction was detected only for the P26-P26 combination. P26 self-interaction domains were mapped using a series of N- and C-terminal truncations. Orthologous P26 proteins from the criniviruses Beet pseudoyellows virus (BPYV), Cucurbit yellow stunting disorder virus (CYSDV), and Lettuce chlorosis virus (LCV) were also tested, and each exhibited strong self-interaction but no interaction with orthologous proteins. Two small putative proteins encoded by LIYV RNA2, P5 and P9, were also tested for interactions with the six aforementioned LIYV proteins and each other. No interactions were detected for P5, but P9-P9 self-interaction was detected. P26- and P9-encoding genes are present in all described members of the genus Crinivirus, but are not present in other members of the family Closteroviridae. LIYV P26 has previously been demonstrated to induce a unique LIYV cytopathology, plasmalemma deposits (PLDs), but no role is yet known for P9.

Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene.

PubMed Central

Rosemblat, S; Durham-Pierre, D; Gardner, J M; Nakatsu, Y; Brilliant, M H; Orlow, S J

1994-01-01

The pink-eyed dilution (p) locus in the mouse is critical to melanogenesis; mutations in the homologous locus in humans, P, are a cause of type II oculocutaneous albinism. Although a cDNA encoded by the p gene has recently been identified, nothing is known about the protein product of this gene. To characterize the protein encoded by the p gene, we performed immunoblot analysis of extracts of melanocytes cultured from wild-type mice with an antiserum from rabbits immunized with a peptide corresponding to amino acids 285-298 of the predicted protein product of the murine p gene. This antiserum recognized a 110-kDa protein. The protein was absent from extracts of melanocytes cultured from mice with two mutations (pcp and p) in which transcripts of the p gene are absent or greatly reduced. Introduction of the cDNA for the p gene into pcp melanocytes by electroporation resulted in expression of the 3.3-kb mRNA and the 110-kDa protein. Upon subcellular fractionation of cultured melanocytes, the 110-kDa protein was found to be present in melanosomes but absent from the vesicular fraction; phase separation performed with the nonionic detergent Triton X-114 confirmed the predicted hydrophobic nature of the protein. These results demonstrate that the p gene encodes a 110-kDa integral melanosomal membrane protein and establish a framework by which mutations at this locus, which diminish pigmentation, can be analyzed at the cellular and biochemical levels. Images PMID:7991586

Protective Vaccination against Blood-Stage Malaria of Plasmodium chabaudi: Differential Gene Expression in the Liver of Balb/c Mice toward the End of Crisis Phase

PubMed Central

Al-Quraishy, Saleh A.; Dkhil, Mohamed A.; Abdel-Baki, Abdel-Azeem A.; Delic, Denis; Wunderlich, Frank

2016-01-01

Protective vaccination induces self-healing of otherwise fatal blood-stage malaria of Plasmodium chabaudi in female Balb/c mice. To trace processes critically involved in self-healing, the liver, an effector against blood-stage malaria, is analyzed for possible changes of its transcriptome in vaccination-protected in comparison to non-protected mice toward the end of the crisis phase. Gene expression microarray analyses reveal that vaccination does not affect constitutive expression of mRNA and lincRNA. However, malaria induces significant (p < 0.01) differences in hepatic gene and lincRNA expression in vaccination-protected vs. non-vaccinated mice toward the end of crisis phase. In vaccination-protected mice, infections induce up-regulations of 276 genes and 40 lincRNAs and down-regulations of 200 genes and 43 lincRNAs, respectively, by >3-fold as compared to the corresponding constitutive expressions. Massive up-regulations, partly by >100-fold, are found for genes as RhD, Add2, Ank1, Ermap, and Slc4a, which encode proteins of erythrocytic surface membranes, and as Gata1 and Gfi1b, which encode transcription factors involved in erythrocytic development. Also, Cldn13 previously predicted to be expressed on erythroblast surfaces is up-regulated by >200-fold, though claudins are known as main constituents of tight junctions acting as paracellular barriers between epithelial cells. Other genes are up-regulated by <100- and >10-fold, which can be subgrouped in genes encoding proteins known to be involved in mitosis, in cell cycle regulation, and in DNA repair. Our data suggest that protective vaccination enables the liver to respond to P. chabaudi infections with accelerated regeneration and extramedullary erythropoiesis during crisis, which contributes to survival of otherwise lethal blood-stage malaria. PMID:27471498

[Cloning, mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R].

PubMed

Li, Yanan; Zeng, Xiaobo; Zhou, Xuejuan; Li, Youguo

2016-12-04

Lipid transfer protein superfamily is involved in lipid transport and metabolism. This study aimed to construct mutants of three lipid transfer protein encoding genes in Mesorhizobium huakuii 7653R, and to study the phenotypes and function of mutations during symbiosis with Astragalus sinicus. We used bioinformatics to predict structure characteristics and biological functions of lipid transfer proteins, and conducted semi-quantitative and fluorescent quantitative real-time PCR to analyze the expression levels of target genes in free-living and symbiotic conditions. Using pK19mob insertion mutagenesis to construct mutants, we carried out pot plant experiments to observe symbiotic phenotypes. MCHK-5577, MCHK-2172 and MCHK-2779 genes encoding proteins belonged to START/RHO alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) superfamily, involved in lipid transport or metabolism, and were identical to M. loti at 95% level. Gene relative transcription level of the three genes all increased compared to free-living condition. We obtained three mutants. Compared with wild-type 7653R, above-ground biomass of plants and nodulenitrogenase activity induced by the three mutants significantly decreased. Results indicated that lipid transfer protein encoding genes of Mesorhizobium huakuii 7653R may play important roles in symbiotic nitrogen fixation, and the mutations significantly affected the symbiotic phenotypes. The present work provided a basis to study further symbiotic function mechanism associated with lipid transfer proteins from rhizobia.

Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification

PubMed Central

Völlenkle, Christine; Weigert, Stefan; Ilk, Nicola; Egelseer, Eva; Weber, Viktoria; Loth, Fritz; Falkenhagen, Dieter; Sleytr, Uwe B.; Sára, Margit

2004-01-01

The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA31-1068/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-μm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm2, whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm2 was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system. PMID:15006773

Three reasons protein disorder analysis makes more sense in the light of collagen

PubMed Central

Oates, Matt E.; Tompa, Peter; Gough, Julian

2016-01-01

Abstract We have identified that the collagen helix has the potential to be disruptive to analyses of intrinsically disordered proteins. The collagen helix is an extended fibrous structure that is both promiscuous and repetitive. Whilst its sequence is predicted to be disordered, this type of protein structure is not typically considered as intrinsic disorder. Here, we show that collagen‐encoding proteins skew the distribution of exon lengths in genes. We find that previous results, demonstrating that exons encoding disordered regions are more likely to be symmetric, are due to the abundance of the collagen helix. Other related results, showing increased levels of alternative splicing in disorder‐encoding exons, still hold after considering collagen‐containing proteins. Aside from analyses of exons, we find that the set of proteins that contain collagen significantly alters the amino acid composition of regions predicted as disordered. We conclude that research in this area should be conducted in the light of the collagen helix. PMID:26941008

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

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

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

PubMed

Siegel, Andrea M; Herskowitz, Jeremy H; Speck, Samuel H

2008-04-04

Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis-identifying a strategy that appears to be conserved between at least EBV and MHV68.

Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection.

PubMed

Tikunova, N V; Morozova, V V

2009-10-01

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099T

PubMed Central

2012-01-01

Background Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival. Results The genome of Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on Blast analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii. Conclusions Based on genomic analyses, Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab. magadii has the genetic potential to adapt to its milieu by intracellular accumulation of inorganic cations and/or neutral organic compounds. The identification of Nab. magadii genes involved in coenzyme biosynthesis is a necessary step toward further reconstruction of the metabolic pathways in halophilic archaea and other extremophiles. The knowledge gained from the genome sequence of this haloalkaliphilic archaeon is highly valuable in advancing the applications of extremophiles and their enzymes. PMID:22559199

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

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

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

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

A RecA Protein Surface Required for Activation of DNA Polymerase V

PubMed Central

Gruber, Angela J.; Erdem, Aysen L.; Sabat, Grzegorz; Karata, Kiyonobu; Jaszczur, Malgorzata M.; Vo, Dan D.; Olsen, Tayla M.; Woodgate, Roger; Goodman, Myron F.; Cox, Michael M.

2015-01-01

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis. PMID:25811184

A conserved fungal glycosyltransferase facilitates pathogenesis of plants by enabling hyphal growth on solid surfaces

PubMed Central

Plummer, Amy; Halsey, Kirstie; Lovegrove, Alison; Hammond-Kosack, Kim

2017-01-01

Pathogenic fungi must extend filamentous hyphae across solid surfaces to cause diseases of plants. However, the full inventory of genes which support this is incomplete and many may be currently concealed due to their essentiality for the hyphal growth form. During a random T-DNA mutagenesis screen performed on the pleomorphic wheat (Triticum aestivum) pathogen Zymoseptoria tritici, we acquired a mutant unable to extend hyphae specifically when on solid surfaces. In contrast “yeast-like” growth, and all other growth forms, were unaffected. The inability to extend surface hyphae resulted in a complete loss of virulence on plants. The affected gene encoded a predicted type 2 glycosyltransferase (ZtGT2). Analysis of >800 genomes from taxonomically diverse fungi highlighted a generally widespread, but discontinuous, distribution of ZtGT2 orthologues, and a complete absence of any similar proteins in non-filamentous ascomycete yeasts. Deletion mutants of the ZtGT2 orthologue in the taxonomically un-related fungus Fusarium graminearum were also severely impaired in hyphal growth and non-pathogenic on wheat ears. ZtGT2 expression increased during filamentous growth and electron microscopy on deletion mutants (ΔZtGT2) suggested the protein functions to maintain the outermost surface of the fungal cell wall. Despite this, adhesion to leaf surfaces was unaffected in ΔZtGT2 mutants and global RNAseq-based gene expression profiling highlighted that surface-sensing and protein secretion was also largely unaffected. However, ΔZtGT2 mutants constitutively overexpressed several transmembrane and secreted proteins, including an important LysM-domain chitin-binding virulence effector, Zt3LysM. ZtGT2 likely functions in the synthesis of a currently unknown, potentially minor but widespread, extracellular or outer cell wall polysaccharide which plays a key role in facilitating many interactions between plants and fungi by enabling hyphal growth on solid matrices. PMID:29020037

Structural and evolutionary analysis of Leishmania Alba proteins.

PubMed

da Costa, Kauê Santana; Galúcio, João Marcos Pereira; Leonardo, Elvis Santos; Cardoso, Guelber; Leal, Élcio; Conde, Guilherme; Lameira, Jerônimo

2017-10-01

The Alba superfamily proteins share a common RNA-binding domain. These proteins participate in a variety of regulatory pathways by controlling developmental gene expression. They also interact with ribosomal subunits, translation factors, and other RNA-binding proteins. The Leishmania infantum genome encodes two Alba-domain proteins, LiAlba1 and LiAlba3. In this work, we used homology modeling, protein-protein docking, and molecular dynamics (MD) simulations to explore the details of the Alba1-Alba3-RNA complex from Leishmania infantum at the molecular level. In addition, we compared the structure of LiAlba3 with the human ribonuclease P component, Rpp20. We also mapped the ligand-binding residues on the Alba3 surface to analyze its druggability and performed mutational analyses in Alba3 using alanine scanning to identify residues involved in its function and structural stability. These results suggest that the RGG-box motif of LiAlba1 is important for protein function and stability. Finally, we discuss the function of Alba proteins in the context of pathogen adaptation to host cells. The data provided herein will facilitate further translational research regarding Alba structure and function. Copyright © 2017 Elsevier B.V. All rights reserved.

The Protein Phosphatases of Synechocystis sp. Strain PCC 6803: Open Reading Frames sll1033 and sll1387 Encode Enzymes That Exhibit both Protein-Serine and Protein-Tyrosine Phosphatase Activity In Vitro.

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

Li, Ruiliang; Potters, M B.; Shi, Liang

2005-09-01

The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp(608) by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product ofmore » ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg(2+) or Mn(2+), for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.« less

Highly thermostable fluorescent proteins

DOEpatents

Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

2011-11-29

Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

Highly thermostable fluorescent proteins

DOEpatents

Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

2011-03-22

Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

Highly thermostable fluorescent proteins

DOEpatents

Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

2012-05-01

Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

Immunity to East Coast fever in cattle induced by a polypeptide fragment of the major surface coat protein of Theileria parva sporozoites.

PubMed

Bishop, Richard; Nene, Vishvanath; Staeyert, Jan; Rowlands, John; Nyanjui, John; Osaso, Julius; Morzaria, Subhash; Musoke, Antony

2003-03-07

Full-length recombinant versions of p67, the 709 amino acid major surface protein of Theileria parva sporozoites, induce immunity to East Coast fever (ECF) in cattle. We show that a soluble Escherichia coli recombinant version of p67 (p67(635)), in which a prokaryotic signal peptide replaces the eukaryotic one, confers protection comparable to that induced by the full-length molecule, but is unstable. Peptides encoding 80 (p67C) and 205 (p67N) amino acid fragments of p67, containing epitopes recognised by sporozoite neutralising monoclonal antibodies, exhibit improved stability in E. coli. Antibodies raised against the central region of p67 (p67M) neutralise sporozoite infectivity in vitro. The p67C peptide induced immunity against ECF in cattle, at a level equivalent to p67(635), suggesting that a synthetic peptide vaccine might be achievable. Copyright 2002 Elsevier Science Ltd.

Compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa) rivals that of animals, fungi, and plants.

PubMed

Gawryluk, Ryan M R; Chisholm, Kenneth A; Pinto, Devanand M; Gray, Michael W

2014-09-23

We present a combined proteomic and bioinformatic investigation of mitochondrial proteins from the amoeboid protist Acanthamoeba castellanii, the first such comprehensive investigation in a free-living member of the supergroup Amoebozoa. This protist was chosen both for its phylogenetic position (as a sister to animals and fungi) and its ecological ubiquity and physiological flexibility. We report 1033 A. castellanii mitochondrial protein sequences, 709 supported by mass spectrometry data (676 nucleus-encoded and 33 mitochondrion-encoded), including two previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Other notable findings include duplicate proteins for all of the enzymes of the tricarboxylic acid (TCA) cycle-which, along with the identification of a mitochondrial malate synthase-isocitrate lyase fusion protein, suggests the interesting possibility that the glyoxylate cycle operates in A. castellanii mitochondria. Additionally, the A. castellanii genome encodes an unusually high number (at least 29) of mitochondrion-targeted pentatricopeptide repeat (PPR) proteins, organellar RNA metabolism factors in other organisms. We discuss several key mitochondrial pathways, including DNA replication, transcription and translation, protein degradation, protein import and Fe-S cluster biosynthesis, highlighting similarities and differences in these pathways in other eukaryotes. In compositional and functional complexity, the mitochondrial proteome of A. castellanii rivals that of multicellular eukaryotes. Comprehensive proteomic surveys of mitochondria have been undertaken in a limited number of predominantly multicellular eukaryotes. This phylogenetically narrow perspective constrains and biases our insights into mitochondrial function and evolution, as it neglects protists, which account for most of the evolutionary and functional diversity within eukaryotes. We report here the first comprehensive investigation of the mitochondrial proteome in a member (A. castellanii) of the eukaryotic supergroup Amoebozoa. Through a combination of tandem mass spectrometry (MS/MS) and in silico data mining, we have retrieved 1033 candidate mitochondrial protein sequences, 709 having MS support. These data were used to reconstruct the metabolic pathways and protein complexes of A. castellanii mitochondria, and were integrated with data from other characterized mitochondrial proteomes to augment our understanding of mitochondrial proteome evolution. Our results demonstrate the power of combining direct proteomic and bioinformatic approaches in the discovery of novel mitochondrial proteins, both nucleus-encoded and mitochondrion-encoded, and highlight the compositional complexity of the A. castellanii mitochondrial proteome, which rivals that of animals, fungi and plants. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein Secondary Structure Prediction Using AutoEncoder Network and Bayes Classifier

NASA Astrophysics Data System (ADS)

Wang, Leilei; Cheng, Jinyong

2018-03-01

Protein secondary structure prediction is belong to bioinformatics,and it's important in research area. In this paper, we propose a new prediction way of protein using bayes classifier and autoEncoder network. Our experiments show some algorithms including the construction of the model, the classification of parameters and so on. The data set is a typical CB513 data set for protein. In terms of accuracy, the method is the cross validation based on the 3-fold. Then we can get the Q3 accuracy. Paper results illustrate that the autoencoder network improved the prediction accuracy of protein secondary structure.

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families.

PubMed

Shah, Shiraz A; Alkhnbashi, Omer S; Behler, Juliane; Han, Wenyuan; She, Qunxin; Hess, Wolfgang R; Garrett, Roger A; Backofen, Rolf

2018-06-19

A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

PubMed Central

Bernasconi, Eric; Germond, Jacques-Edouard; Delley, Michèle; Fritsché, Rodolphe; Corthésy, Blaise

2002-01-01

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB∂-BLG yielded up to 170 μg per 109 CFU in the culture supernatant and 9 μg per 109 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue. PMID:12039750

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells.

PubMed

Wu, Yaqin; Zhuang, Jiabao; Zhao, Dan; Zhang, Fuqiang; Ma, Jiayin; Xu, Chun

2017-10-01

This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells. After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24 h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84 key genes encoding cytoskeletal regulators after 6 and 24 h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes. The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6 h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24 h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated. The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.

Structural studies of the HIV-1 accessory protein Vpu in langmuir monolayers: synchrotron X-ray reflectivity.

PubMed Central

Zheng, S; Strzalka, J; Ma, C; Opella, S J; Ocko, B M; Blasie, J K

2001-01-01

Vpu is an 81 amino acid integral membrane protein encoded by the HIV-1 genome with a N-terminal hydrophobic domain and a C-terminal hydrophilic domain. It enhances the release of virus from the infected cell and triggers degradation of the virus receptor CD4. Langmuir monolayers of mixtures of Vpu and the phospholipid 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (DLgPC) at the water-air interface were studied by synchrotron radiation-based x-ray reflectivity over a range of mole ratios at constant surface pressure and for several surface pressures at a maximal mole ratio of Vpu/DLgPC. Analysis of the x-ray reflectivity data by both slab model-refinement and model-independent box-refinement methods firmly establish the monolayer electron density profiles. The electron density profiles as a function of increasing Vpu/DLgPC mole ratio at a constant, relatively high surface pressure indicated that the amphipathic helices of the cytoplasmic domain lie on the surface of the phospholipid headgroups and the hydrophobic transmembrane helix is oriented approximately normal to the plane of monolayer within the phospholipid hydrocarbon chain layer. At maximal Vpu/DLgPC mole ratio, the tilt of the transmembrane helix with respect to the monolayer normal decreases with increasing surface pressure and the conformation of the cytoplasmic domain varies substantially with surface pressure. PMID:11259297

Similarity and functional analyses of expressed parasitism genes in Heterodera schachtii and Heterodera glycines

USDA-ARS?s Scientific Manuscript database

The secreted proteins encoded by “parasitism genes” expressed within the esophageal glands cells of cyst nematodes play important roles in plant parasitism. Homologous transcripts and encoded proteins of the Heterodera glycines pioneer parasitism genes Hgsyv46, Hg4e02 and Hg5d08 were identified and ...

Influence of energy supply on expression of genes encoding for lipogenic enzymes and regulatory proteins in growing beef steers

USDA-ARS?s Scientific Manuscript database

Forty crossbred beef steers were used to determine the effects metabolizable energy (ME) intake and of site and complexity of carbohydrate (CHO) infusion on expression of genes encoding lipogenic enzymes and regulatory proteins in subcutaneous (SC), mesenteric (MES) and omental (OM) adipose. Treatm...

Nucleic acids encoding human trithorax protein

DOEpatents

Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

2001-01-01

In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

Nip the HPV encoded evil in the cancer bud: HPV reshapes TRAILs and signaling landscapes

PubMed Central

2013-01-01

HPV encoded proteins can elicit ectopic protein–protein interactions that re-wire signaling pathways, in a mode that promotes malignancy. Moreover, accumulating data related to HPV is now providing compelling substantiation of a central role played by HPV in escaping immunosurveillance and impairment of apoptotic response. What emerges is an intricate network of Wnt, TGF, Notch signaling cascades that forms higher-order ligand–receptor complexes routing downstream signaling in HPV infected cells. These HPV infected cells are regulated both extracellularly by ligand receptor axis and intracellularly by HPV encoded proteins and impair TRAIL mediated apoptosis. We divide this review into different sections addressing how linear signaling pathways integrate to facilitate carcinogenesis and compounds that directly or indirectly reverse these aberrant interactions offer new possibilities for therapy in cancer. Although HPV encoded proteins mediated misrepresentation of pathways is difficult to target, improved drug-discovery platforms and new technologies have facilitated the discovery of agents that can target dysregulated pathways in HPV infected cervical cancer cells, thus setting the stage for preclinical models and clinical trials. PMID:23773282

Ubiquitin--conserved protein or selfish gene?

PubMed

Catic, André; Ploegh, Hidde L

2005-11-01

The posttranslational modifier ubiquitin is encoded by a multigene family containing three primary members, which yield the precursor protein polyubiquitin and two ubiquitin moieties, Ub(L40) and Ub(S27), that are fused to the ribosomal proteins L40 and S27, respectively. The gene encoding polyubiquitin is highly conserved and, until now, those encoding Ub(L40) and Ub(S27) have been generally considered to be equally invariant. The evolution of the ribosomal ubiquitin moieties is, however, proving to be more dynamic. It seems that the genes encoding Ub(L40) and Ub(S27) are actively maintained by homologous recombination with the invariant polyubiquitin locus. Failure to recombine leads to deterioration of the sequence of the ribosomal ubiquitin moieties in several phyla, although this deterioration is evidently constrained by the structural requirements of the ubiquitin fold. Only a few amino acids in ubiquitin are vital for its function, and we propose that conservation of all three ubiquitin genes is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as a 'selfish gene'.

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

PubMed Central

Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

1986-01-01

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins.

PubMed

Ahi, Yadvinder S; Zhang, Shu; Thappeta, Yashna; Denman, Audrey; Feizpour, Amin; Gummuluru, Suryaram; Reinhard, Bjoern; Muriaux, Delphine; Fivash, Matthew J; Rein, Alan

2016-11-22

Gammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called "glycogag" (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes. Many murine leukemia viruses (MLVs) encode a protein called "glycogag." The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5. Copyright © 2016 Ahi et al.

Prediction of the protein components of a putative Calanus finmarchicus (Crustacea, Copepoda) circadian signaling system using a de novo assembled transcriptome

PubMed Central

Christie, Andrew E.; Fontanilla, Tiana M.; Nesbit, Katherine T.; Lenz, Petra H.

2013-01-01

Diel vertical migration and seasonal diapause are critical life history events for the copepod Calanus finmarchicus. While much is known about these behaviors phenomenologically, little is known about their molecular underpinnings. Recent studies in insects suggest that some circadian genes/proteins also contribute to the establishment of seasonal diapause. Thus, it is possible that in Calanus these distinct timing regimes share some genetic components. To begin to address this possibility, we used the well-established Drosophila melanogaster circadian system as a reference for mining clock transcripts from a 200,000+ sequence Calanus transcriptome; the proteins encoded by the identified transcripts were also deduced and characterized. Sequences encoding homologs of the Drosophila core clock proteins CLOCK, CYCLE, PERIOD and TIMELESS were identified, as was one encoding CRYPTOCHROME 2, a core clock protein in ancestral insect systems, but absent in Drosophila. Calanus transcripts encoding proteins known to modulate the Drosophila core clock were also identified and characterized, e.g. CLOCKWORK ORANGE, DOUBLETIME, SHAGGY and VRILLE. Alignment and structural analyses of the deduced Calanus proteins with their Drosophila counterparts revealed extensive sequence conservation, particularly in functional domains. Interestingly, reverse BLAST analyses of these sequences against all arthropod proteins typically revealed non-Drosophila isoforms to be most similar to the Calanus queries. This, in combination with the presence of both CRYPTOCHROME 1 (a clock input pathway protein) and CRYPTOCHROME 2 in Calanus, suggests that the organization of the copepod circadian system is an ancestral one, more similar to that of insects like Danaus plexippus than to that of Drosophila. PMID:23727418

Surface Functionalization Methods to Enhance Bioconjugation in Metal-Labeled Polystyrene Particles

PubMed Central

Abdelrahman, Ahmed I.; Thickett, Stuart C.; Liang, Yi; Ornatsky, Olga; Baranov, Vladimir; Winnik, Mitchell A.

2011-01-01

Lanthanide-encoded polystyrene particles synthesized by dispersion polymerization are excellent candidates for mass cytometry based immunoassays, however they have previously lacked the ability to conjugate biomolecules to the particle surface. We present here three approaches to post-functionalize these particles, enabling the covalent attachment of proteins. Our first approach used partially hydrolyzed poly(N-vinylpyrrolidone) as a dispersion polymerization stabilizer to synthesize particles with high concentration of -COOH groups on the particle surface. In an alternative strategy to provide -COOH functionality to the lanthanide-encoded particles, we employed seeded emulsion polymerization to graft poly(methacrylic acid) (PMAA) chains onto the surface of these particles. However, these two approaches gave little to no improvement in the extent of bioconjugation. In our third approach, seeded emulsion polymerization was subsequently used as a method to grow a functional polymer shell (in this case, poly(glycidyl methacrylate) (PGMA)) onto the surface of these particles, which proved highly successful. The epoxide-rich PGMA shell permitted extensive surface bioconjugation of NeutrAvidin, as probed by an Lu-labeled biotin reporter (ca. 7 × 105 binding events per particle with a very low amount of non-specific binding) and analyzed by mass cytometry. It was shown that coupling agents such as EDC were not needed, such was the reactivity of the particle surface. These particles were stable and the addition of a polymeric shell was shown did not affect the narrow lanthanide ion distribution within the particle interior as analyzed by mass cytometry. These particles represent the most promising candidates for the development of a highly multiplexed bioassay based on lanthanide-labeled particles to date. PMID:21799543

PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

PubMed Central

Mitra, Avishek; Speer, Alexander; Lin, Kan; Ehrt, Sabine

2017-01-01

ABSTRACT Iron is essential for replication of Mycobacterium tuberculosis, but iron is efficiently sequestered in the human host during infection. Heme constitutes the largest iron reservoir in the human body and is utilized by many bacterial pathogens as an iron source. While heme acquisition is well studied in other bacterial pathogens, little is known in M. tuberculosis. To identify proteins involved in heme utilization by M. tuberculosis, a transposon mutant library was screened for resistance to the toxic heme analog gallium(III)-porphyrin (Ga-PIX). Inactivation of the ppe36, ppe62, and rv0265c genes resulted in resistance to Ga-PIX. Growth experiments using isogenic M. tuberculosis deletion mutants showed that PPE36 is essential for heme utilization by M. tuberculosis, while the functions of PPE62 and Rv0265c are partially redundant. None of the genes restored growth of the heterologous M. tuberculosis mutants, indicating that the proteins encoded by the genes have separate functions. PPE36, PPE62, and Rv0265c bind heme as shown by surface plasmon resonance spectroscopy and are associated with membranes. Both PPE36 and PPE62 proteins are cell surface accessible, while the Rv0265c protein is probably located in the periplasm. PPE36 and PPE62 are, to our knowledge, the first proline-proline-glutamate (PPE) proteins of M. tuberculosis that bind small molecules and are involved in nutrient acquisition. The absence of a virulence defect of the ppe36 deletion mutant indicates that the different iron acquisition pathways of M. tuberculosis may substitute for each other during growth and persistence in mice. The emerging model of heme utilization by M. tuberculosis as derived from this study is substantially different from those of other bacteria. PMID:28119467

Characterization of the Eimeria maxima sporozoite surface protein IMP1.

PubMed

Jenkins, M C; Fetterer, R; Miska, K; Tuo, W; Kwok, O; Dubey, J P

2015-07-30

The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection. Published by Elsevier B.V.

Hybrid organic-inorganic system for producing biofuels

DOEpatents

Yeh, Yi-Chun; Singer, Steven W.; Chhabra, Swapnil R.; Beller, Harry R.; Mueller, Jana

2017-10-03

The present invention provides for a system for converting CO.sub.2 and H.sub.2 to one or more biologically derived compounds. In some embodiments, the system comprises a host cell comprising one or more nucleic acids encoding genes for a recombinant surface display protein which is capable of tethering an electrocatalyst molecule, such as a cobalt(II) complex supported by tetradentate polypyridyl ligand 2-bis(2-pyridyl)(methoxy)methyl-6-pyridylpyridine (PY4), and enzymes for synthesizing a biologically derived compound, such as an alkane, alcohol, fatty acid, ester, or isoprenoid.

Modular protein switches derived from antibody mimetic proteins.

PubMed

Nicholes, N; Date, A; Beaujean, P; Hauk, P; Kanwar, M; Ostermeier, M

2016-02-01

Protein switches have potential applications as biosensors and selective protein therapeutics. Protein switches built by fusion of proteins with the prerequisite input and output functions are currently developed using an ad hoc process. A modular switch platform in which existing switches could be readily adapted to respond to any ligand would be advantageous. We investigated the feasibility of a modular protein switch platform based on fusions of the enzyme TEM-1 β-lactamase (BLA) with two different antibody mimetic proteins: designed ankyrin repeat proteins (DARPins) and monobodies. We created libraries of random insertions of the gene encoding BLA into genes encoding a DARPin or a monobody designed to bind maltose-binding protein (MBP). From these libraries, we used a genetic selection system for β-lactamase activity to identify genes that conferred MBP-dependent ampicillin resistance to Escherichia coli. Some of these selected genes encoded switch proteins whose enzymatic activity increased up to 14-fold in the presence of MBP. We next introduced mutations into the antibody mimetic domain of these switches that were known to cause binding to different ligands. To different degrees, introduction of the mutations resulted in switches with the desired specificity, illustrating the potential modularity of these platforms. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Glycosylphosphatidylinositol-Anchored Proteins in Fusarium graminearum: Inventory, Variability, and Virulence

PubMed Central

Rittenour, William R.; Harris, Steven D.

2013-01-01

The contribution of cell surface proteins to plant pathogenicity of fungi is not well understood. As such, the objective of this study was to investigate the functions and importance of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the wheat pathogen F. graminearum. GPI-APs are surface proteins that are attached to either the membrane or cell wall. In order to simultaneously disrupt several GPI-APs, a phosphoethanolamine transferase-encoding gene gpi7 was deleted and the resultant mutant characterized in terms of growth, development, and virulence. The Δgpi7 mutants exhibited slower radial growth rates and aberrantly shaped macroconidia. Furthermore, virulence tests and microscopic analyses indicated that Gpi7 is required for ramification of the fungus throughout the rachis of wheat heads. In parallel, bioinformatics tools were utilized to predict and inventory GPI-APs within the proteome of F. graminearum. Two of the genes identified in this screen (FGSG_01588 and FGSG_08844) displayed isolate-specific length variability as observed for other fungal cell wall adhesion genes. Nevertheless, deletion of these genes failed to reveal obvious defects in growth, development, or virulence. This research demonstrates the global importance of GPI-APs to in planta proliferation in F. graminearum, and also highlights the potential of individual GPI-APs as diagnostic markers. PMID:24312325

Single-Molecule Encoders for Tracking Motor Proteins on DNA

NASA Astrophysics Data System (ADS)

Lipman, Everett A.

2012-02-01

Devices such as inkjet printers and disk drives track position and velocity using optical encoders, which produce periodic signals precisely synchronized with linear or rotational motion. We have implemented this technique at the nanometer scale by labeling DNA with regularly spaced fluorescent dyes. The resulting molecular encoders can be used in several ways for high-resolution continuous tracking of individual motor proteins. These measurements do not require mechanical coupling to macroscopic instrumentation, are automatically calibrated by the underlying structure of DNA, and depend on signal periodicity rather than absolute level. I will describe the synthesis of single-molecule encoders, data from and modeling of experiments on a helicase and a DNA polymerase, and some ideas for future work.

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

PubMed Central

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants. PMID:27602024

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

PubMed

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants.

Characterization of the Genes Encoding the Cytosolic and Plastidial Forms of ADP-Glucose Pyrophosphorylase in Wheat Endosperm1

PubMed Central

Burton, Rachel A.; Johnson, Philip E.; Beckles, Diane M.; Fincher, Geoffrey B.; Jenner, Helen L.; Naldrett, Mike J.; Denyer, Kay

2002-01-01

In most species, the synthesis of ADP-glucose (Glc) by the enzyme ADP-Glc pyrophosphorylase (AGPase) occurs entirely within the plastids in all tissues so far examined. However, in the endosperm of many, if not all grasses, a second form of AGPase synthesizes ADP-Glc outside the plastid, presumably in the cytosol. In this paper, we show that in the endosperm of wheat (Triticum aestivum), the cytosolic form accounts for most of the AGPase activity. Using a combination of molecular and biochemical approaches to identify the cytosolic and plastidial protein components of wheat endosperm AGPase we show that the large and small subunits of the cytosolic enzyme are encoded by genes previously thought to encode plastidial subunits, and that a gene, Ta.AGP.S.1, which encodes the small subunit of the cytosolic form of AGPase, also gives rise to a second transcript by the use of an alternate first exon. This second transcript encodes an AGPase small subunit with a transit peptide. However, we could not find a plastidial small subunit protein corresponding to this transcript. The protein sequence of the purified plastidial small subunit does not match precisely to that encoded by Ta.AGP.S.1 or to the predicted sequences of any other known gene from wheat or barley (Hordeum vulgare). Instead, the protein sequence is most similar to those of the plastidial small subunits from chickpea (Cicer arietinum) and maize (Zea mays) and rice (Oryza sativa) seeds. These data suggest that the gene encoding the major plastidial small subunit of AGPase in wheat endosperm has yet to be identified. PMID:12428011

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

2011-03-08

The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding an F-box protein that interacts with a EIN3 involved in an ethylene response of plants, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding an F-box protein. The inventions also relates to methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein.