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Sample records for encoded receptor protein

  1. Cloning of human genes encoding novel G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  2. A macrophage inflammatory protein homolog encoded by guinea pig cytomegalovirus signals via CC chemokine receptor 1

    International Nuclear Information System (INIS)

    Penfold, Mark; Miao Zhenhua; Wang Yu; Haggerty, Shannon; Schleiss, Mark R.

    2003-01-01

    Cytomegaloviruses encode homologs of cellular immune effector proteins, including chemokines (CKs) and CK receptor-like G protein-coupled receptors (GPCRs). Sequence of the guinea pig cytomegalovirus (GPCMV) genome identified an open reading frame (ORF) which predicted a 101 amino acid (aa) protein with homology to the macrophage inflammatory protein (MIP) subfamily of CC (β) CKs, designated GPCMV-MIP. To assess functionality of this CK, recombinant GPCMV-MIP was expressed in HEK293 cells and assayed for its ability to bind to and functionally interact with a variety of GPCRs. Specific signaling was observed with the hCCR1 receptor, which could be blocked with hMIP -1α in competition experiments. Migration assays revealed that GPCMV-MIP was able to induce chemotaxis in hCCR1-L1.2 cells. Antisera raised against a GST-MIP fusion protein immunoprecipitated species of ∼12 and 10 kDa from GPCMV-inoculated tissue culture lysates, and convalescent antiserum from GPCMV-infected animals was immunoreactive with GST-MIP by ELISA assay. These results represent the first substantive in vitro characterization of a functional CC CK encoded by a cytomegalovirus

  3. Virally encoded 7TM receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Waldhoer, M; Lüttichau, H R

    2001-01-01

    expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets.......A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion...

  4. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Heiber, M.; Marchese, A.; O`Dowd, B.F. [Univ. of Toronto, Ontario (Canada)] [and others

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  5. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

    Science.gov (United States)

    Jones, Matthew L; Norman, Jane E; Morgan, Neil V; Mundell, Stuart J; Lordkipanidzé, Marie; Lowe, Gillian C; Daly, Martina E; Simpson, Michael A; Drake, Sian; Watson, Steve P; Mumford, Andrew D

    2015-04-01

    Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

  6. Tracking G-protein-coupled receptor activation using genetically encoded infrared probes.

    Science.gov (United States)

    Ye, Shixin; Zaitseva, Ekaterina; Caltabiano, Gianluigi; Schertler, Gebhard F X; Sakmar, Thomas P; Deupi, Xavier; Vogel, Reiner

    2010-04-29

    Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

  7. rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development.

    Science.gov (United States)

    Gates, Julie; Lam, Geanette; Ortiz, José A; Losson, Régine; Thummel, Carl S

    2004-01-01

    Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more

  8. NMDA receptors and memory encoding.

    Science.gov (United States)

    Morris, Richard G M

    2013-11-01

    It is humbling to think that 30 years have passed since the paper by Collingridge, Kehl and McLennan showing that one of Jeff Watkins most interesting compounds, R-2-amino-5-phosphonopentanoate (d-AP5), blocked the induction of long-term potentiation in vitro at synapses from area CA3 of the hippocampus to CA1 without apparent effect on baseline synaptic transmission (Collingridge et al., 1983). This dissociation was one of the key triggers for an explosion of interest in glutamate receptors, and much has been discovered since that collectively contributes to our contemporary understanding of glutamatergic synapses - their biophysics and subunit composition, of the agonists and antagonists acting on them, and their diverse functions in different networks of the brain and spinal cord. It can be fairly said that Collingridge et al.'s (1983) observation was the stimulus that has led, on the one hand, to structural biological work at the atomic scale describing the key features of NMDA receptors that enables their coincidence function to happen; and, on the other, to work with whole animals investigating the contributions that calcium signalling via this receptor can have on rhythmical activities controlled by spinal circuits, memory encoding in the hippocampus (the topic of this article), visual cortical plasticity, sensitization in pain, and other functions. In this article, I lay out how my then interest in long-term potentiation (LTP) as a model of memory enabled me to recognise the importance of Collingridge et al.'s discovery - and how I and my colleagues endeavoured to take things forward in the area of learning and memory. This is in some respects a personal story, and I tell it as such. The idea that NMDA receptor activation is essential for memory encoding, though not for storage, took time to develop and to be accepted. Along the way, there have been confusions, challenges, and surprises surrounding the idea that activation of NMDA receptors can

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

    International Nuclear Information System (INIS)

    Tao Yongguang; Song Xing; Deng Xiyun; Xie Daxin; Lee, Leo M.; Liu Yiping; Li Wei; Li Lili; Deng Lin; Wu Qiao; Gong Jianping; Cao Ya

    2005-01-01

    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 could 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

  10. Superfamily of genes encoding G protein-coupled receptors in the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae).

    Science.gov (United States)

    Wu, S-F; Yu, H-Y; Jiang, T-T; Gao, C-F; Shen, J-L

    2015-08-01

    G protein-coupled receptors (GPCRs) are the largest and most versatile superfamily of cell membrane proteins, which mediate various physiological processes including reproduction, development and behaviour. The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is one of the most notorious insect pests, preferentially feeding on cruciferous plants. P. xylostella is not only one of the world's most widespread lepidopteran insects, but has also developed resistance to nearly all classes of insecticides. Although the mechanisms of insecticide resistance have been studied extensively in many insect species, few investigations have been carried out on GPCRs in P. xylostella. In the present study, we identified 95 putative GPCRs in the P. xylostella genome. The identified GPCRs were compared with their homologues in Bombyx mori and Drosophila melanogaster. Our results suggest that GPCRs in different insect species may have evolved by a birth-and-death process. One of the differences among compared insects is the duplication of short neuropeptide F receptor and adipokinetic hormone receptors in P. xylostella and B. mori. Another divergence is the decrease in quantity and diversity of the stress-tolerance gene, Mth, in P. xylostella. The evolution by the birth-and-death process is probably involved in adaptation to the feeding behaviour, reproduction and stress responses of P. xylostella. Some of the genes identified in the present study could be potential targets for the development of novel pesticides. © 2015 The Royal Entomological Society.

  11. cDNA for the human β2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor

    International Nuclear Information System (INIS)

    Kobilka, B.K.; Dixon, R.A.F.; Frielle, T.

    1987-01-01

    The authors have isolated and sequenced a cDNA encoding the human β 2 -adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster β 2 -adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. They have localized the gene for the β 2 -adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor

  12. Chimeric Feline Coronaviruses That Encode Type II Spike Protein on Type I Genetic Background Display Accelerated Viral Growth and Altered Receptor Usage▿

    Science.gov (United States)

    Tekes, Gergely; Hofmann-Lehmann, Regina; Bank-Wolf, Barbara; Maier, Reinhard; Thiel, Heinz-Jürgen; Thiel, Volker

    2010-01-01

    Persistent infection of domestic cats with feline coronaviruses (FCoVs) can lead to a highly lethal, immunopathological disease termed feline infectious peritonitis (FIP). Interestingly, there are two serotypes, type I and type II FCoVs, that can cause both persistent infection and FIP, even though their main determinant of host cell tropism, the spike (S) protein, is of different phylogeny and displays limited sequence identity. In cell culture, however, there are apparent differences. Type II FCoVs can be propagated to high titers by employing feline aminopeptidase N (fAPN) as a cellular receptor, whereas the propagation of type I FCoVs is usually difficult, and the involvement of fAPN as a receptor is controversial. In this study we have analyzed the phenotypes of recombinant FCoVs that are based on the genetic background of type I FCoV strain Black but encode the type II FCoV strain 79-1146 S protein. Our data demonstrate that recombinant FCoVs expressing a type II FCoV S protein acquire the ability to efficiently use fAPN for host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein. PMID:19906918

  13. The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase

    NARCIS (Netherlands)

    Beisser, P.S.; Verzijl, D.; Gruijthuijsen, Y.K.; Beuken, E.V.; Smit, M.J.; Leurs, R.; Bruggeman, C.A.; Vink, C.

    2005-01-01

    Epstein-Barr vires (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis and Burkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesvirases. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has

  14. Epstein-Barr virus-encoded BARF1 protein is a decoy receptor for macrophage colony stimulating factor and interferes with macrophage differentiation and activation

    NARCIS (Netherlands)

    Hoebe, Eveline K.; Le Large, Tessa Y. S.; Tarbouriech, Nicolas; Oosterhoff, Dinja; de Gruijl, Tanja D.; Middeldorp, Jaap M.; Greijer, Astrid E.

    2012-01-01

    Epstein-Barr virus (EBV), like many other persistent herpes viruses, has acquired numerous mechanisms for subverting or evading immune surveillance. This study investigates the role of secreted EBV-encoded BARF1 protein (sBARF1) in creating an immune evasive microenvironment. Wild-type consensus

  15. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase

    Science.gov (United States)

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 famil...

  16. Molecular mechanisms for protein-encoded inheritance

    Science.gov (United States)

    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

  17. A common W556S mutation in the LDL receptor gene of Danish patients with familial hypercholesterolemia encodes a transport-defective protein

    DEFF Research Database (Denmark)

    Jensen, H K; Holst, H; Jensen, L G

    1997-01-01

    -Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein...

  18. The CXC chemokine receptor encoded by herpesvirus saimiri, ECRF3, shows ligand-regulated signaling through Gi, Gq, and G12/13 proteins but constitutive signaling only through Gi and G12/13 proteins

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; McLean, Katherine A; Holst, Peter J

    2004-01-01

    Open reading frame 74 (ORF74) of many gamma(2)-herpesviruses encodes a CXC chemokine receptor. The molecular pharmacological profile of ORF74 from herpesvirus saimiri, ECRF3, is characterized here and compared with that of the well known ORF74 from human herpesvirus 8 (HHV8). The ECRF3 receptor...... ligand selectivity of ECRF3 among ORF74 receptors could reflect differences in the cellular tropism of the gamma(2)-herpesviruses....

  19. Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Smit, M J; Waldhoer, M

    2008-01-01

    A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting...... pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we...

  20. Virus-encoded chemokine receptors--putative novel antiviral drug targets

    DEFF Research Database (Denmark)

    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...... 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...

  1. α/sub i/-3 cDNA encodes the α subunit of G/sub k/, the stimulatory G protein of receptor-regulated K+ channels

    International Nuclear Information System (INIS)

    Codina, J.; Olate, J.; Abramowitz, J.; Mattera, R.; Cook, R.G.; Birnbaumer, L.

    1988-01-01

    cDNA cloning has identified the presence in the human genome of three genes encoding α subunits of pertussis toxin substrates, generically called G/sub i/. They are named α/sub i/-1, α/sub i/-2 and α/sub i/-3. However, none of these genes has been functionally identified with any of the α subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A 2 , G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K + channels. The authors now report the nucleotide sequence and the complete predicted amino acid sequence of human liver α/sub i/-3 and the partial amino acid sequence of proteolytic fragments of the α subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of α/sub i/-3, thus identifying it as α/sub k/. The probable identity of α/sub i/-1 with α/sub p/ and possible roles for α/sub i/-2, as well as additional roles for α/sub i/-1 and α/sub i/-3 (α/sub k/) are discussed

  2. Bioinformatic prediction of G protein-coupled receptor encoding sequences from the transcriptome of the foreleg, including the Haller's organ, of the cattle tick, Rhipicephalus australis.

    Directory of Open Access Journals (Sweden)

    Sergio Munoz

    Full Text Available The cattle tick of Australia, Rhipicephalus australis, is a vector for microbial parasites that cause serious bovine diseases. The Haller's organ, located in the tick's forelegs, is crucial for host detection and mating. To facilitate the development of new technologies for better control of this agricultural pest, we aimed to sequence and annotate the transcriptome of the R. australis forelegs and associated tissues, including the Haller's organ. As G protein-coupled receptors (GPCRs are an important family of eukaryotic proteins studied as pharmaceutical targets in humans, we prioritized the identification and classification of the GPCRs expressed in the foreleg tissues. The two forelegs from adult R. australis were excised, RNA extracted, and pyrosequenced with 454 technology. Reads were assembled into unigenes and annotated by sequence similarity. Python scripts were written to find open reading frames (ORFs from each unigene. These ORFs were analyzed by different GPCR prediction approaches based on sequence alignments, support vector machines, hidden Markov models, and principal component analysis. GPCRs consistently predicted by multiple methods were further studied by phylogenetic analysis and 3D homology modeling. From 4,782 assembled unigenes, 40,907 possible ORFs were predicted. Using Blastp, Pfam, GPCRpred, TMHMM, and PCA-GPCR, a basic set of 46 GPCR candidates were compiled and a phylogenetic tree was constructed. With further screening of tertiary structures predicted by RaptorX, 6 likely GPCRs emerged and the strongest candidate was classified by PCA-GPCR to be a GABAB receptor.

  3. Tyrosylprotein sulfotransferase-1 and tyrosine sulfation of chemokine receptor 4 are induced by Epstein-Barr virus encoded latent membrane protein 1 and associated with the metastatic potential of human nasopharyngeal carcinoma.

    Directory of Open Access Journals (Sweden)

    Juan Xu

    Full Text Available The latent membrane protein 1 (LMP1, which is encoded by the Epstein-Barr virus (EBV, is an important oncogenic protein that is closely related to carcinogenesis and metastasis of nasopharyngeal carcinoma (NPC, a prevalent cancer in China. We previously reported that the expression of the functional chemokine receptor CXCR4 is associated with human NPC metastasis. In this study, we show that LMP1 induces tyrosine sulfation of CXCR4 through tyrosylprotein sulfotransferase-1 (TPST-1, an enzyme that is responsible for catalysis of tyrosine sulfation in vivo, which is likely to contribute to the highly metastatic character of NPC. LMP1 could induce tyrosine sulfation of CXCR4 and its associated cell motility and invasiveness in a NPC cell culture model. In contrast, the expression of TPST-1 small interfering RNA reversed LMP1-induced tyrosine sulfation of CXCR4. LMP1 conveys signals through the epidermal growth factor receptor (EGFR pathway, and EGFR-targeted siRNA inhibited the induction of TPST-1 by LMP1. We used a ChIP assay to show that EGFR could bind to the TPST-1 promoter in vivo under the control of LMP1. A reporter gene assay indicated that the activity of the TPST-1 promoter could be suppressed by deleting the binding site between EGFR and TPST-1. Finally, in human NPC tissues, the expression of TPST-1 and LMP1 was directly correlated and clinically, the expression of TPST-1 was associated with metastasis. These results suggest the up-regulation of TPST-1 and tyrosine sulfation of CXCR4 by LMP1 might be a potential mechanism contributing to NPC metastasis.

  4. Modeling structure of G protein-coupled receptors in huan genome

    KAUST Repository

    Zhang, Yang

    2016-01-01

    G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due

  5. Nucleic acid compositions and the encoding proteins

    Science.gov (United States)

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  6. Fluorescence-Based Multiplex Protein Detection Using Optically Encoded Microbeads

    Directory of Open Access Journals (Sweden)

    Dae Hong Jeong

    2012-03-01

    Full Text Available Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.

  7. The Expression of Genes Encoding Secreted Proteins in Medicago truncatula A17 Inoculated Roots

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    LUCIA KUSUMAWATI

    2013-09-01

    Full Text Available Subtilisin-like serine protease (MtSBT, serine carboxypeptidase (MtSCP, MtN5, non-specific lipid transfer protein (MtnsLTP, early nodulin2-like protein (MtENOD2-like, FAD-binding domain containing protein (MtFAD-BP1, and rhicadhesin receptor protein (MtRHRE1 were among 34 proteins found in the supernatant of M. truncatula 2HA and sickle cell suspension cultures. This study investigated the expression of genes encoding those proteins in roots and developing nodules. Two methods were used: quantitative real time RT-PCR and gene expression analysis (with promoter:GUS fusion in roots. Those proteins are predicted as secreted proteins which is indirectly supported by the findings that promoter:GUS fusions of six of the seven genes encoding secreted proteins were strongly expressed in the vascular bundle of transgenic hairy roots. All six genes have expressed in 14-day old nodule. The expression levels of the selected seven genes were quantified in Sinorhizobium-inoculated and control plants using quantitative real time RT-PCR. In conclusion, among seven genes encoding secreted proteins analyzed, the expression level of only one gene, MtN5, was up-regulated significantly in inoculated root segments compared to controls. The expression of MtSBT1, MtSCP1, MtnsLTP, MtFAD-BP1, MtRHRE1 and MtN5 were higher in root tip than in other tissues examined.

  8. Synthesis and structure-activity relationship of the first nonpeptidergic inverse agonists for the human cytomegalovirus encoded chemokine receptor US28

    NARCIS (Netherlands)

    Hulshof, Janneke W; Casarosa, Paola; Menge, Wiro M P B; Kuusisto, Leena M S; van der Goot, Henk; Smit, Martine J; de Esch, Iwan J P; Leurs, Rob

    2005-01-01

    US28 is a human cytomegalovirus (HCMV) encoded G-protein-coupled receptor that signals in a constitutively active manner. Recently, we identified 1 [5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile] as the first reported nonpeptidergic inverse agonist for a viral-encoded

  9. Direct Pathogenic Effects of HERV-encoded Proteins

    DEFF Research Database (Denmark)

    Hansen, Dorte Tranberg; Møller-Larsen, Anné; Petersen, Thor

    Background: Multiple sclerosis (MS) is a demyelinating, inflammatory disease of the central nervous system (CNS). MS is mediated by the immune system but the etiology of the disease remains unknown. Retroviral envelope (Env) proteins, encoded by human endogenous retroviruses (HERVs), are expressed...... in increased amounts on B cells from MS patients. Furthermore, the amount of anti-HERV antibodies in serum and cerebrospinal fluid from patients with MS is increased when compared with healthy controls. Aim: The overall aim of this project is to investigate the potential role of HERVs in the development of MS...... and the possible direct pathogenic effects of HERV-encoded Env proteins on the CNS. Methods: Construction and characterization of a panel of recombinant Env-proteins is initiated and their pathogenic potential will be investigated: Fusiogenic potential analyzed by flow cytometry and confocal microscopy. Analysis...

  10. Functional properties of Virus-Encoded and Virus-Regulated 7TM Receptors

    DEFF Research Database (Denmark)

    Spiess, Katja; Rosenkilde, Mette Marie

    2014-01-01

    During co-evolution with their hosts, viruses have developed several survival strategies that involve exploitation of 7TM receptors. These include virus-encoded 7TM receptors and ligands and viral regulation of endogenous receptors. Many functional properties have been ascribed to virus-exploited...

  11. Exhaustive search of linear information encoding protein-peptide recognition.

    Science.gov (United States)

    Kelil, Abdellali; Dubreuil, Benjamin; Levy, Emmanuel D; Michnick, Stephen W

    2017-04-01

    High-throughput in vitro methods have been extensively applied to identify linear information that encodes peptide recognition. However, these methods are limited in number of peptides, sequence variation, and length of peptides that can be explored, and often produce solutions that are not found in the cell. Despite the large number of methods developed to attempt addressing these issues, the exhaustive search of linear information encoding protein-peptide recognition has been so far physically unfeasible. Here, we describe a strategy, called DALEL, for the exhaustive search of linear sequence information encoded in proteins that bind to a common partner. We applied DALEL to explore binding specificity of SH3 domains in the budding yeast Saccharomyces cerevisiae. Using only the polypeptide sequences of SH3 domain binding proteins, we succeeded in identifying the majority of known SH3 binding sites previously discovered either in vitro or in vivo. Moreover, we discovered a number of sites with both non-canonical sequences and distinct properties that may serve ancillary roles in peptide recognition. We compared DALEL to a variety of state-of-the-art algorithms in the blind identification of known binding sites of the human Grb2 SH3 domain. We also benchmarked DALEL on curated biological motifs derived from the ELM database to evaluate the effect of increasing/decreasing the enrichment of the motifs. Our strategy can be applied in conjunction with experimental data of proteins interacting with a common partner to identify binding sites among them. Yet, our strategy can also be applied to any group of proteins of interest to identify enriched linear motifs or to exhaustively explore the space of linear information encoded in a polypeptide sequence. Finally, we have developed a webserver located at http://michnick.bcm.umontreal.ca/dalel, offering user-friendly interface and providing different scenarios utilizing DALEL.

  12. Poxvirus-encoded TNF decoy receptors inhibit the biological activity of transmembrane TNF.

    Science.gov (United States)

    Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

    2015-10-01

    Poxviruses encode up to four different soluble TNF receptors, named cytokine response modifier B (CrmB), CrmC, CrmD and CrmE. These proteins mimic the extracellular domain of the cellular TNF receptors to bind and inhibit the activity of TNF and, in some cases, other TNF superfamily ligands. Most of these ligands are released after the enzymic cleavage of a membrane precursor. However, transmembrane TNF (tmTNF) is not only a precursor of soluble TNF but also exerts specific pro-inflammatory and immunological activities. Here, we report that viral TNF receptors bound and inhibited tmTNF and describe some interesting differences in their activity against the soluble cytokine. Thus, CrmE, which does not inhibit mouse soluble TNF, could block murine tmTNF-induced cytotoxicity. We propose that this anti-tmTNF effect should be taken into consideration when assessing the role of viral TNF decoy receptors in the pathogenesis of poxvirus.

  13. Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

    DEFF Research Database (Denmark)

    Jacobsen, Linda; Madsen, P; Moestrup, S K

    1996-01-01

    the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine...... density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis......The 39-40-kDa receptor-associated protein (RAP) binds to the members of the low density lipoprotein receptor gene family and functions as a specialized endoplasmic reticulum/Golgi chaperone. Using RAP affinity chromatography, we have purified a novel approximately 250-kDa brain protein and isolated...

  14. The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues.

    Science.gov (United States)

    Ghysels, Bart; Ochsner, Urs; Möllman, Ute; Heinisch, Lothar; Vasil, Michael; Cornelis, Pierre; Matthijs, Sandra

    2005-05-15

    Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two

  15. Analyses of pea necrotic yellow dwarf virus-encoded proteins.

    Science.gov (United States)

    Krenz, Björn; Schießl, Ingrid; Greiner, Eva; Krapp, Susanna

    2017-06-01

    Pea necrotic yellow dwarf virus (PNYDV) is a multipartite, circular, single-stranded DNA plant virus. PNYDV encodes eight proteins and the function of three of which remains unknown-U1, U2, and U4. PNYDV proteins cellular localization was analyzed by GFP tagging and bimolecular fluorescence complementation (BiFC) studies. The interactions of all eight PNYDV proteins were tested pairwise in planta (36 combinations in total). Seven interactions were identified and two (M-Rep with CP and MP with U4) were characterized further. MP and U4 complexes appeared as vesicle-like spots and were localized at the nuclear envelope and cell periphery. These vesicle-like spots were associated with the endoplasmatic reticulum. In addition, a nuclear localization signal (NLS) was mapped for U1, and a mutated U1 with NLS disrupted localized at plasmodesmata and therefore might also have a role in movement. Taken together, this study provides evidence for previously undescribed nanovirus protein-protein interactions and their cellular localization with novel findings not only for those proteins with unknown function, but also for characterized proteins such as the CP.

  16. Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

    Science.gov (United States)

    Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

    2008-12-01

    Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

  17. Genotype-Phenotype Associations of the CD-Associated Single Nucleotide Polymorphism within the Gene Locus Encoding Protein Tyrosine Phosphatase Non-Receptor Type 22 in Patients of the Swiss IBD Cohort.

    Directory of Open Access Journals (Sweden)

    Marianne R Spalinger

    Full Text Available Protein tyrosine phosphatase non-receptor type 22 (PTPN22 plays an important role in immune cell function and intestinal homeostasis. The single nucleotide polymorphism (SNP rs2476601 within the PTPN22 gene locus results in aberrant function of PTPN22 protein and protects from Crohn's disease (CD. Here, we investigated associations of PTPN22 SNP rs2476601 in inflammatory bowel disease (IBD patients in the Swiss IBD Cohort Study (SIBDCS.2'028 SIBDCS patients (1173 CD and 855 ulcerative colitis (UC patients were included. The clinical characteristics were analysed for an association with the presence of the PTPN22 SNP rs2476601 genotypes 'homozygous variant' (AA, 'heterozygous' (GA and 'homozygous wild-type' (GG.13 patients (0.6% were homozygous variant (AA for the PTPN22 polymorphism, 269 (13.3% heterozygous variant (GA and 1'746 (86.1% homozygous wild-type (GG. In CD, AA and GA genotypes were associated with less use of steroids and antibiotics, and reduced prevalence of vitamin D and calcium deficiency. In UC the AA and GA genotype was associated with increased use of azathioprine and anti-TNF antibodies, but significantly less patients with the PTPN22 variant featured malabsorption syndrome (p = 0.026.Our study for the first time addressed how presence of SNP rs2476601 within the PTPN22 gene affects clinical characteristics in IBD-patients. Several factors that correlate with more severe disease were found to be less common in CD patients carrying the A-allele, pointing towards a protective role for this variant in affected CD patients. In UC patients however, we found the opposite trend, suggesting a disease-promoting effect of the A-allele.

  18. Synthesis and pharmacological characterization of novel inverse agonists acting on the viral-encoded chemokine receptor US28.

    Science.gov (United States)

    Hulshof, Janneke W; Vischer, Henry F; Verheij, Mark H P; Fratantoni, Silvina A; Smit, Martine J; de Esch, Iwan J P; Leurs, Rob

    2006-11-01

    G-protein coupled receptors encoded by viruses represent an unexplored class of potential drug targets. In this study, we describe the synthesis and pharmacological characterization of the first class of inverse agonists acting on the HCMV-encoded receptor US28. It is shown that replacement of the 4-hydroxy group of lead compound 1 with a methylamine group results in a significant 6-fold increase in affinity. Interestingly, increasing the rigidity of the spacer by the introduction of a double bond also leads to a significant increase in binding affinity compared to 1. These novel inverse agonists serve as valuable tools to elucidate the role of constitutive signaling in the pathogenesis of viral infection and may have therapeutic potential as leads for new antiviral drugs.

  19. Properties of virion transactivator proteins encoded by primate cytomegaloviruses

    Directory of Open Access Journals (Sweden)

    Barry Peter A

    2009-05-01

    Full Text Available Abstract Background Human cytomegalovirus (HCMV is a betaherpesvirus that causes severe disease in situations where the immune system is immature or compromised. HCMV immediate early (IE gene expression is stimulated by the virion phosphoprotein pp71, encoded by open reading frame (ORF UL82, and this transactivation activity is important for the efficient initiation of viral replication. It is currently recognized that pp71 acts to overcome cellular intrinsic defences that otherwise block viral IE gene expression, and that interactions of pp71 with the cell proteins Daxx and ATRX are important for this function. A further property of pp71 is the ability to enable prolonged gene expression from quiescent herpes simplex virus type 1 (HSV-1 genomes. Non-human primate cytomegaloviruses encode homologs of pp71, but there is currently no published information that addresses their effects on gene expression and modes of action. Results The UL82 homolog encoded by simian cytomegalovirus (SCMV, strain Colburn, was identified and cloned. This ORF, named S82, was cloned into an HSV-1 vector, as were those from baboon, rhesus monkey and chimpanzee cytomegaloviruses. The use of an HSV-1 vector enabled expression of the UL82 homologs in a range of cell types, and permitted investigation of their abilities to direct prolonged gene expression from quiescent genomes. The results show that all UL82 homologs activate gene expression, and that neither host cell type nor promoter target sequence has major effects on these activities. Surprisingly, the UL82 proteins specified by non-human primate cytomegaloviruses, unlike pp71, did not direct long term expression from quiescent HSV-1 genomes. In addition, significant differences were observed in the intranuclear localization of the UL82 homologs, and in their effects on Daxx. Strikingly, S82 mediated the release of Daxx from nuclear domain 10 substructures much more rapidly than pp71 or the other proteins tested. All

  20. Protein tyrosine phosphatase receptor type R deficient mice exhibit increased exploration in a new environment and impaired novel object recognition memory

    NARCIS (Netherlands)

    Erkens, M.; Bakker, B.; Duijn, L.M. van; Hendriks, W.J.A.J.; Zee, C.E.E.M. van der

    2014-01-01

    Mouse gene Ptprr encodes multiple protein tyrosine phosphatase receptor type R (PTPRR) isoforms that negatively regulate mitogen-activated protein kinase (MAPK) signaling pathways. In the mouse brain, PTPRR proteins are expressed in cerebellum, olfactory bulb, hippocampus, amygdala and perirhinal

  1. Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins.

    Science.gov (United States)

    Ramírez-Sánchez, Obed; Pérez-Rodríguez, Paulino; Delaye, Luis; Tiessen, Axel

    2016-12-01

    Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylogenetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa), average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ∼81aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ∼10 exons of small size [∼176 nucleotides (nt)]. Streptophyta have on average only ∼5.7 exons of medium size (∼230nt). Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400nt). Among subcellular compartments, membrane proteins are the largest (∼520aa), whereas the smallest proteins correspond to the gene ontology group of ribosome (∼240aa). Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ∼34% more but ∼20% smaller proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  2. Plant Proteins Are Smaller Because They Are Encoded by Fewer Exons than Animal Proteins

    Directory of Open Access Journals (Sweden)

    Obed Ramírez-Sánchez

    2016-12-01

    Full Text Available Protein size is an important biochemical feature since longer proteins can harbor more domains and therefore can display more biological functionalities than shorter proteins. We found remarkable differences in protein length, exon structure, and domain count among different phylogenetic lineages. While eukaryotic proteins have an average size of 472 amino acid residues (aa, average protein sizes in plant genomes are smaller than those of animals and fungi. Proteins unique to plants are ∼81 aa shorter than plant proteins conserved among other eukaryotic lineages. The smaller average size of plant proteins could neither be explained by endosymbiosis nor subcellular compartmentation nor exon size, but rather due to exon number. Metazoan proteins are encoded on average by ∼10 exons of small size [∼176 nucleotides (nt]. Streptophyta have on average only ∼5.7 exons of medium size (∼230 nt. Multicellular species code for large proteins by increasing the exon number, while most unicellular organisms employ rather larger exons (>400 nt. Among subcellular compartments, membrane proteins are the largest (∼520 aa, whereas the smallest proteins correspond to the gene ontology group of ribosome (∼240 aa. Plant genes are encoded by half the number of exons and also contain fewer domains than animal proteins on average. Interestingly, endosymbiotic proteins that migrated to the plant nucleus became larger than their cyanobacterial orthologs. We thus conclude that plants have proteins larger than bacteria but smaller than animals or fungi. Compared to the average of eukaryotic species, plants have ∼34% more but ∼20% smaller proteins. This suggests that photosynthetic organisms are unique and deserve therefore special attention with regard to the evolutionary forces acting on their genomes and proteomes.

  3. Protein Connectivity in Chemotaxis Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Stephan Eismann

    2015-12-01

    Full Text Available The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.

  4. The herpesvirus 8-encoded chemokine vMIP-II, but not the poxvirus-encoded chemokine MC148, inhibits the CCR10 receptor

    DEFF Research Database (Denmark)

    Lüttichau, H R; Lewis, I C; Gerstoft, J

    2001-01-01

    The viral chemokine antagonist vMIP-II encoded by human herpesvirus 8 (HHV8) and MC148 encoded by the poxvirus - Molluscum contagiosum - were tested against the newly identified chemokine receptor CCR10. As the CCR10 ligand ESkine / CCL27 had the highest identity to MC148 and because both...

  5. The cytomegalovirus-encoded chemokine receptor US28 promotes intestinal neoplasia in transgenic mice

    NARCIS (Netherlands)

    Bongers, Gerold; Maussang, David; Muniz, Luciana R; Noriega, Vanessa M; Fraile-Ramos, Alberto; Barker, Nick; Marchesi, Federica; Thirunarayanan, Nanthakumar; Vischer, Henry F; Qin, Lihui; Mayer, Lloyd; Harpaz, Noam; Leurs, Rob; Furtado, Glaucia C; Clevers, Hans; Tortorella, Domenico; Smit, Martine J; Lira, Sergio A

    2010-01-01

    US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice)

  6. Internalization of G-protein-coupled receptors: Implication in receptor function, physiology and diseases.

    Science.gov (United States)

    Calebiro, Davide; Godbole, Amod

    2018-04-01

    G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and mediate the effects of numerous hormones and neurotransmitters. The nearly 1000 GPCRs encoded by the human genome regulate virtually all physiological functions and are implicated in the pathogenesis of prevalent human diseases such as thyroid disorders, hypertension or Parkinson's disease. As a result, 30-50% of all currently prescribed drugs are targeting these receptors. Once activated, GPCRs induce signals at the cell surface. This is often followed by internalization, a process that results in the transfer of receptors from the plasma membrane to membranes of the endosomal compartment. Internalization was initially thought to be mainly implicated in signal desensitization, a mechanism of adaptation to prolonged receptor stimulation. However, several unexpected functions have subsequently emerged. Most notably, accumulating evidence indicates that internalization can induce prolonged receptor signaling on intracellular membranes, which is apparently required for at least some biological effects of hormones like TSH, LH and adrenaline. These findings reveal an even stronger connection between receptor internalization and signaling than previously thought. Whereas new studies are just beginning to reveal an important physiological role for GPCR signaling after internalization and ways to exploit it for therapeutic purposes, future investigations will be required to explore its involvement in human disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

    Directory of Open Access Journals (Sweden)

    Bryan D Moyer

    Full Text Available BACKGROUND: Using fungiform (FG and circumvallate (CV taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive, sour cells (PKD2L1-positive, as well as other taste cell populations. Transmembrane protein 44 (TMEM44, a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1, a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1, a calcium-binding transmembrane protein; and anoctamin 7 (ANO7, a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B, a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins

  8. Chromosome locations of genes encoding human signal transduction adapter proteins, Nck (NCK), Shc (SHC1), and Grb2 (GRB2)

    DEFF Research Database (Denmark)

    Huebner, K; Kastury, K; Druck, T

    1994-01-01

    "adapter" proteins, which are involved in transducing signals from receptor tyrosine kinases to downstream signal recipients such as ras, because adaptor protein genes could also, logically, serve as targets of mutation, rearrangement, or other aberration in disease. Therefore, DNAs from panels of rodent-human......Abnormalities due to chromosomal aberration or point mutation in gene products of growth factor receptors or in ras gene products, which lie on the same signaling pathway, can cause disease in animals and humans. Thus, it can be important to determine chromosomal map positions of genes encoding...... hybrids carrying defined complements of human chromosomes were assayed for the presence of the cognate genes for NCK, SHC, and GRB2, three SH2 or SH2/SH3 (Src homology 2 and 3) domain-containing adapter proteins. Additionally, NCK and SHC genes were more narrowly localized by chromosomal in situ...

  9. G-protein coupling of cannabinoid receptors

    International Nuclear Information System (INIS)

    Glass, M.

    2001-01-01

    Full text: Since the cloning of the cannabinoid CB1 and CB2 receptors in the early 1990's extensive research has focused on understanding their signal transduction pathways. While it has been known for sometime that both receptors can couple to intracellular signalling via pertussis toxin sensitive G-proteins (Gi/Go), the specificity and kinetics of these interactions have only recently been elucidated. We have developed an in situ reconstitution approach to investigating receptor-G-protein interactions. This approach involves chaotropic extraction of receptor containing membranes in order to inactivate or remove endogenous G-proteins. Recombinant or isolated brain G-proteins can then be added back to the receptors, and their activation monitored through the binding of [ 35 S]-GTPγS. This technique has been utilised for an extensive study of cannabinoid receptor mediated activation of G-proteins. In these studies we have established that CB1 couples with high affinity to both Gi and Go type G-proteins. In contrast, CB2 couples strongly to Gi, but has a very low affinity for Go. This finding correlated well with the previous findings that while CB1 and CB2 both couple to the inhibition of adenylate cyclase, CB1 but not CB2 could also inhibit calcium channels. We then examined the ability of a range of cannabinoid agonists to activate the Gi and Go via CB1. Conventional receptor theory suggests that a receptor is either active or inactive with regard to a G-protein and that the active receptor activates all relevant G-proteins equally. However, in this study we found that agonists could produce different degrees of activation, depending on which G-protein was present. Further studies have compared the ability of the two endocannabinoids to drive the activation of Gi or Go. These studies show that agonists can induce multiple forms of activated receptor that differ in their ability to catalyse the activation of Gi or Go. The ability of an agonist to drive a receptor

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

    International Nuclear Information System (INIS)

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

    2017-01-01

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

  11. Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

    Science.gov (United States)

    Freimuth, Paul I.

    2010-04-06

    The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

  12. TARM1 Is a Novel Leukocyte Receptor Complex-Encoded ITAM Receptor That Costimulates Proinflammatory Cytokine Secretion by Macrophages and Neutrophils

    DEFF Research Database (Denmark)

    Radjabova, Valeria; Mastroeni, Piero; Skjødt, Karsten

    2015-01-01

    We identified a novel, evolutionarily conserved receptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell-interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residu...

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

    Science.gov (United States)

    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.

  14. The implications of alternative splicing in the ENCODE protein complement

    DEFF Research Database (Denmark)

    Tress, Michael L.; Martelli, Pier Luigi; Frankish, Adam

    2007-01-01

    suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has...

  15. Receptor-interacting protein (RIP) kinase family

    OpenAIRE

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

  16. Amtyr1: characterization of a gene from honeybee (Apis mellifera) brain encoding a functional tyramine receptor.

    Science.gov (United States)

    Blenau, W; Balfanz, S; Baumann, A

    2000-03-01

    Biogenic amine receptors are involved in the regulation and modulation of various physiological and behavioral processes in both vertebrates and invertebrates. We have cloned a member of this gene family from the CNS of the honeybee, Apis mellifera. The deduced amino acid sequence is homologous to tyramine receptors cloned from Locusta migratoria and Drosophila melanogaster as well as to an octopamine receptor cloned from Heliothis virescens. Functional properties of the honeybee receptor were studied in stably transfected human embryonic kidney 293 cells. Tyramine reduced forskolin-induced cyclic AMP production in a dose-dependent manner with an EC50 of approximately 130 nM. A similar effect of tyramine was observed in membrane homogenates of honeybee brains. Octopamine also reduced cyclic AMP production in the transfected cell line but was both less potent (EC50 of approximately 3 microM) and less efficacious than tyramine. Receptor-encoding mRNA has a wide-spread distribution in the brain and subesophageal ganglion of the honeybee, suggesting that this tyramine receptor is involved in sensory signal processing as well as in higher-order brain functions.

  17. The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus

    NARCIS (Netherlands)

    Boot, H.J.; Kolen, C.P.A.M.; Pot, B.; Kersters, K.; Pouwels, P.H.

    1996-01-01

    Previously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this

  18. An α-Helical Core Encodes the Dual Functions of the Chlamydial Protein IncA*

    Science.gov (United States)

    Ronzone, Erik; Wesolowski, Jordan; Bauler, Laura D.; Bhardwaj, Anshul; Hackstadt, Ted; Paumet, Fabienne

    2014-01-01

    Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2. PMID:25324548

  19. An α-helical core encodes the dual functions of the chlamydial protein IncA.

    Science.gov (United States)

    Ronzone, Erik; Wesolowski, Jordan; Bauler, Laura D; Bhardwaj, Anshul; Hackstadt, Ted; Paumet, Fabienne

    2014-11-28

    Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.

    Directory of Open Access Journals (Sweden)

    John G Yamauchi

    2011-01-01

    Full Text Available We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC by developing sensor cells stably expressing a Ca(2+ permeable LGIC and a genetically encoded Förster (or fluorescence resonance energy transfer (FRET-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT(3A serotonin receptors and a chimera of human α7/mouse 5-HT(3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

  1. Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74

    DEFF Research Database (Denmark)

    McLean, Katherine A; Holst, Peter J; Martini, Lene

    2004-01-01

    The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding...

  2. Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

    Science.gov (United States)

    Fischer, J A; Muff, R; Born, W

    2002-08-01

    The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

  3. Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

    Science.gov (United States)

    Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

    2012-04-01

    The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

  4. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    Science.gov (United States)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  5. Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae)

    OpenAIRE

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2014-01-01

    The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino a...

  6. RTA, a candidate G protein-coupled receptor: Cloning, sequencing, and tissue distribution

    International Nuclear Information System (INIS)

    Ross, P.C.; Figler, R.A.; Corjay, M.H.; Barber, C.M.; Adam, N.; Harcus, D.R.; Lynch, K.R.

    1990-01-01

    Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M 1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. TRA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. They conclude that RTA is not an angiotensin receptor; to date, they have been unable to identify its ligand

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

    Science.gov (United States)

    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

  8. Molecular quantification of genes encoding for green-fluorescent proteins

    DEFF Research Database (Denmark)

    Felske, A; Vandieken, V; Pauling, B V

    2003-01-01

    A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification...... PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant....

  9. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

    Science.gov (United States)

    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

    The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Mnemons: encoding memory by protein super-assembly

    Directory of Open Access Journals (Sweden)

    Fabrice Caudron

    2015-02-01

    Full Text Available Memory is mainly understood as the recollection of past events. The human brain and its simplest unit, the synapse, belong to the places in which such memories are physically stored. From an experimental point of view, memory can be tested in humans by recall. However, in other organisms, memory is reflected in its use by individuals to learn about and adapt their behavior to their environment. Under this criterion, even unicellular organisms are able to learn from their environments and show the ability to adapt their responses to repeating stimuli. This indicates that they are able to keep track of their histories and use these traces to elaborate adapted responses, making these traces akin to memory encodings. Understanding these phenomena may even help us to dissect part of the rather complex molecular orchestration happening in our synapses. When exposed unsuccessfully to mating pheromone, i.e. when mating does not happen, budding yeast cells become refractory to the mating signal. This refractory state is restricted to the mother cell and not inherited by the daughter cells, even though it is stable for most if not the entire life span of the mother cell. Interestingly, both stability and asymmetric segregation of the acquired state are explained by the molecular mechanism underlying its establishment, which shows important analogies and distinctions to prions. Here we discuss these similarities and differences

  11. Mnemons: encoding memory by protein super-assembly.

    Science.gov (United States)

    Caudron, Fabrice; Barral, Yves

    2014-02-25

    Memory is mainly understood as the recollection of past events. The human brain and its simplest unit, the synapse, belong to the places in which such memories are physically stored. From an experimental point of view, memory can be tested in humans by recall. However, in other organisms, memory is reflected in its use by individuals to learn about and adapt their behavior to their environment. Under this criterion, even unicellular organisms are able to learn from their environments and show the ability to adapt their responses to repeating stimuli. This indicates that they are able to keep track of their histories and use these traces to elaborate adapted responses, making these traces akin to memory encodings. Understanding these phenomena may even help us to dissect part of the rather complex molecular orchestration happening in our synapses. When exposed unsuccessfully to mating pheromone, i.e. when mating does not happen, budding yeast cells become refractory to the mating signal. This refractory state is restricted to the mother cell and not inherited by the daughter cells, even though it is stable for most if not the entire life span of the mother cell. Interestingly, both stability and asymmetric segregation of the acquired state are explained by the molecular mechanism underlying its establishment, which shows important analogies and distinctions to prions. Here we discuss these similarities and differences.

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

    Science.gov (United States)

    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

  13. High-performance fluorescence-encoded magnetic microbeads as microfluidic protein chip supports for AFP detection

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Xiaoqun [School of Life Sciences, Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072 (China); Yan, Huan; Yang, Jiumin [Department of Laboratory Medicine, Tianjin Medical University General Hospital, Tianjin, 300052 (China); Wu, Yudong; Zhang, Jian; Yao, Yingyi [School of Life Sciences, Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072 (China); Liu, Ping [Bioscience (Tianjin) Diagnostic Technology CO., LTD, Tianjin, 300300 (China); Wang, Huiquan [Department of Biomedical Engineering, School of Electronics and Information Engineering, Tianjin Polytechnic University, Tianjin, 300387 (China); Hu, Zhidong, E-mail: huzhidong27@163.com [Department of Laboratory Medicine, Tianjin Medical University General Hospital, Tianjin, 300052 (China); Chang, Jin, E-mail: jinchang@tju.edu.cn [School of Life Sciences, Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072 (China)

    2016-10-05

    Fluorescence-encoded magnetic microbeads (FEMMs), with the fluorescence encoding ability of quantum dots (QDs) and magnetic enrichment and separation functions of Fe{sub 3}O{sub 4} nanoparticles, have been widely used for multiple biomolecular detection as microfluidic protein chip supports. However, the preparation of FEMMs with long-term fluorescent encoding and immunodetection stability is still a challenge. In this work, we designed a novel high-temperature chemical swelling strategy. The QDs and Fe{sub 3}O{sub 4} nanoparticles were effectively packaged into microbeads via the thermal motion of the polymer chains and the hydrophobic interaction between the nanoparticles and microbeads. The FEMMs obtained a highly uniform fluorescent property and long-term encoding and immunodetection stability and could be quickly magnetically separated and enriched. Then, the QD-encoded magnetic microbeads were applied to alpha fetoprotein (AFP) detection via sandwich immunoreaction. The properties of the encoded microspheres were characterized using a self-designed detecting apparatus, and the target molecular concentration in the sample was also quantified. The results suggested that the high-performance FEMMs have great potential in the field of biomolecular detection. - Graphical abstract: We designed a novel strategy to prepare a kind of high-performance fluorescence-encoded magnetic microbeads as microfluidic protein chip support with long-time fluorescent encoding and immunodetection stability for AFP detection. - Highlights: • A novel strategy combined the high temperature with chemical swelling technology is designed. • Based on hydrophobic interaction and polymer thermal motion, QDs and Fe{sub 3}O{sub 4} were effectively packaged into microbeads. • The fluorescence-encoded magnetic microbeads show long-term fluorescent encoding and immunodetection stability.

  14. Cellular and molecular biology of orphan G protein-coupled receptors.

    Science.gov (United States)

    Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

    2006-01-01

    The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

  15. The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis

    DEFF Research Database (Denmark)

    Waldhoer, Maria; Casarosa, Paola; Rosenkilde, Mette M

    2003-01-01

    are separable entities in this viral chemokine receptor. We generated chimeric and mutant US28 proteins that were altered in either their constitutive endocytic (US28 Delta 300, US28 Delta 317, US28-NK1-ctail, and US28-ORF74-ctail) or signaling properties (US28R129A). By using this series of mutants, we show...... further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX3CL1 and show that in the absence of the US28 C terminus, fractalkine/CX3CL1 acts as an agonist on US28. This demonstrates for the first time that the endocytic properties of a 7TM......US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28...

  16. Gardenia jasminoides Encodes an Inhibitor-2 Protein for Protein Phosphatase Type 1

    Science.gov (United States)

    Gao, Lan; Li, Hao-Ming

    2017-08-01

    Protein phosphatase-1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. Inhibitor-2 (I-2) can inhibit the activity of PP1 and has been found in diverse organisms. In this work, a Gardenia jasminoides fruit cDNA library was constructed, and the GjI-2 cDNA was isolated from the cDNA library by sequencing method. The GjI-2 cDNA contains a predicted 543 bp open reading frame that encodes 180 amino acids. The bioinformatics analysis suggested that the GjI-2 has conserved PP1c binding motif, and contains a conserved phosphorylation site, which is important in regulation of its activity. The three-dimensional model structure of GjI-2 was buite, its similar with the structure of I-2 from mouse. The results suggest that GjI-2 has relatively conserved RVxF, FxxR/KxR/K and HYNE motif, and these motifs are involved in interaction with PP1.

  17. Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.

    Science.gov (United States)

    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.

  18. Novel nuclear-encoded proteins interacting with a plastid sigma factor, Sig1, in Arabidopsis thaliana.

    Science.gov (United States)

    Morikawa, Kazuya; Shiina, Takashi; Murakami, Shinya; Toyoshima, Yoshinori

    2002-03-13

    Sigma factor binding proteins are involved in modifying the promoter preferences of the RNA polymerase in bacteria. We found the nuclear encoded protein (SibI) that is transported into chloroplasts and interacts specifically with the region 4 of Sig1 in Arabidopsis. SibI and its homologue, T3K9.5 are novel proteins, which are not homologous to any protein of known function. The expression of sibI was tissue specific, light dependent, and developmentally timed. We suggest the transcriptional regulation by sigma factor binding proteins to function in the plastids of higher plant.

  19. Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B

    International Nuclear Information System (INIS)

    Brown-Shimer, S.; Johnson, K.A.; Bruskin, A.; Green, N.R.; Hill, D.E.; Lawrence, J.B.; Johnson, C.

    1990-01-01

    The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatases function as growth suppressors, the authors have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1,305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B. A genomic clone has been isolated and used in an in situ hybridization to banded metaphase chromosomes to determine that the gene encoding protein phosphotyrosyl phosphatase 1B maps as a single-copy gene to the long arm of chromosome 20 in the region q13.1-q13.2

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  1. The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.

    NARCIS (Netherlands)

    de Munnik, S.M.; van der Lee, R.; Velders, D.M.; van Offenbeek, J.; Smits-de Vries, L.; Leurs, R.; Smit, M.J.; Vischer, H.F.

    2016-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth

  2. Identification of physicochemical selective pressure on protein encoding nucleotide sequences

    Directory of Open Access Journals (Sweden)

    Sainudiin Raazesh

    2006-03-01

    Full Text Available Abstract Background Statistical methods for identifying positively selected sites in protein coding regions are one of the most commonly used tools in evolutionary bioinformatics. However, they have been limited by not taking the physiochemical properties of amino acids into account. Results We develop a new codon-based likelihood model for detecting site-specific selection pressures acting on specific physicochemical properties. Nonsynonymous substitutions are divided into substitutions that differ with respect to the physicochemical properties of interest, and those that do not. The substitution rates of these two types of changes, relative to the synonymous substitution rate, are then described by two parameters, γ and ω respectively. The new model allows us to perform likelihood ratio tests for positive selection acting on specific physicochemical properties of interest. The new method is first used to analyze simulated data and is shown to have good power and accuracy in detecting physicochemical selective pressure. We then re-analyze data from the class-I alleles of the human Major Histocompatibility Complex (MHC and from the abalone sperm lysine. Conclusion Our new method allows a more flexible framework to identify selection pressure on particular physicochemical properties.

  3. Receptor-interacting protein (RIP) kinase family

    Science.gov (United States)

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

  4. Molecular evolution of the Paramyxoviridae and Rhabdoviridae multiple-protein-encoding P gene.

    Science.gov (United States)

    Jordan, I K; Sutter, B A; McClure, M A

    2000-01-01

    Presented here is an analysis of the molecular evolutionary dynamics of the P gene among 76 representative sequences of the Paramyxoviridae and Rhabdoviridae RNA virus families. In a number of Paramyxoviridae taxa, as well as in vesicular stomatitis viruses of the Rhabdoviridae, the P gene encodes multiple proteins from a single genomic RNA sequence. These products include the phosphoprotein (P), as well as the C and V proteins. The complexity of the P gene makes it an intriguing locus to study from an evolutionary perspective. Amino acid sequence alignments of the proteins encoded at the P and N loci were used in independent phylogenetic reconstructions of the Paramyxoviridae and Rhabdoviridae families. P-gene-coding capacities were mapped onto the Paramyxoviridae phylogeny, and the most parsimonious path of multiple-coding-capacity evolution was determined. Levels of amino acid variation for Paramyxoviridae and Rhabdoviridae P-gene-encoded products were also analyzed. Proteins encoded in overlapping reading frames from the same nucleotides have different levels of amino acid variation. The nucleotide architecture that underlies the amino acid variation was determined in order to evaluate the role of selection in the evolution of the P gene overlapping reading frames. In every case, the evolution of one of the proteins encoded in the overlapping reading frames has been constrained by negative selection while the other has evolved more rapidly. The integrity of the overlapping reading frame that represents a derived state is generally maintained at the expense of the ancestral reading frame encoded by the same nucleotides. The evolution of such multicoding sequences is likely a response by RNA viruses to selective pressure to maximize genomic information content while maintaining small genome size. The ability to evolve such a complex genomic strategy is intimately related to the dynamics of the viral quasispecies, which allow enhanced exploration of the adaptive

  5. Ixodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins.

    Science.gov (United States)

    Daix, V; Schroeder, H; Praet, N; Georgin, J-P; Chiappino, I; Gillet, L; de Fays, K; Decrem, Y; Leboulle, G; Godfroid, E; Bollen, A; Pastoret, P-P; Gern, L; Sharp, P M; Vanderplasschen, A

    2007-04-01

    The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures. Recently, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., 2000; J. Biol. Chem. 275, 18717-18723). Here, we isolated two different sequences, similar to Isac, from the transcriptome of I. ricinus salivary glands. Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins, called I. ricinus anticomplement (IRAC) protein I and II, are coexpressed constitutively in I. ricinus salivary glands and are upregulated during blood feeding. Also, we demonstrated that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses demonstrate that ticks belonging to the Ixodes ricinus complex encode a family of relatively small anticomplement molecules undergoing diversification by positive Darwinian selection.

  6. Protein Secondary Structure Prediction Using AutoEncoder Network and Bayes Classifier

    Science.gov (United States)

    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.

  7. Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis.

    NARCIS (Netherlands)

    Hollander, A.I. den; Koenekoop, R.K.; Mohamed, M.D.; Arts, H.H.; Boldt, K.; Towns, K.V.; Sedmak, T.; Beer, M. de; Nagel-Wolfrum, K.; McKibbin, M.; Dharmaraj, S.; Lopez, I.; Ivings, L.; Williams, G.A.; Springell, K.; Woods, C.G.; Jafri, H.; Rashid, Y.; Strom, T.M.; Zwaag, B. van der; Gosens, I.; Kersten, F.F.J.; Wijk, E. van; Veltman, J.A.; Zonneveld, M.N.; Beersum, S.E.C. van; Maumenee, I.H.; Wolfrum, U.; Cheetham, M.E.; Ueffing, M.; Cremers, F.P.M.; Inglehearn, C.F.; Roepman, R.

    2007-01-01

    Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We

  8. Identification of Genes Encoding the Folate- and Thiamine-Binding Membrane Proteins in Firmicutes

    NARCIS (Netherlands)

    Eudes, Aymerick; Erkens, Guus B.; Slotboom, Dirk J.; Rodionov, Dmitry A.; Naponelli, Valeria; Hanson, Andrew D.

    Genes encoding high-affinity folate- and thiamine-binding proteins (FolT, ThiT) were identified in the Lactobacillus casei genome, expressed in Lactococcus lactis, and functionally characterized. Similar genes occur in many Firmicutes, sometimes next to folate or thiamine salvage genes. Most thiT

  9. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells

    Directory of Open Access Journals (Sweden)

    Gin-Wen Chang

    2016-05-01

    Full Text Available Natural killer (NK cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

  10. Identification and functional comparison of seven-transmembrane G-protein-coupled BILF1 receptors in recently discovered nonhuman primate lymphocryptoviruses

    DEFF Research Database (Denmark)

    Spiess, Katja; Fares, Suzan; Sparre-Ulrich, Alexander H

    2015-01-01

    Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immuno...

  11. Upconversion Nanoparticles-Encoded Hydrogel Microbeads-Based Multiplexed Protein Detection

    Science.gov (United States)

    Shikha, Swati; Zheng, Xiang; Zhang, Yong

    2018-06-01

    Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex's xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

  12. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

  13. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1

    DEFF Research Database (Denmark)

    Morland, Cecilie; Lauritzen, Knut Huso; Puchades, Maja

    2015-01-01

    We have proposed that lactate is a “volume transmitter” in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes......, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress...

  14. G Protein-Coupled Receptors in Cancer

    Directory of Open Access Journals (Sweden)

    Rachel Bar-Shavit

    2016-08-01

    Full Text Available Despite the fact that G protein-coupled receptors (GPCRs are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.

  15. Resistance to β-Lactams in Neisseria ssp Due to Chromosomally Encoded Penicillin-Binding Proteins.

    Science.gov (United States)

    Zapun, André; Morlot, Cécile; Taha, Muhamed-Kheir

    2016-09-28

    Neisseria meningitidis and Neisseria gonorrhoeae are human pathogens that cause a variety of life-threatening systemic and local infections, such as meningitis or gonorrhoea. The treatment of such infection is becoming more difficult due to antibiotic resistance. The focus of this review is on the mechanism of reduced susceptibility to penicillin and other β-lactams due to the modification of chromosomally encoded penicillin-binding proteins (PBP), in particular PBP2 encoded by the penA gene. The variety of penA alleles and resulting variant PBP2 enzymes is described and the important amino acid substitutions are presented and discussed in a structural context.

  16. The multiligand α2-macroglobulin receptor/low density lipoprotein receptor-related protein

    DEFF Research Database (Denmark)

    Gliemann, Jørgen; Nykjær, Anders; Petersen, Claus Munck

    1994-01-01

    The fusion of separate lines of research has greatly helped in elucidating the function of the giant members of the low density lipoprotein (LDL) receptor (LDLR) supergene family. The cDNA encoding a large protein structurally closely related to LDLR, and hence named LDLR-related protein (LRP......), was cloned by Herz et al. in 1988.'Evidence was provided demonstrating that LRP can function as a receptor for chylomicron remnants@-migrating very low density lipoproteins (P-VLDL) rich in apolipoprotein E (apoE)?' The a2-macroglobulin (a2M) receptor (a2MR) was purified from rat livep and human p l a~e n t...... from the observation that affinity-purified a2MR/LRP contains a 40-kDa5.8 or 39-kDa6.' protein, designated a2MRAP, in addition to the a2MFULRP a- and P-chains. cDNA cloning" disclosed the 323-residue protein as both the human homologue of mouse heparin binding protein 44 (see reference 11) and...

  17. Integrated cannabinoid CB1 receptor transmission within the amygdala-prefrontal cortical pathway modulates neuronal plasticity and emotional memory encoding.

    Science.gov (United States)

    Tan, Huibing; Lauzon, Nicole M; Bishop, Stephanie F; Bechard, Melanie A; Laviolette, Steven R

    2010-06-01

    The cannabinoid CB1 receptor system is functionally involved in the processing and encoding of emotionally salient sensory information, learning and memory. The CB1 receptor is found in high concentrations in brain structures that are critical for emotional processing, including the basolateral amygdala (BLA) and the medial prefrontal cortex (mPFC). In addition, synaptic plasticity in the form of long-term potentiation (LTP) within the BLA > mPFC pathway is an established correlate of exposure to emotionally salient events. We performed a series of in vivo LTP studies by applying tetanic stimulation to the BLA combined with recordings of local field potentials within prelimbic cortical (PLC) region of the rat mPFC. Systemic pretreatment with AM-251 dose dependently blocked LTP along the BLA-PLC pathway and also the behavioral acquisition of conditioned fear memories. We next performed a series of microinfusion experiments wherein CB1 receptor transmission within the BLA > PLC circuit was pharmacologically blocked. Asymmetrical, interhemispheric blockade of CB1 receptor transmission along the BLA > PLC pathway prevented the acquisition of emotionally salient associative memory. Our results indicate that coordinated CB1 receptor transmission within the BLA > PLC pathway is critically involved in the encoding of emotional fear memories and modulates neural plasticity related to the encoding of emotionally salient associative learning.

  18. Identification of protein features encoded by alternative exons using Exon Ontology.

    Science.gov (United States)

    Tranchevent, Léon-Charles; Aubé, Fabien; Dulaurier, Louis; Benoit-Pilven, Clara; Rey, Amandine; Poret, Arnaud; Chautard, Emilie; Mortada, Hussein; Desmet, François-Olivier; Chakrama, Fatima Zahra; Moreno-Garcia, Maira Alejandra; Goillot, Evelyne; Janczarski, Stéphane; Mortreux, Franck; Bourgeois, Cyril F; Auboeuf, Didier

    2017-06-01

    Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insight into the protein features encoded by alternative exons and investigating whether coregulated exons contain the same biological information. © 2017 Tranchevent et al.; Published by Cold Spring Harbor Laboratory Press.

  19. Agrobacterium T-DNA-encoded protein Atu6002 interferes with the host auxin response

    Science.gov (United States)

    Lacroix, Benoît; Gizatullina, Diana I.; Babst, Benjamin A.; Gifford, Andrew N.; Citovsky, Vitaly

    2013-01-01

    Summary Several genes in the Agrobacterium tumefaciens transferred (T) DNA encode proteins that are involved in developmental alterations leading to the formation of tumors in infected plants. We investigated the role of the protein encoded by the Atu6002 gene, the function of which is completely unknown. The Atu6002 expression occurs in Agrobacterium-induced tumors, and is also activated upon activation of plant cell division by growth hormones. Within the expressing plant cells, the Atu6002 protein is targeted to the plasma membrane. Interestingly, constitutive ectopic expression of Atu6002 in transgenic tobacco plants lead to a severe developmental phenotype characterized by stunted growth, shorter internodes, lanceolate leaves, increased branching, and modified flower morphology. These Atu6002-expressing plants also displayed impaired response to auxin. However, auxin cellular uptake and polar transport were not significantly inhibited in these plants, suggesting that Atu6002 interferes with auxin perception or signaling pathways. PMID:24128370

  20. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    Science.gov (United States)

    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).

  1. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    International Nuclear Information System (INIS)

    Ostlund, Cecilia; Guan, Tinglu; Figlewicz, Denise A.; Hays, Arthur P.; Worman, Howard J.; Gerace, Larry; Schirmer, Eric C.

    2009-01-01

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  2. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ostlund, Cecilia [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Guan, Tinglu [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Figlewicz, Denise A. [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Hays, Arthur P. [Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Worman, Howard J. [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Gerace, Larry [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Schirmer, Eric C., E-mail: e.schirmer@ed.ac.uk [Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037 (United States); Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR (United Kingdom)

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  3. Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold

    Science.gov (United States)

    Li, Yizhou; De Luca, Roberto; Cazzamalli, Samuele; Pretto, Francesca; Bajic, Davor; Scheuermann, Jörg; Neri, Dario

    2018-03-01

    In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

  4. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

    International Nuclear Information System (INIS)

    Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Kitai, Yuichi; Kon, Shigeyuki; Oritani, Kenji; Matsuda, Tadashi

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR

  5. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

    Energy Technology Data Exchange (ETDEWEB)

    Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Kitai, Yuichi; Kon, Shigeyuki [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan); Oritani, Kenji [Department of Hematology and Oncology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Matsuda, Tadashi, E-mail: tmatsuda@pharm.hokudai.ac.jp [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan)

    2015-07-31

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR.

  6. A novel, mouse mammary tumor virus encoded protein with Rev-like properties

    International Nuclear Information System (INIS)

    Indik, Stanislav; Guenzburg, Walter H.; Salmons, Brian; Rouault, Francoise

    2005-01-01

    We have identified a novel, multiple spliced, subgenomic mRNA species in MMTV producing cells of different origin containing an open reading frame encoding a 39-kDa Rev-like protein, Rem (regulator of expression of MMTV). An EGFP-Rem fusion protein is shown to be predominantly in the nucleolus. Further leptomycin B inhibits the nuclear export of nonspliced MMTV transcripts, implicating Rem in nuclear export by the Crm1 pathway in MMTV. Rem is thus reminiscent of the Rec protein from the related endogenous human retrovirus, HERV-K

  7. RESEARCH ARTICLE Ne2 encodes protein(s) and the altered ...

    Indian Academy of Sciences (India)

    friendly method for increasing sustainable global food security. .... This qualitative difference suggests that Ne2 could encode one or two or three of ... things, common wheat must have at least three types of chloroplast-genomes (A, D, and B).

  8. Genome-Wide Identification and Analysis of Genes Encoding PHD-Finger Protein in Tomato

    International Nuclear Information System (INIS)

    Hayat, S.; Cheng, Z.; Chen, X.

    2016-01-01

    The PHD-finger proteins are conserved in eukaryotic organisms and are involved in a variety of important functions in different biological processes in plants. However, the function of PHD fingers are poorly known in tomato (Solanum lycopersicum L.). In current study, we identified 45 putative genes coding Phd finger protein in tomato distributed on 11 chromosomes except for chromosome 8. Some of the genes encode other conserved key domains besides Phd-finger. Phylogenetic analysis of these 45 proteins resulted in seven clusters. Most Phd finger proteins were predicted to PML body location. These PHD-finger genes displayed differential expression either in various organs, at different development stages and under stresses in tomato. Our study provides the first systematic analysis of PHD-finger genes and proteins in tomato. This preliminary study provides a very useful reference information for Phd-finger proteins in tomato. They will be helpful for cloning and functional study of tomato PHD-finger genes. (author)

  9. Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger.

    Science.gov (United States)

    Liu, Qili; Tabuchi, Masashi; Liu, Sha; Kodama, Lay; Horiuchi, Wakako; Daniels, Jay; Chiu, Lucinda; Baldoni, Daniel; Wu, Mark N

    2017-05-05

    Free-living animals must not only regulate the amount of food they consume but also choose which types of food to ingest. The shifting of food preference driven by nutrient-specific hunger can be essential for survival, yet little is known about the underlying mechanisms. We identified a dopamine circuit that encodes protein-specific hunger in Drosophila The activity of these neurons increased after substantial protein deprivation. Activation of this circuit simultaneously promoted protein intake and restricted sugar consumption, via signaling to distinct downstream neurons. Protein starvation triggered branch-specific plastic changes in these dopaminergic neurons, thus enabling sustained protein consumption. These studies reveal a crucial circuit mechanism by which animals adjust their dietary strategy to maintain protein homeostasis. Copyright © 2017, American Association for the Advancement of Science.

  10. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  11. Polymorphisms in genes encoding leptin, ghrelin and their receptors in German multiple sclerosis patients.

    Science.gov (United States)

    Rey, Linda K; Wieczorek, Stefan; Akkad, Denis A; Linker, Ralf A; Chan, Andrew; Hoffjan, Sabine

    2011-01-01

    Multiple sclerosis (MS) is a neuro-inflammatory, autoimmune disease influenced by environmental and polygenic components. There is growing evidence that the peptide hormone leptin, known to regulate energy homeostasis, as well as its antagonist ghrelin play an important role in inflammatory processes in autoimmune diseases, including MS. Recently, single nucleotide polymorphisms (SNPs) in the genes encoding leptin, ghrelin and their receptors were evaluated, amongst others, in Wegener's granulomatosis and Churg-Strauss syndrome. The Lys656Asn SNP in the LEPR gene showed a significant but contrasting association with these vasculitides. We therefore aimed at investigating these polymorphisms in a German MS case-control cohort. Twelve SNPs in the LEP, LEPR, GHRL and GHSR genes were genotyped in 776 MS patients and 878 control subjects. We found an association of a haplotype in the GHSR gene with MS that could not be replicated in a second cohort. Otherwise, no significant differences in allele or genotype frequencies were observed between patients and controls in this particular cohort. Thus, the present results do not support the hypothesis that genetic variation in the leptin/ghrelin system contributes substantially to the pathogenesis of MS. However, a modest effect of GHSR variation cannot be ruled out and needs to be further evaluated in future studies. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Encoded novel forms of HSP70 or a cytolytic protein increase DNA vaccine potency.

    Science.gov (United States)

    Garrod, Tamsin; Grubor-Bauk, Branka; Yu, Stanley; Gargett, Tessa; Gowans, Eric J

    2014-01-01

    In humans, DNA vaccines have failed to demonstrate the equivalent levels of immunogenicity that were shown in smaller animals. Previous studies have encoded adjuvants, predominantly cytokines, within these vaccines in an attempt to increase antigen-specific immune responses. However, these strategies have lacked breadth of innate immune activation and have led to disappointing results in clinical trials. Damage associated molecular patterns (DAMPs) have been identified as pattern recognition receptor (PRR) agonists. DAMPs can bind to a wide range of PRRs on dendritic cells (DCs) and thus our studies have aimed to utilize this characteristic to act as an adjuvant in a DNA vaccine approach. Specifically, HSP70 has been identified as a DAMP, but has been limited by its lack of accessibility to PRRs in and on DCs. Here, we discuss the promising results achieved with the inclusion of membrane-bound or secreted HSP70 into a DNA vaccine encoding HIV gag as the model immunogen.

  13. Synthesis and structure-activity relationship of the first nonpeptidergic inverse agonists for the human cytomegalovirus encoded chemokine receptor US28.

    Science.gov (United States)

    Hulshof, Janneke W; Casarosa, Paola; Menge, Wiro M P B; Kuusisto, Leena M S; van der Goot, Henk; Smit, Martine J; de Esch, Iwan J P; Leurs, Rob

    2005-10-06

    US28 is a human cytomegalovirus (HCMV) encoded G-protein-coupled receptor that signals in a constitutively active manner. Recently, we identified 1 [5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile] as the first reported nonpeptidergic inverse agonist for a viral-encoded chemokine receptor. Interestingly, this compound is able to partially inhibit the viral entry of HIV-1. In this study we describe the synthesis of 1 and several of its analogues and unique structure-activity relationships for this first class of small-molecule ligands for the chemokine receptor US28. Moreover, the compounds have been pharmacologically characterized as inverse agonists on US28. By modification of lead structure 1, it is shown that a 4-phenylpiperidine moiety is essential for affinity and activity. Other structural features of 1 are shown to be of less importance. These compounds define the first SAR of ligands on a viral GPCR (US28) and may therefore serve as important tools to investigate the significance of US28-mediated constitutive activity during viral infection.

  14. Polypeptide structure and encoding location of the adenovirus serotype 2 late, nonstructural 33K protein

    International Nuclear Information System (INIS)

    Oosterom-Dragon, E.A.; Anderson, C.W.

    1983-01-01

    Radiochemical microsequence analysis of selected tryptic peptides of the adenovirus type 2 33K nonstructural protein has revealed the precise region of the genomic nucleotide sequence that encodes this protein. The initiation codon for the 33K protein lies 606 nucleotides to the right of the EcoRI restriction site at 70.7 map units and 281 nucleotides to the left of the postulated carboxyterminal codon of the adenovirus 100K protein. The coding regions for these two proteins thus overlap; however, the 33K protein is derived from the +1 frame with respect to the postulated 100K reading frame. Our results contradict an earlier published report suggesting that these two proteins share extensive amino acid sequence homology. The published nucleotide sequence of the Ad2 EcoRI-F fragment (70.7 to 75.9 map units) cannot accomodate in a single reading frame the peptide sequences of the 33K protein that we have determined. Sequence analysis of DNA fragments derived from virus has confirmed the published nucleotide sequence in all critical regions with respect to the coding region for the 33K protein. Consequently, our data are only consistent with the existence of an mRNA splice within the coding for 33K. Consensus donor and acceptor splice sequences have been located that would predict the removal of 202 nucleotides from the transcripts for the 33K protein. Removal of these nucleotides would explain the structure of a peptide that cannot otherwise be directly encoded by the EcoRI-F fragment. Identification of the precise splice points by peptide sequencing has permitted a prediction of the complete amino acid sequence for the 33K protein

  15. The Drosophila melanogaster DmCK2beta transcription unit encodes for functionally non-redundant protein isoforms.

    Science.gov (United States)

    Jauch, Eike; Wecklein, Heike; Stark, Felix; Jauch, Mandy; Raabe, Thomas

    2006-06-07

    Genes encoding for the two evolutionary highly conserved subunits of a heterotetrameric protein kinase CK2 holoenzyme are present in all examined eukaryotic genomes. Depending on the organism, multiple transcription units encoding for a catalytically active CK2alpha subunit and/or a regulatory CK2beta subunit may exist. The phosphotransferase activity of members of the protein kinase CK2alpha family is thought to be independent of second messengers but is modulated by interaction with CK2beta-like proteins. In the genome of Drosophila melanogaster, one gene encoding for a CK2alpha subunit and three genes encoding for CK2beta-like proteins are present. The X-linked DmCK2beta transcription unit encodes for several CK2beta protein isoforms due to alternative splicing of its primary transcript. We addressed the question whether CK2beta-like proteins are redundant in function. Our in vivo experiments show that variations of the very C-terminal tail of CK2beta isoforms encoded by the X-linked DmCK2beta transcription unit influence their functional properties. In addition, we find that CK2beta-like proteins encoded by the autosomal D. melanogaster genes CK2betates and CK2beta' cannot fully substitute for a loss of CK2beta isoforms encoded by DmCK2beta.

  16. G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

    Science.gov (United States)

    Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2015-04-24

    G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Cloning an artificial gene encoding angiostatic anginex: From designed peptide to functional recombinant protein

    International Nuclear Information System (INIS)

    Brandwijk, Ricardo J.M.G.E.; Nesmelova, Irina; Dings, Ruud P.M.; Mayo, Kevin H.; Thijssen, Victor L.J.L.; Griffioen, Arjan W.

    2005-01-01

    Anginex, a designed peptide 33-mer, is a potent angiogenesis inhibitor and anti-tumor agent in vivo. Anginex functions by inhibiting endothelial cell (EC) proliferation and migration leading to detachment and apoptosis of activated EC's. To better understand tumor endothelium targeting properties of anginex and enable its use in gene therapy, we constructed an artificial gene encoding the biologically exogenous peptide and produced the protein recombinantly in Pichia pastoris. Mass spectrometry shows recombinant anginex to be a dimer and circular dichroism shows the recombinant protein folds with β-strand structure like the synthetic peptide. Moreover, like parent anginex, the recombinant protein is active at inhibiting EC growth and migration, as well as inhibiting angiogenesis in vivo in the chorioallantoic membrane of the chick embryo. This study demonstrated that it is possible to produce a functionally active protein version of a rationally designed peptide, using an artificial gene and the recombinant protein approach

  18. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel

    2015-01-01

    at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...... involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.......Background FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-07-15

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

  20. Surfactant Protein-D-Encoding Gene Variant Polymorphisms Are Linked to Respiratory Outcome in Premature Infants

    DEFF Research Database (Denmark)

    Sorensen, Grith Lykke; Dahl, Marianne; Tan, Qihua

    2014-01-01

    OBJECTIVE: Associations between the genetic variation within or downstream of the surfactant protein-D-encoding gene (SFTPD), which encodes the collectin surfactant protein-D (SP-D) and may lead to respiratory distress syndrome or bronchopulmonary dysplasia, recently were reported. Our aim...... were used to associate genetic variation to SP-D, respiratory distress (RD), oxygen requirement, and respiratory support. RESULTS: The 5'-upstream SFTPD SNP rs1923534 and the 3 structural SNPs rs721917, rs2243639, and rs3088308 were associated with the SP-D level. The same SNPs were associated with RD......, a requirement for supplemental oxygen, and a requirement for respiratory support. Haplotype analyses identified 3 haplotypes that included the minor alleles of rs1923534, rs721917, and rs3088308 that exhibited highly significant associations with decreased SP-D levels and decreased ORs for RD, oxygen...

  1. Cellulolytic (cel) genes of Clostridium thermocellum F7 and the proteins encoded by them

    International Nuclear Information System (INIS)

    Piruzyan, E.S.; Mogutov, M.A.; Velikodvorskaya, G.A.; Pushkarskaya, T.A.

    1988-01-01

    This study is concerned with genes cell, ce12, and ce13 encoding the endoglucanase of the cellulolytic complex of the anaerobic thermophilic Clostridium thermocellum F7 bacteria, these genes having been closed by us earlier. The authors present the characteristics of proteins synthesized by the cel genes in the minicell system of the strain Escherichia coli K-12 X925. The molecular weights of the proteins encoded by genes cell, ce12, and ce13 are 30,000, 45,000, and 50,000 dalton, respectively. The study of the homology of the cloned section of the C. thermocellum DNA containing the endoglucanase genes, using Southern's blot-hybridization method, did not reveal their physical linkage in the genome. The authors detected a plasmid with a size of about 30 kb in the cells of the C. thermocellum F7 strain investigated

  2. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

    Directory of Open Access Journals (Sweden)

    Nordlund Henri R

    2005-03-01

    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  3. A family of related proteins is encoded by the major Drosophila heat shock gene family

    International Nuclear Information System (INIS)

    Wadsworth, S.C.

    1982-01-01

    At least four proteins of 70,000 to 75,000 molecular weight (70-75K) were synthesized from mRNA which hybridized with a cloned heat shock gene previously shown to be localized to the 87A and 87C heat shock puff sites. These in vitro-synthesized proteins were indistinguishable from in vivo-synthesized heat shock-induced proteins when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of the pattern of this group of proteins synthesized in vivo during a 5-min pulse or during continuous labeling indicates that the 72-75K proteins are probably not kinetic precursors to the major 70K heat shock protein. Partial digestion products generated with V8 protease indicated that the 70-75K heat shock proteins are closely related, but that there are clear differences between them. The partial digestion patterns obtained from heat shock proteins from the Kc cell line and from the Oregon R strain of Drosophila melanogaster are very similar. Genetic analysis of the patterns of 70-75K heat shock protein synthesis indicated that the genes encoding at least two of the three 72-75K heat shock proteins are located outside of the major 87A and 87C puff sites

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

    Science.gov (United States)

    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

  5. An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

    Science.gov (United States)

    Wittenberger, T; Schaller, H C; Hellebrand, S

    2001-03-30

    We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families. Copyright 2001 Academic Press.

  6. System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

    Science.gov (United States)

    Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

    2013-02-05

    The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

  7. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher (Stanford); (Stanford-MED); (Whitehead); (MIT)

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  8. Over-expressed Testis-specific Protein Y-encoded 1 as a novel biomarker for male hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Shan Li

    Full Text Available Hepatocellular carcinoma (HCC is a male-predominant cancer. Previous studies have focused on the sex-related disparity in HCC, but the underlying mechanism remains unclear. Here, we aimed to discover characteristic biomarkers for male HCC. Clinical samples were subjected to iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. Seventy-three differential proteins containing 16 up-regulated and 57 down-regulated proteins were screened out in the male HCC group compared to that in female HCC group. Testis-specific Protein Y-encoded 1(TSPY1 is characteristically present in male HCC and was chosen for further investigation. The data from the functional effects of TSPY1 indicated that over-expression of TSPY1 could potentiate HCC cell proliferation, increase soft agar colonization, induce higher cell invasive ability and correlate with the metastatic potential of the HCC cell lines. In addition, TSPY1 and androgen receptor (AR were co-expressed simultaneously in HCC cell lines as well as in HCC tissue. TSPY1 up- or down-regulation could lead to a high or low level expression of AR. These results implied that TSPY1 may be included in the regulation of AR expression involved in male HCC and it may act as a novel biomarker for male HCC.

  9. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  10. RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates

    International Nuclear Information System (INIS)

    Reynolds, P.; Weber, S.; Prakash, L.

    1985-01-01

    The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. The authors have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which the authors have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids. 48 references, 6 figures

  11. Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima.

    Science.gov (United States)

    Clark, A M; Jacobsen, K R; Bostwick, D E; Dannenhoffer, J M; Skaggs, M I; Thompson, G A

    1997-07-01

    Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lactin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lactin, further suggesting an interaction between these phloem-specific proteins.

  12. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

    1990-01-01

    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 extracellular 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)

  13. Detailed analysis of putative genes encoding small proteins in legume genomes

    Directory of Open Access Journals (Sweden)

    Gabriel eGuillén

    2013-06-01

    Full Text Available Diverse plant genome sequencing projects coupled with powerful bioinformatics tools have facilitated massive data analysis to construct specialized databases classified according to cellular function. However, there are still a considerable number of genes encoding proteins whose function has not yet been characterized. Included in this category are small proteins (SPs, 30-150 amino acids encoded by short open reading frames (sORFs. SPs play important roles in plant physiology, growth, and development. Unfortunately, protocols focused on the genome-wide identification and characterization of sORFs are scarce or remain poorly implemented. As a result, these genes are underrepresented in many genome annotations. In this work, we exploited publicly available genome sequences of Phaseolus vulgaris, Medicago truncatula, Glycine max and Lotus japonicus to analyze the abundance of annotated SPs in plant legumes. Our strategy to uncover bona fide sORFs at the genome level was centered in bioinformatics analysis of characteristics such as evidence of expression (transcription, presence of known protein regions or domains, and identification of orthologous genes in the genomes explored. We collected 6170, 10461, 30521, and 23599 putative sORFs from P. vulgaris, G. max, M. truncatula, and L. japonicus genomes, respectively. Expressed sequence tags (ESTs available in the DFCI Gene Index database provided evidence that ~one-third of the predicted legume sORFs are expressed. Most potential SPs have a counterpart in a different plant species and counterpart regions or domains in larger proteins. Potential functional sORFs were also classified according to a reduced set of GO categories, and the expression of 13 of them during P. vulgaris nodule ontogeny was confirmed by qPCR. This analysis provides a collection of sORFs that potentially encode for meaningful SPs, and offers the possibility of their further functional evaluation.

  14. Role of protein dynamics in transmembrane receptor signalling

    DEFF Research Database (Denmark)

    Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

    2018-01-01

    Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function....

  15. Involvement of hippocampal NMDA receptors in encoding and consolidation, but not retrieval, processes of spontaneous object location memory in rats.

    Science.gov (United States)

    Yamada, Kazuo; Arai, Misaki; Suenaga, Toshiko; Ichitani, Yukio

    2017-07-28

    The hippocampus is thought to be involved in object location recognition memory, yet the contribution of hippocampal NMDA receptors to the memory processes, such as encoding, retention and retrieval, is unknown. First, we confirmed that hippocampal infusion of a competitive NMDA receptor antagonist, AP5 (2-amino-5-phosphonopentanoic acid, 20-40nmol), impaired performance of spontaneous object location recognition test but not that of novel object recognition test in Wistar rats. Next, the effects of hippocampal AP5 treatment on each process of object location recognition memory were examined with three different injection times using a 120min delay-interposed test: 15min before the sample phase (Time I), immediately after the sample phase (Time II), and 15min before the test phase (Time III). The blockade of hippocampal NMDA receptors before and immediately after the sample phase, but not before the test phase, markedly impaired performance of object location recognition test, suggesting that hippocampal NMDA receptors play an important role in encoding and consolidation/retention, but not retrieval, of spontaneous object location memory. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. G protein-coupled receptor mutations and human genetic disease.

    Science.gov (United States)

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

    2014-01-01

    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  17. Biased and g protein-independent signaling of chemokine receptors

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie

    2014-01-01

    ), different receptors (with the same ligand), or different tissues or cells (for the same ligand-receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may...

  18. Semiotic Selection of Mutated or Misfolded Receptor Proteins

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio; Maggio, Roberto

    2013-01-01

    contention that the plasma membrane acts as the locus where several contextual cues may be integrated. As such it allows the semiotic selection of those receptor configurations that provide cells with the minimum essential requirements for agency. The occurrence of protein misfolding makes it impossible...... focused on the significance and semiotic nature of the interplay between membrane receptors and the epigenetic control of gene expression, as mediated by the control of mismatched repairing and protein folding mechanisms....

  19. Sequence and expression pattern of a novel human orphan G-protein-coupled receptor, GPRC5B, a family C receptor with a short amino-terminal domain

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Krogsgaard-Larsen, P

    2000-01-01

    Query of GenBank with the amino acid sequence of human metabotropic glutamate receptor subtype 2 (mGluR2) identified a predicted gene product of unknown function on BAC clone CIT987SK-A-69G12 (located on chromosome band 16p12) as a homologous protein. The transcript, entitled GPRC5B, was cloned f...... from an expressed sequence tag clone that contained the entire open reading frame of the transcript encoding a protein of 395 amino acids. Analysis of the protein sequence reveal that GPRC5B contains a signal peptide and seven transmembrane alpha-helices, which is a hallmark of G...

  20. Characterization and expression of genes encoding three small heat shock proteins in Sesamia inferens (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2014-12-12

    The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

  1. Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    Meng Sun

    2014-12-01

    Full Text Available The pink stem borer, Sesamia inferens (Walker, is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

  2. A Genetically Encoded pH Sensor for Tracking Surface Proteins through Endocytosis**

    OpenAIRE

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

    2012-01-01

    We have combined our fluorogen activating peptide[1] with a new tandem dye molecule to develop a biosensor that labels a cell-surface protein and displays an easily detectable pH dependent emission color change by efficient intramolecular Förster resonant energy transfer. This probe has demonstrated pH variations in β2-adrenergic receptor trafficking and revealed a process of surface to endosome inter-cellular transfer in dendritic cells with potential significance in antigen transfer.

  3. Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics

    Directory of Open Access Journals (Sweden)

    María S. Aymerich

    2011-01-01

    Full Text Available Understanding the trafficking of G-protein-coupled receptors (GPCRs and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

  4. Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites

    International Nuclear Information System (INIS)

    Lenz, J.; Okenquist, S.A.; LoSardo, J.E.; Hamilton, K.K.; Doetsch, P.W.

    1990-01-01

    Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of β-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins

  5. Centromere pairing by a plasmid-encoded type I ParB protein

    DEFF Research Database (Denmark)

    Ringgaard, Simon; Löwe, Jan; Gerdes, Kenn

    2007-01-01

    The par2 locus of Escherichia coli plasmid pB171 encodes two trans-acting proteins, ParA and ParB, and two cis-acting sites, parC1 and parC2, to which ParB binds cooperatively. ParA is related to MinD and oscillates in helical structures and thereby positions ParB/parC-carrying plasmids regularly......, hence identifying the N terminus of ParB as a requirement for ParB-mediated centromere pairing. These observations suggest that centromere pairing is an important intermediate step in plasmid partitioning mediated by the common type I loci....

  6. Expression analysis of the Theileria parva subtelomere-encoded variable secreted protein gene family.

    Directory of Open Access Journals (Sweden)

    Jacqueline Schmuckli-Maurer

    Full Text Available The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm.We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals.Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic

  7. Receptor oligomerization in family B1 of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Roed, Sarah Norklit; Ørgaard, Anne; Jørgensen, Rasmus

    2012-01-01

    , the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this......, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality......The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30-40% of all drug targets. During the last two decades...

  8. Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

    Directory of Open Access Journals (Sweden)

    Kornbluth Richard S

    2009-08-01

    Full Text Available Abstract Background Targeting of protein antigens to dendritic cells (DC via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR and CD40 ligands (CD40L as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen. Results Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Conclusion Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.

  9. A plastome mutation affects processing of both chloroplast and nuclear DNA-encoded plastid proteins.

    Science.gov (United States)

    Johnson, E M; Schnabelrauch, L S; Sears, B B

    1991-01-01

    Immunoblotting of a chloroplast mutant (pm7) of Oenothera showed that three proteins, cytochrome f and the 23 kDa and 16 kDa subunits of the oxygen-evolving subcomplex of photosystem II, were larger than the corresponding mature proteins of the wild type and, thus, appear to be improperly processed in pm7. The mutant is also chlorotic and has little or no internal membrane development in the plastids. The improperly processed proteins, and other proteins that are completely missing, represent products of both the plastid and nuclear genomes. To test for linkage of these defects, a green revertant of pm7 was isolated from cultures in which the mutant plastids were maintained in a nuclear background homozygous for the plastome mutator (pm) gene. In this revertant, all proteins analyzed co-reverted to the wild-type condition, indicating that a single mutation in a plastome gene is responsible for the complex phenotype of pm7. These results suggest that the defect in pm7 lies in a gene that affects a processing protease encoded in the chloroplast genome.

  10. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions.

    Science.gov (United States)

    Luo, Yonglun; Blechingberg, Jenny; Fernandes, Ana Miguel; Li, Shengting; Fryland, Tue; Børglum, Anders D; Bolund, Lars; Nielsen, Anders Lade

    2015-11-14

    FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.

  11. Receptor-like proteins involved in plant disease resistance

    NARCIS (Netherlands)

    Kruijt, M.; Kock, de M.J.D.; Wit, de P.J.G.M.

    2005-01-01

    Race-specific resistance in plants against microbial pathogens is governed by several distinct classes of resistance (R) genes. This review focuses on the class that consists of the plasma membrane-bound leucine-rich repeat proteins known as receptor-like proteins (RLPs). The first isolated

  12. The structure and function of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Rosenbaum, Daniel M; Rasmussen, Søren Gøgsig Faarup; Kobilka, Brian K

    2009-01-01

    G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein...

  13. Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Tian, He; Naganathan, Saranga; Kazmi, Manija A

    2014-01-01

    Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal...

  14. A monoclonal antibody for G protein-coupled receptor crystallography

    DEFF Research Database (Denmark)

    Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural inf...

  15. Association of connexin43 with a receptor protein tyrosine phosphatase

    NARCIS (Netherlands)

    Giepmans, Ben N G; Feiken, Elles; Gebbink, Martijn F B G; Moolenaar, Wouter H

    2003-01-01

    Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap

  16. A Drosophila gene encoding a protein resembling the human β-amyloid protein precursor

    International Nuclear Information System (INIS)

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K.

    1989-01-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human β-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human β-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development

  17. Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

    Science.gov (United States)

    Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

    2012-01-01

    Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

  18. Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

    Science.gov (United States)

    Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

    2009-02-01

    Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

  19. Protein-induced satiation and the calcium-sensing receptor

    OpenAIRE

    Ojha,Utkarsh

    2018-01-01

    Utkarsh Ojha Faculty of Medicine, Imperial College School of Medicine, Imperial College London, London, UK Abstract: Obesity is a major global health issue. High-protein diets have been shown to be associated with weight loss and satiety. The precise mechanism by which protein-rich diets promote weight loss remains unclear. Evidence suggests amino acids, formed as a consequence of protein digestion, are sensed by specific receptors on L-cells in the gastrointestinal (GI) tract. These L-cells ...

  20. Presynaptic G Protein-Coupled Receptors: Gatekeepers of Addiction?

    Directory of Open Access Journals (Sweden)

    Kari A Johnson

    2016-11-01

    Full Text Available Drug abuse and addiction cause widespread social and public health problems, and the neurobiology underlying drug actions and drug use and abuse is an area of intensive research. Drugs of abuse alter synaptic transmission, and these actions contribute to acute intoxication as well as the chronic effects of abused substances. Transmission at most mammalian synapses involves neurotransmitter activation of two receptor subtypes, ligand-gated ion channels that mediate fast synaptic responses, and G protein-coupled receptors (GPCRs that have slower neuromodulatory actions. The GPCRs represent a large proportion of neurotransmitter receptors involved in almost all facets of nervous system function. In addition, these receptors are targets for many pharmacotherapeutic agents. Drugs of abuse directly or indirectly affect neuromodulation mediated by GPCRs, with important consequences for intoxication, drug taking and responses to prolonged drug exposure, withdrawal and addiction. Among the GPCRs are several subtypes involved in presynaptic inhibition, most of which are coupled to the Gi/o class of G protein. There is increasing evidence that these presynaptic Gi/o-coupled GPCRs have important roles in the actions of drugs of abuse, as well as behaviors related to these drugs. This topic will be reviewed, with particular emphasis on receptors for three neurotransmitters, dopamine (D1- and D2-like receptors, endocannabinoids (CB1 receptors and glutamate (group II metabotropic glutamate (mGlu receptors. The focus is on recent evidence from laboratory animal models (and some evidence in humans implicating these receptors in the acute and chronic effects of numerous abused drugs, as well as in the control of drug seeking and taking. The ability of drugs targeting these receptors to modify drug seeking behavior has raised the possibility of using compounds targeting these receptors for addiction pharmacotherapy. This topic is also discussed, with emphasis on

  1. Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor.

    Science.gov (United States)

    Pakhomov, Alexey A; Martynov, Vladimir I; Orsa, Alexander N; Bondarenko, Alena A; Chertkova, Rita V; Lukyanov, Konstantin A; Petrenko, Alexander G; Deyev, Igor E

    2017-12-02

    Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F

    DEFF Research Database (Denmark)

    Helin, K; Lees, J A; Vidal, M

    1992-01-01

    The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3...

  3. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    International Nuclear Information System (INIS)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H.

    1988-01-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10 6 independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I

  4. Synaptic proteins and receptors defects in autism spectrum disorders

    Directory of Open Access Journals (Sweden)

    Jianling eChen

    2014-09-01

    Full Text Available Recent studies have found that hundreds of genetic variants, including common and rare variants, rare and de novo mutations, and common polymorphisms have contributed to the occurrence of autism spectrum disorders (ASDs. The mutations in a number of genes such as neurexin, neuroligin, postsynaptic density protein 95 (PSD-95, SH3 and multiple ankyrin repeat domains 3 (SHANK3, synapsin, gephyrin, cadherin (CDH and protocadherin (PCDH, thousand-and-one-amino acid 2 kinase (TAOK2, and contactin (CNTN, have been shown to play important roles in the development and function of synapses. In addition, synaptic receptors, such as gamma-aminobutyric acid (GABA receptors and glutamate receptors, have also been associated with ASDs. This review will primarily focus on the defects of synaptic proteins and receptors associated with ASDs and their roles in the pathogenesis of ASDs via synaptic pathways.

  5. Genetically encoded pH sensor for tracking surface proteins through endocytosis.

    Science.gov (United States)

    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.

  6. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  7. G protein-coupled receptors in Anopheles gambiae.

    Science.gov (United States)

    Hill, Catherine A; Fox, A Nicole; Pitts, R Jason; Kent, Lauren B; Tan, Perciliz L; Chrystal, Mathew A; Cravchik, Anibal; Collins, Frank H; Robertson, Hugh M; Zwiebel, Laurence J

    2002-10-04

    We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

  8. The G protein-coupled receptors deorphanization landscape.

    Science.gov (United States)

    Laschet, Céline; Dupuis, Nadine; Hanson, Julien

    2018-07-01

    G protein-coupled receptors (GPCRs) are usually highlighted as being both the largest family of membrane proteins and the most productive source of drug targets. However, most of the GPCRs are understudied and hence cannot be used immediately for innovative therapeutic strategies. Besides, there are still around 100 orphan receptors, with no described endogenous ligand and no clearly defined function. The race to discover new ligands for these elusive receptors seems to be less intense than before. Here, we present an update of the various strategies employed to assign a function to these receptors and to discover new ligands. We focus on the recent advances in the identification of endogenous ligands with a detailed description of newly deorphanized receptors. Replication being a key parameter in these endeavors, we also discuss the latest controversies about problematic ligand-receptor pairings. In this context, we propose several recommendations in order to strengthen the reporting of new ligand-receptor pairs. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

    DEFF Research Database (Denmark)

    Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...

  10. Sequence of a cloned cDNA encoding human ribosomal protein S11

    Energy Technology Data Exchange (ETDEWEB)

    Lott, J B; Mackie, G A

    1988-02-11

    The authors have isolated a cloned cDNA that encodes human ribosomal protein (rp) S11 by screening a human fibroblast cDNA library with a labelled 204 bp DNA fragment encompassing residues 212-416 of pRS11, a rat rp Sll cDNA clone. The human rp S11 cloned cDNA consists of 15 residues of the 5' leader, the entire coding sequence and all 51 residues of the 3' untranslated region. The predicted amino acid sequence of 158 residues is identical to rat rpS11. The nucleotide sequence in the coding region differs, however, from that in rat in the first position in two codons and in the third position in 44 codons.

  11. Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor.

    Science.gov (United States)

    Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

    2011-07-06

    Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.

  12. Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein.

    Science.gov (United States)

    Allison, A B; Palacios, G; Travassos da Rosa, A; Popov, V L; Lu, L; Xiao, S Y; DeToy, K; Briese, T; Lipkin, W I; Keel, M K; Stallknecht, D E; Bishop, G R; Tesh, R B

    2011-01-01

    The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic (SH) protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase protein indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein

    Science.gov (United States)

    Allison, A. B.; Palacios, G.; Rosa, A. Travassos da; Popov, V. L.; Lu, L.; Xiao, S. Y.; DeToy, K.; Briese, T.; Lipkin, W. Ian; Keel, M. K.; Stallknecht, D. E.; Bishop, G. R.; Tesh, R. B.

    2010-01-01

    The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase proteins indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses. PMID:20863863

  14. Regulation of dsr genes encoding proteins responsible for the oxidation of stored sulfur in Allochromatium vinosum.

    Science.gov (United States)

    Grimm, Frauke; Dobler, Nadine; Dahl, Christiane

    2010-03-01

    Sulfur globules are formed as obligatory intermediates during the oxidation of reduced sulfur compounds in many environmentally important photo- and chemolithoautotrophic bacteria. It is well established that the so-called Dsr proteins are essential for the oxidation of zero-valent sulfur accumulated in the globules; however, hardly anything is known about the regulation of dsr gene expression. Here, we present a closer look at the regulation of the dsr genes in the phototrophic sulfur bacterium Allochromatium vinosum. The dsr genes are expressed in a reduced sulfur compound-dependent manner and neither sulfite, the product of the reverse-acting dissimilatory sulfite reductase DsrAB, nor the alternative electron donor malate inhibit the gene expression. Moreover, we show the oxidation of sulfur to sulfite to be the rate-limiting step in the oxidation of sulfur to sulfate as sulfate production starts concomitantly with the upregulation of the expression of the dsr genes. Real-time RT-PCR experiments suggest that the genes dsrC and dsrS are additionally expressed from secondary internal promoters, pointing to a special function of the encoded proteins. Earlier structural analyses indicated the presence of a helix-turn-helix (HTH)-like motif in DsrC. We therefore assessed the DNA-binding capability of the protein and provide evidence for a possible regulatory function of DsrC.

  15. Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.

    Directory of Open Access Journals (Sweden)

    Zhou Han

    Full Text Available ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein. Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼ 1%. The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

  16. Viroids: how to infect a host and cause disease without encoding proteins.

    Science.gov (United States)

    Navarro, Beatriz; Gisel, Andreas; Rodio, Maria-Elena; Delgado, Sonia; Flores, Ricardo; Di Serio, Francesco

    2012-07-01

    Despite being composed by a single-stranded, circular, non-protein-coding RNA of just 246-401 nucleotides (nt), viroids can incite in their host plants symptoms similar to those caused by DNA and RNA viruses, which have genomes at least 20-fold bigger and encode proteins. On the other hand, certain non-protein-coding plant satellite RNAs display structural similarities with viroids but for replication and transmission they need to parasitize specific helper viruses (modifying concomitantly the symptoms they induce). While phenotypic alterations accompanying infection by viruses may partly result from expressing the proteins they code for, how the non-protein-coding viroids (and satellite RNAs) cause disease remains a conundrum. Initial ideas on viroid pathogenesis focused on a direct interaction of the genomic RNA with host proteins resulting in their malfunction. With the advent of RNA silencing, it was alternatively proposed that symptoms could be produced by viroid-derived small RNAs (vd-sRNAs) -generated by the host defensive machinery- targeting specific host mRNA or DNA sequences for post-transcriptional or transcriptional gene silencing, respectively, a hypothesis that could also explain pathogenesis of non-protein-coding satellite RNAs. Evidence sustaining this view has been circumstantial, but recent data provide support for it in two cases: i) the yellow symptoms associated with a specific satellite RNA result from a 22-nt small RNA (derived from the 24-nt fragment of the satellite genome harboring the pathogenic determinant), which is complementary to a segment of the mRNA of the chlorophyll biosynthetic gene CHLI and targets it for cleavage by the RNA silencing machinery, and ii) two 21-nt vd-sRNAS containing the pathogenic determinant of the albino phenotype induced by a chloroplast-replicating viroid target for cleavage the mRNA coding for the chloroplastic heat-shock protein 90 via RNA silencing too. This evidence, which is compelling for the

  17. The 1p-encoded protein stathmin and resistance of malignant gliomas to nitrosoureas.

    Science.gov (United States)

    Ngo, Teri-T B; Peng, Tien; Liang, Xing-Jie; Akeju, Oluwaseun; Pastorino, Sandra; Zhang, Wei; Kotliarov, Yuri; Zenklusen, Jean C; Fine, Howard A; Maric, Dragan; Wen, Patrick Y; De Girolami, Umberto; Black, Peter McL; Wu, Wells W; Shen, Rong-Fong; Jeffries, Neal O; Kang, Dong-Won; Park, John K

    2007-04-18

    Malignant gliomas are generally resistant to all conventional therapies. Notable exceptions are anaplastic oligodendrogliomas with loss of heterozygosity on chromosome 1p (1p+/-). Patients with 1p+/- anaplastic oligodendroglioma frequently respond to procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and vincristine. Because the underlying biologic basis for this clinical finding is unclear, we evaluated differentially expressed 1p-encoded proteins in 1p+/- and 1p+/+ malignant glioma cell lines and then examined whether their expression was associated with outcome of patients with anaplastic oligodendroglioma. We used a comparative proteomic screen of A172 (1p+/-) and U251 (1p+/+) malignant glioma cell lines to identify differentially expressed 1p-encoded proteins, including stathmin, a microtubule-associated protein. 1p+/- and 1p+/+ anaplastic oligodendroglioma specimens from 24 patients were assessed for stathmin expression by immunohistochemistry. The relationship between stathmin expression and clinical outcome was assessed with Kaplan-Meier analyses. RNA inhibition and cDNA transfection experiments tested effects of stathmin under- and overexpression, respectively, on the in vitro and in vivo resistance of malignant glioma cells to treatment with nitrosourea. For in vivo resistance studies, 36 mice with intracranial and 16 mice with subcutaneous xenograft tumor implants were used (one tumor per mouse). Flow cytometry was used for cell cycle analysis. Immunoblotting was used to assess protein expression. All statistical tests were two-sided. Decreased stathmin expression in tumors was statistically significantly associated with loss of heterozygosity in 1p (Pnitrosourea-treated mice carrying xenograft tumors. Median survival of mice with stathmin+/- tumors was 95 days (95% CI = 68.7 to 121.3 days) and that of mice with stathmin+/+ tumors was 64 days (95% CI = 58.2 to 69.8 days) (difference = 31 days, 95% CI = 4.1 to 57.9 days; PNitrosoureas induced

  18. Functional characterization of viral tumor necrosis factor receptors encoded by cyprinid herpesvirus 3 (CyHV3) genome.

    Science.gov (United States)

    Yi, Yang; Qi, Hemei; Yuan, Jimin; Wang, Rui; Weng, Shaoping; He, Jianguo; Dong, Chuanfu

    2015-08-01

    Cyprinid herpesvirus 3 (CyHV3) is a large double-stranded DNA virus of Alloherpesviridae family in the order Herpesvirales. It causes significant morbidity and mortality in common carp and its ornamental koi variety, and threatens the aquaculture industries worldwide. Mimicry of cytokines and cytokine receptors is a particular strategy for large DNA viruses in modulating the host immune response. Here, we report the identification and characterization of two novel viral homologues of tumor necrosis factor receptor (TNFR) encoded by CyHV3-ORF4 and -ORF12, respectively. CyHV3-ORF4 was identified as a homologue of HVEM and CyHV3-ORF12 as a homologue of TNFRSF1. Overexpression of ORF4 and ORF12 in zebrafish embryos results in embryonic lethality, morphological defects and increased apoptosis. Although we failed to identify any interaction between the two vTNFRs and their potential ligands in zebrafish TNF superfamily by yeast two-hybrid system, the expression of some genes in TNF superfamily or TNFR superfamily were mis-regulated in ORF4 or ORF12-overexpressing embryos, especially the death receptor zHDR and its cognate ligand DL1b. Further studies showed that the apoptosis induced by the both CyHV3 vTNFRs is mainly activated through the intrinsic apoptotic pathway and requires the crosstalk between the intrinsic and extrinsic apoptotic pathway. Additionally, using RT-qPCR and Western blot assays, the expression patterns of the both vTNFRs were also analyzed during CyHV3 productive infection. Collectively, this is the first functional study of two unique vTNFRs encoded by a herpesvirus infecting non-mammalian vertebrates, which may provide novel insights into viral immune regulation mechanism and the pathogenesis of CyHV3 infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Indirect Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Reactive with a Recombinant Protein Expressed from the Gene Encoding the 116-Kilodalton Protein of Mycoplasma pneumoniae

    OpenAIRE

    Duffy, Michael F.; Whithear, Kevin G.; Noormohammadi, Amir H.; Markham, Philip F.; Catton, Michael; Leydon, Jennie; Browning, Glenn F.

    1999-01-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactiv...

  20. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

    Science.gov (United States)

    Gainetdinov, R R; Bohn, L M; Walker, J K; Laporte, S A; Macrae, A D; Caron, M G; Lefkowitz, R J; Premont, R T

    1999-12-01

    G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

  1. Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity

    DEFF Research Database (Denmark)

    Holst, P J; Rosenkilde, M M; Manfra, D

    2001-01-01

    sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does...... not respond to agonist stimulation have a much lower incidence of angiogenic lesions and tumors. These results indicate that induction of the KS-like disease in transgenic mice by ORF74 requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines....

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

    International Nuclear Information System (INIS)

    Higgins, Matthew K.

    2008-01-01

    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, purified and crystallized. Native data sets extending to 1.9 Å resolution have been collected and phasing is under way

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

    Energy Technology Data Exchange (ETDEWEB)

    Higgins, Matthew K., E-mail: mkh20@cam.ac.uk [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom)

    2008-03-01

    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, purified and crystallized. Native data sets extending to 1.9 Å resolution have been collected and phasing is under way.

  4. Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor.

    Science.gov (United States)

    Armour, S L; Foord, S; Kenakin, T; Chen, W J

    1999-12-01

    Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.

  5. Applications of molecular replacement to G protein-coupled receptors

    International Nuclear Information System (INIS)

    Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K.; Weis, William I.

    2013-01-01

    The use of molecular replacement in solving the structures of G protein-coupled receptors is discussed, with specific examples being described in detail. G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed

  6. Differential Involvement of Amygdala and Cortical NMDA Receptors Activation upon Encoding in Odor Fear Memory

    Science.gov (United States)

    Hegoburu, Chloé; Parrot, Sandrine; Ferreira, Guilaume; Mouly, Anne-Marie

    2014-01-01

    Although the basolateral amygdala (BLA) plays a crucial role for the acquisition of fear memories, sensory cortices are involved in their long-term storage in rats. However, the time course of their respective involvement has received little investigation. Here we assessed the role of the glutamatergic N-methyl-D-aspartate (NMDA) receptors in the…

  7. Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs

    Directory of Open Access Journals (Sweden)

    Kai Yang

    2014-02-01

    Full Text Available G Protein Coupled Receptors (GPCRs are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs, which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity.

  8. Oligomerization of G protein-coupled receptors: computational methods.

    Science.gov (United States)

    Selent, J; Kaczor, A A

    2011-01-01

    Recent research has unveiled the complexity of mechanisms involved in G protein-coupled receptor (GPCR) functioning in which receptor dimerization/oligomerization may play an important role. Although the first high-resolution X-ray structure for a likely functional chemokine receptor dimer has been deposited in the Protein Data Bank, the interactions and mechanisms of dimer formation are not yet fully understood. In this respect, computational methods play a key role for predicting accurate GPCR complexes. This review outlines computational approaches focusing on sequence- and structure-based methodologies as well as discusses their advantages and limitations. Sequence-based approaches that search for possible protein-protein interfaces in GPCR complexes have been applied with success in several studies, but did not yield always consistent results. Structure-based methodologies are a potent complement to sequence-based approaches. For instance, protein-protein docking is a valuable method especially when guided by experimental constraints. Some disadvantages like limited receptor flexibility and non-consideration of the membrane environment have to be taken into account. Molecular dynamics simulation can overcome these drawbacks giving a detailed description of conformational changes in a native-like membrane. Successful prediction of GPCR complexes using computational approaches combined with experimental efforts may help to understand the role of dimeric/oligomeric GPCR complexes for fine-tuning receptor signaling. Moreover, since such GPCR complexes have attracted interest as potential drug target for diverse diseases, unveiling molecular determinants of dimerization/oligomerization can provide important implications for drug discovery.

  9. VPAC receptors: structure, molecular pharmacology and interaction with accessory proteins.

    Science.gov (United States)

    Couvineau, Alain; Laburthe, Marc

    2012-05-01

    The vasoactive intestinal peptide (VIP) is a neuropeptide with wide distribution in both central and peripheral nervous systems, where it plays important regulatory role in many physiological processes. VIP displays a large biological functions including regulation of exocrine secretions, hormone release, fetal development, immune responses, etc. VIP appears to exert beneficial effect in neuro-degenerative and inflammatory diseases. The mechanism of action of VIP implicates two subtypes of receptors (VPAC1 and VPAC2), which are members of class B receptors belonging to the super-family of GPCR. This article reviews the current knowledge regarding the structure and molecular pharmacology of VPAC receptors. The structure-function relationship of VPAC1 receptor has been extensively studied, allowing to understand the molecular basis for receptor affinity, specificity, desensitization and coupling to adenylyl cyclase. Those studies have clearly demonstrated the crucial role of the N-terminal ectodomain (N-ted) of VPAC1 receptor in VIP recognition. By using different approaches including directed mutagenesis, photoaffinity labelling, NMR, molecular modelling and molecular dynamic simulation, it has been shown that the VIP molecule interacts with the N-ted of VPAC1 receptor, which is itself structured as a 'Sushi' domain. VPAC1 receptor also interacts with a few accessory proteins that play a role in cell signalling of receptors. Recent advances in the structural characterization of VPAC receptor and more generally of class B GPCRs will lead to the design of new molecules, which could have considerable interest for the treatment of inflammatory and neuro-degenerative diseases. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  10. Saccharomyces cerevisiae ribosomal protein L37 is encoded by duplicate genes that are differentially expressed.

    Science.gov (United States)

    Tornow, J; Santangelo, G M

    1994-06-01

    A duplicate copy of the RPL37A gene (encoding ribosomal protein L37) was cloned and sequenced. The coding region of RPL37B is very similar to that of RPL37A, with only one conservative amino-acid difference. However, the intron and flanking sequences of the two genes are extremely dissimilar. Disruption experiments indicate that the two loci are not functionally equivalent: disruption of RPL37B was insignificant, but disruption of RPL37A severely impaired the growth rate of the cell. When both RPL37 loci are disrupted, the cell is unable to grow at all, indicating that rpL37 is an essential protein. The functional disparity between the two RPL37 loci could be explained by differential gene expression. The results of two experiments support this idea: gene fusion of RPL37A to a reporter gene resulted in six-fold higher mRNA levels than was generated by the same reporter gene fused to RPL37B, and a modest increase in gene dosage of RPL37B overcame the lack of a functional RPL37A gene.

  11. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

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

    Science.gov (United States)

    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.

  13. Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins.

    Science.gov (United States)

    Tollefson, A E; Toth, K; Doronin, K; Kuppuswamy, M; Doronina, O A; Lichtenstein, D L; Hermiston, T W; Smith, C A; Wold, W S

    2001-10-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus

  14. Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

    International Nuclear Information System (INIS)

    Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

    1988-01-01

    Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is ∼9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged

  15. Crystal Structure of a Lipid G Protein-Coupled Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C [Scripps; (Receptos)

    2012-03-01

    The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

  16. Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

    International Nuclear Information System (INIS)

    Sege, R.D.; Kozarsky, K.F.; Krieger, M.

    1986-01-01

    The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene

  17. Protein kinase C alpha controls erythropoietin receptor signaling.

    NARCIS (Netherlands)

    M.M. von Lindern (Marieke); M. Parren-Van Amelsvoort (Martine); T.B. van Dijk (Thamar); E. Deiner; B. Löwenberg (Bob); E. van den Akker (Emile); S. van Emst-de Vries (Sjenet); P.J. Willems (Patrick); H. Beug (Hartmut)

    2000-01-01

    textabstractProtein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We

  18. Protein kinase C alpha controls erythropoietin receptor signaling

    NARCIS (Netherlands)

    von Lindern, M.; Parren-van Amelsvoort, M.; van Dijk, T.; Deiner, E.; van den Akker, E.; van Emst-de Vries, S.; Willems, P.; Beug, H.; Löwenberg, B.

    2000-01-01

    Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors

  19. The alpha7 nicotinic receptor agonist SSR180711 increases activity regulated cytoskeleton protein (Arc) gene expression in the prefrontal cortex of the rat

    DEFF Research Database (Denmark)

    Kristensen, Søren E; Thomsen, Morten S; Hansen, Henrik H

    2007-01-01

    Nicotinic alpha7 acetylcholine receptors (alpha7 nAChR) have been shown to enhance attentional function and aspects of memory function in experimental models and in man. The protein Arc encoded by the effector immediate early gene arc or arg3.1 has been shown to be strongly implicated in long...

  20. Cell transformation mediated by the Epstein-Barr virus G protein-coupled receptor BILF1 is dependent on constitutive signaling

    DEFF Research Database (Denmark)

    Lyngaa, Rikke Birgitte; Nørregaard, K.; Kristensen, Martin

    2010-01-01

    Epstein-Barr virus (EBV) open reading frame BILF1 encodes a seven trans-membrane (TM) G protein-coupled receptor that signals with high constitutive activity through G alpha(i) (Beisser et al., 2005; Paulsen et al., 2005). In this paper, the transforming potential of BILF1 is investigated in vitro...

  1. The viral G protein-coupled receptor ORF74 hijacks β-arrestins for endocytic trafficking in response to human chemokines

    NARCIS (Netherlands)

    De Munnik, Sabrina M.; Kooistra, Albert J.; Van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, C.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of

  2. Genomics and physiology of a marine flavobacterium encoding a proteorhodopsin and a xanthorhodopsin-like protein.

    Directory of Open Access Journals (Sweden)

    Thomas Riedel

    Full Text Available Proteorhodopsin (PR photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete β-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition.

  3. Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus

    Directory of Open Access Journals (Sweden)

    Chakrabarti Mrinmay

    2010-08-01

    Full Text Available Abstract Background Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV, a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11 in its genome. Some of its genome segments (S2 and S6-S11 have been previously characterized but genome segments encoding viral capsid have not been characterized. Results In this study genome segments 1 (S1 and 3 (S3 of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV, Lymantria dispar CPV (LdCPV, and Dendrolimus punctatus CPV (DpCPV. The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein. Conclusion Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3

  4. Macrophage colony-stimulating factor, CSF-1, and its proto-oncogene-encoded receptor

    International Nuclear Information System (INIS)

    Sherr, C.J.; Rettenmier, C.W.; Roussel, M.F.

    1988-01-01

    The macrophage colony-stimulating factor, CSF-1, or M-CSF, is one of a family of hematopoietic growth factors that stimulates the proliferation of monocytes, macrophages, and their committed bone marrow progenitors. Unlike pluripotent hemopoietins such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3 or multi-CSF), which affect the growth of myeloid cells of several different hematopoietic lineages, CSF-1 acts only on cells of the mononuclear phagocyte series to stimulate their growth and enhance their survival. Retroviral transduction of the feline c-fms gene in the Susan McDonough and Hardy Zuckerman-5 (HZ-5) strains of feline sarcoma virus (FeSV) led to genetic alterations that endowed the recombined viral oncogene (v-fms) with the ability to transform cells in culture morphologically and to induce firbrosarcomas and hematopoietic neoplasms in susceptible animals. The v-fms oncogene product differs from the normal CSF-1 receptor in certain of its cardinal biochemical properties, most notably in exhibiting constitutively high basal levels of tyrosine kinase activity in the absence of its ligand. Comparative studies of the c-fms and v-fms genes coupled with analyses of engineered mutants and receptor chimeras have begun to pinpoint pertinent genetic alterations in the normal receptor gene that unmask its latent oncogenic potential. In addition, the availability of biologically active c-fms, v-fms, and CSF-1 cDNAs has allowed these genes to be mobilized and expressed in naive cells, thereby facilitating assays for receptor coupling with downstream components of the mitogenic pathway in diverse cell types

  5. Protein-induced satiation and the calcium-sensing receptor

    Directory of Open Access Journals (Sweden)

    Ojha U

    2018-03-01

    Full Text Available Utkarsh Ojha Faculty of Medicine, Imperial College School of Medicine, Imperial College London, London, UK Abstract: Obesity is a major global health issue. High-protein diets have been shown to be associated with weight loss and satiety. The precise mechanism by which protein-rich diets promote weight loss remains unclear. Evidence suggests amino acids, formed as a consequence of protein digestion, are sensed by specific receptors on L-cells in the gastrointestinal (GI tract. These L-cells respond by secreting gut hormones that subsequently induce satiety. In recent years, the calcium-sensing receptor has been identified in several cells of the GI tract, including L-cells, and suggested to sense specific amino acids. This review evaluates the evidence for protein-rich diets in inducing weight loss and how the calcium-sensing receptor may be implicated in this phenomenon. Commandeering the mechanisms by which elements of a protein-rich diet suppress appetite may provide another successful avenue for developing anti-obesity drugs. Keywords: amino acids, energy regulation, obesity therapy, glucagon-like-peptide-1, peptide YY

  6. Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Holliday, Nicholas D; Hansen, Jakob L

    2007-01-01

    To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive muta......To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase...

  7. Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; Peters, Stephan L. M.; Michel, Martin C.; Alewijnse, Astrid E.

    2008-01-01

    G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization

  8. Nucleotide sequence of a human cDNA encoding a ras-related protein (rap1B)

    Energy Technology Data Exchange (ETDEWEB)

    Pizon, V; Lerosey, I; Chardin, P; Tavitian, A [INSERM, Paris (France)

    1988-08-11

    The authors have previously characterized two human ras-related genes rap1 and rap2. Using the rap1 clone as probe they isolated and sequenced a new rap cDNA encoding the 184aa rap1B protein. The rap1B protein is 95% identical to rap1 and shares several properties with the ras protein suggesting that it could bind GTP/GDP and have a membrane location. As for rap1, the structural characteristics of rap1B suggest that the rap and ras proteins might interact on the same effector.

  9. G-Protein Coupled Receptors: Surface Display and Biosensor Technology

    Science.gov (United States)

    McMurchie, Edward; Leifert, Wayne

    Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

  10. Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same

    Science.gov (United States)

    Tsien, Roger Y [La Jolla, CA; Wang, Lei [San Diego, CA

    2008-07-01

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  11. [Cloning, mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R].

    Science.gov (United States)

    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.

  12. Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4

    DEFF Research Database (Denmark)

    Jen, Angela; Parkyn, Celia J; Mootoosamy, Roy C

    2010-01-01

    For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low......-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor...... both prion and LRP1 biology....

  13. Translational Control of Host Gene Expression by a Cys-Motif Protein Encoded in a Bracovirus.

    Directory of Open Access Journals (Sweden)

    Eunseong Kim

    Full Text Available Translational control is a strategy that various viruses use to manipulate their hosts to suppress acute antiviral response. Polydnaviruses, a group of insect double-stranded DNA viruses symbiotic to some endoparasitoid wasps, are divided into two genera: ichnovirus (IV and bracovirus (BV. In IV, some Cys-motif genes are known as host translation-inhibitory factors (HTIF. The genome of endoparasitoid wasp Cotesia plutellae contains a Cys-motif gene (Cp-TSP13 homologous to an HTIF known as teratocyte-secretory protein 14 (TSP14 of Microplitis croceipes. Cp-TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value of 7.928. Genomic DNA region encoding its open reading frame has three introns. Cp-TSP13 possesses six conserved cysteine residues as other Cys-motif genes functioning as HTIF. Cp-TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV was purified and injected into non-parasitized P. xylostella that expressed Cp-TSP13. Cp-TSP13 was cloned into a eukaryotic expression vector and used to infect Sf9 cells to transiently express Cp-TSP13. The synthesized Cp-TSP13 protein was detected in culture broth. An overlaying experiment showed that the purified Cp-TSP13 entered hemocytes. It was localized in the cytosol. Recombinant Cp-TSP13 significantly inhibited protein synthesis of secretory proteins when it was added to in vitro cultured fat body. In addition, the recombinant Cp-TSP13 directly inhibited the translation of fat body mRNAs in in vitro translation assay using rabbit reticulocyte lysate. Moreover, the recombinant Cp-TSP13 significantly suppressed cellular immune responses by inhibiting hemocyte-spreading behavior. It also exhibited significant insecticidal activities by both injection and feeding routes. These results indicate that Cp-TSP13 is a viral HTIF.

  14. The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ian G Woods

    2005-03-01

    Full Text Available Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.

  15. Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies.

    Science.gov (United States)

    Vinck, Arman; de Bekker, Charissa; Ossin, Adam; Ohm, Robin A; de Vries, Ronald P; Wösten, Han A B

    2011-01-01

    Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the α-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  16. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    Science.gov (United States)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  17. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed

    Science.gov (United States)

    Rocca, Jennifer D.; Hall, Edward K.; Lennon, Jay T.; Evans, Sarah E.; Waldrop, Mark P.; Cotner, James B.; Nemergut, Diana R.; Graham, Emily B.; Wallenstein, Matthew D.

    2015-01-01

    For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes.

  18. Diverse circular replication-associated protein encoding viruses circulating in invertebrates within a lake ecosystem.

    Science.gov (United States)

    Dayaram, Anisha; Galatowitsch, Mark L; Argüello-Astorga, Gerardo R; van Bysterveldt, Katherine; Kraberger, Simona; Stainton, Daisy; Harding, Jon S; Roumagnac, Philippe; Martin, Darren P; Lefeuvre, Pierre; Varsani, Arvind

    2016-04-01

    Over the last five years next-generation sequencing has become a cost effective and efficient method for identifying known and unknown microorganisms. Access to this technique has dramatically changed the field of virology, enabling a wide range of environmental viral metagenome studies to be undertaken of organisms and environmental samples from polar to tropical regions. These studies have led to the discovery of hundreds of highly divergent single stranded DNA (ssDNA) virus-like sequences encoding replication-associated proteins. Yet, few studies have explored how viruses might be shared in an ecosystem through feeding relationships. Here we identify 169 circular molecules (160 CRESS DNA molecules, nine circular molecules) recovered from a New Zealand freshwater lake, that we have tentatively classified into 51 putatively novel species and five previously described species (DflaCV-3, -5, -6, -8, -10). The CRESS DNA viruses identified in this study were recovered from molluscs (Echyridella menzeisii, Musculium novaezelandiae, Potamopyrgus antipodarum and Physella acuta) and insect larvae (Procordulia grayi, Xanthocnemis zealandica, and Chironomus zealandicus) collected from Lake Sarah, as well as from the lake water and benthic sediments. Extensive diversity was observed across most CRESS DNA molecules recovered. The putative capsid protein of one viral species was found to be most similar to those of members of the Tombusviridae family, thus expanding the number of known RNA-DNA hybrid viruses in nature. We noted a strong association between the CRESS DNA viruses and circular molecules identified in the water and browser organisms (C. zealandicus, P. antipodarum and P. acuta), and between water sediments and undefended prey species (C. zealandicus). However, we were unable to find any significant correlation of viral assemblages to the potential feeding relationships of the host aquatic invertebrates. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Interaction of Hepatitis C virus proteins with pattern recognition receptors

    Directory of Open Access Journals (Sweden)

    Imran Muhammad

    2012-06-01

    Full Text Available Abstract Hepatitis C virus (HCV is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I as the receptors for intracellular viral double stranded RNA (dsRNA, and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C, non structural 3/4 A (NS3/4A and non structural 5A (NS5A have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

  20. Mechanisms of Disease: the first kiss-a crucial role for kisspeptin-1 and its receptor, G-protein-coupled receptor 54, in puberty and reproduction.

    Science.gov (United States)

    Seminara, Stephanie B

    2006-06-01

    Although the hypothalamic secretion of gonadotropin-releasing hormone (GnRH) is the defining hormonal event of puberty, the physiologic mechanisms that drive secretion of GnRH at the time of sexual maturation have been difficult to identify. After puberty is initiated, the factors that modulate the frequency and amplitude of GnRH secretion in rapidly changing sex-steroid environments (i.e. the female menstrual cycle) also remain unknown. The discovery that, in both humans and mouse models, loss-of-function mutations in the gene that encodes G-protein-coupled receptor 54 result in phenotypes of hypogonadotropic hypogonadism with an absence of pubertal development has unearthed a novel pathway regulating GnRH secretion. Ligands for G-protein-coupled receptor 54 (KiSS-1R), including metastin (derived from the parent compound, kisspeptin-1) and metastin's C-terminal peptide fragments, have been shown to be powerful stimulants for GnRH release in vivo via their stimulation of G-protein-coupled receptor 54. This article reviews the discovery of the GPR54 gene, places it into the appropriate biological context, and explores the data from in vitro and in vivo studies that point to this ligand-receptor system as a major driver of GnRH secretion.

  1. Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2006-12-01

    Full Text Available Abstract Background The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV 229E. The prototype virus has a split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU in Salisbury, UK. Results All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%. Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates carry a single ORF, 660 nt in size, encoding a single protein of 219 amino acids, which is a homologue of the ORF3 proteins encoded by HCoV-NL63 and PEDV. Conclusion Thus, the genome organization of the group 1b coronaviruses HCoV-NL63, PEDV and HCoV-229E is identical. It is possible that extensive culturing of the HCoV-229E laboratory strain resulted in truncation of ORF4. This may indicate that the protein is not essential in cell culture, but the highly conserved amino acid sequence of the ORF4 protein among clinical isolates suggests that the protein plays an important role in vivo.

  2. DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

  3. Common variants of the genes encoding erythropoietin and its receptor modulate cognitive performance in schizophrenia

    DEFF Research Database (Denmark)

    Kästner, Anne; Grube, Sabrina; El-Kordi, Ahmed

    2012-01-01

    -term memory readouts, with one particular combination of genotypes superior to all others (p 800), these associations were confirmed. A matching preclinical study with mice demonstrated cognitive processing speed and memory enhanced upon transgenic......Erythropoietin (EPO) improves cognitive performance in clinical studies and rodent experiments. We hypothesized that an intrinsic role of EPO for cognition exists, with particular relevance in situations of cognitive decline, which is reflected by associations of EPO and EPO receptor (EPOR......) genotypes with cognitive functions. To prove this hypothesis, schizophrenic patients (N > 1000) were genotyped for 5' upstream-located gene variants, EPO SNP rs1617640 (T/G) and EPORSTR(GA)(n). Associations of these variants were obtained for cognitive processing speed, fine motor skills and short...

  4. G-protein-coupled receptor 137 accelerates proliferation of urinary bladder cancer cells in vitro.

    Science.gov (United States)

    Du, Yiheng; Bi, Wenhuan; Zhang, Fei; Wu, Wenbo; Xia, Shujie; Liu, Haitao

    2015-01-01

    Urinary bladder cancer is a worldwide concern because of its level of incidence and recurrence. To search an effective therapeutic strategy for urinary bladder cancer, it is important to identify proteins involved in tumorigenesis that could serve as potential targets for diagnosis and treatment. G-protein-coupled receptors (GPRs) constitute a large protein family of receptors that sense molecules outside the cell and activate signal transduction pathways and cellular responses inside the cell. GPR137 is a newly discovered human gene encoding orphan GPRs. In this study, we aimed to investigate the physiological role of GPR137 in urinary bladder cancer. The effect of GPR137 on cell growth was examined via an RNA interference (RNAi) lentivirus system in two human urinary bladder cancer cell lines BT5637 and T24. Lentivirus-mediated RNAi could specifically suppressed GPR137 expression in vitro, resulting in alleviated cell viability and impaired colony formation, as well as blocks G0/G1 and S phases of the cell cycle. These results suggested GPR137 as an essential player in urinary bladder cancer cell growth, and it may serve as a potential target for gene therapy in the treatment of urinary bladder cancer. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  5. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

    Science.gov (United States)

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

    G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

  6. Cyanobacterial flv4-2 Operon-Encoded Proteins Optimize Light Harvesting and Charge Separation in Photosystem II.

    Science.gov (United States)

    Chukhutsina, Volha; Bersanini, Luca; Aro, Eva-Mari; van Amerongen, Herbert

    2015-05-01

    Photosystem II (PSII) complexes drive the water-splitting reaction necessary to transform sunlight into chemical energy. However, too much light can damage and disrupt PSII. In cyanobacteria, the flv4-2 operon encodes three proteins (Flv2, Flv4, and Sll0218), which safeguard PSII activity under air-level CO2 and in high light conditions. However, the exact mechanism of action of these proteins has not been clarified yet. We demonstrate that the PSII electron transfer properties are influenced by the flv4-2 operon-encoded proteins. Accelerated secondary charge separation kinetics was observed upon expression/overexpression of the flv4-2 operon. This is likely induced by docking of the Flv2/Flv4 heterodimer in the vicinity of the QB pocket of PSII, which, in turn, increases the QB redox potential and consequently stabilizes forward electron transfer. The alternative electron transfer route constituted by Flv2/Flv4 sequesters electrons from QB(-) guaranteeing the dissipation of excess excitation energy in PSII under stressful conditions. In addition, we demonstrate that in the absence of the flv4-2 operon-encoded proteins, about 20% of the phycobilisome antenna becomes detached from the reaction centers, thus decreasing light harvesting. Phycobilisome detachment is a consequence of a decreased relative content of PSII dimers, a feature observed in the absence of the Sll0218 protein. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  7. Motif analysis unveils the possible co-regulation of chloroplast genes and nuclear genes encoding chloroplast proteins.

    Science.gov (United States)

    Wang, Ying; Ding, Jun; Daniell, Henry; Hu, Haiyan; Li, Xiaoman

    2012-09-01

    Chloroplasts play critical roles in land plant cells. Despite their importance and the availability of at least 200 sequenced chloroplast genomes, the number of known DNA regulatory sequences in chloroplast genomes are limited. In this paper, we designed computational methods to systematically study putative DNA regulatory sequences in intergenic regions near chloroplast genes in seven plant species and in promoter sequences of nuclear genes in Arabidopsis and rice. We found that -35/-10 elements alone cannot explain the transcriptional regulation of chloroplast genes. We also concluded that there are unlikely motifs shared by intergenic sequences of most of chloroplast genes, indicating that these genes are regulated differently. Finally and surprisingly, we found five conserved motifs, each of which occurs in no more than six chloroplast intergenic sequences, are significantly shared by promoters of nuclear-genes encoding chloroplast proteins. By integrating information from gene function annotation, protein subcellular localization analyses, protein-protein interaction data, and gene expression data, we further showed support of the functionality of these conserved motifs. Our study implies the existence of unknown nuclear-encoded transcription factors that regulate both chloroplast genes and nuclear genes encoding chloroplast protein, which sheds light on the understanding of the transcriptional regulation of chloroplast genes.

  8. Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

    Directory of Open Access Journals (Sweden)

    Randall Marcelo Chin

    2018-03-01

    Full Text Available Summary: Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression—the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3′ UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene. : Allotopic expression of proteins normally encoded by mtDNA is a promising therapy for mitochondrial disease. Chin et al. use an unbiased and high-content imaging-based screening platform to optimize allotopic expression. Modified mRNAs encoding for the optimized allotopic expression constructs rescued the respiration and growth of mtATP6-deficient cells. Keywords: mitochondria, mitochondrial disease, mRNA, modified mRNA, ATP6, allotopic expression, rare disease, gene therapy, screening, high content imaging

  9. A lepidopteran-specific gene family encoding valine-rich midgut proteins.

    Directory of Open Access Journals (Sweden)

    Jothini Odman-Naresh

    Full Text Available Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM, an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps, which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran

  10. G protein-coupled receptors: the inside story.

    Science.gov (United States)

    Jalink, Kees; Moolenaar, Wouter H

    2010-01-01

    Recent findings necessitate revision of the traditional view of G protein-coupled receptor (GPCR) signaling and expand the diversity of mechanisms by which receptor signaling influences cell behavior in general. GPCRs elicit signals at the plasma membrane and are then rapidly removed from the cell surface by endocytosis. Internalization of GPCRs has long been thought to serve as a mechanism to terminate the production of second messengers such as cAMP. However, recent studies show that internalized GPCRs can continue to either stimulate or inhibit cAMP production in a sustained manner. They do so by remaining associated with their cognate G protein subunit and adenylyl cyclase at endosomal compartments. Once internalized, the GPCRs produce cellular responses distinct from those elicited at the cell surface.

  11. Modeling structure of G protein-coupled receptors in huan genome

    KAUST Repository

    Zhang, Yang

    2016-01-26

    G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due to difficulties in crystallization, experimental structure determination remains extremely difficult for human GPCRs, which have been a major barrier in modern structure-based drug discovery. We proposed a new hybrid protocol, GPCR-I-TASSER, to construct GPCR structure models by integrating experimental mutagenesis data with ab initio transmembrane-helix assembly simulations, assisted by the predicted transmembrane-helix interaction networks. The method was tested in recent community-wide GPCRDock experiments and constructed models with a root mean square deviation 1.26 Å for Dopamine-3 and 2.08 Å for Chemokine-4 receptors in the transmembrane domain regions, which were significantly closer to the native than the best templates available in the PDB. GPCR-I-TASSER has been applied to model all 1,026 putative GPCRs in the human genome, where 923 are found to have correct folds based on the confidence score analysis and mutagenesis data comparison. The successfully modeled GPCRs contain many pharmaceutically important families that do not have previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin and Neuropeptide Y receptors. All the human GPCR models have been made publicly available through the GPCR-HGmod database at http://zhanglab.ccmb.med.umich.edu/GPCR-HGmod/ The results demonstrate new progress on genome-wide structure modeling of transmembrane proteins which should bring useful impact on the effort of GPCR-targeted drug discovery.

  12. The urokinase receptor associated protein (uPARAP/endo180)

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Danø, K

    2001-01-01

    The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components of...... and their substrate degradation products and thus may add to the complicated interplay between several cell types in governing restricted tissue degradation....

  13. The gene encoding the melanin-concentrating hormone receptor 1 is associated with schizophrenia in a Danish case-control sample

    DEFF Research Database (Denmark)

    Demontis, Ditte; Nyegaard, Mette; Christensen, Jane H

    2012-01-01

    OBJECTIVE: The MCHR1 gene encoding the melanin-concentrating hormone receptor 1 is located on chromosome 22q13.2 and has previously been associated with schizophrenia in a study of cases and controls from the Faroe Islands and Scotland. Herein we report an association between variations in the MCHR...

  14. Imaging Intracellular pH in Live Cells with a Genetically-Encoded Red Fluorescent Protein Sensor

    OpenAIRE

    Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

    2011-01-01

    Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically-encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at...

  15. Isolation and characterisation of the cDNA encoding a glycosylated accessory protein of pea chloroplast DNA polymerase.

    OpenAIRE

    Gaikwad, A; Tewari, K K; Kumar, D; Chen, W; Mukherjee, S K

    1999-01-01

    The cDNA encoding p43, a DNA binding protein from pea chloroplasts (ct) that binds to cognate DNA polymerase and stimulates the polymerase activity, has been cloned and characterised. The characteristic sequence motifs of hydroxyproline-rich glyco-proteins (HRGP) are present in the cDNA corres-ponding to the N-terminal domain of the mature p43. The protein was found to be highly O-arabinosylated. Chemically deglycosylated p43 (i.e. p29) retains its binding to both DNA and pea ct-DNA polymeras...

  16. Nuclear-encoded mitochondrial MTO1 and MRPL41 are regulated in an opposite epigenetic mode based on estrogen receptor status in breast cancer

    International Nuclear Information System (INIS)

    Kim, Tae Woo; Kim, Byungtak; Kim, Ju Hee; Kang, Seongeun; Park, Sung-Bin; Jeong, Gookjoo; Kang, Han-Sung; Kim, Sun Jung

    2013-01-01

    MTO1 and MRPL41 are nuclear-encoded mitochondrial genes encoding a mitochondrial tRNA-modifying enzyme and a mitochondrial ribosomal protein, respectively. Although both genes have been known to have potential roles in cancer, little is known about their molecular regulatory mechanism, particularly from an epigenetic approach. In this study, we aimed to address their epigenetic regulation through the estrogen receptor (ER) in breast cancer. Digital differential display (DDD) was conducted to identify mammary gland-specific gene candidates including MTO1 and MRPL41. Promoter CpG methylation and expression in breast cancer cell lines and tissues were examined by methylation-specific PCR and real time RT-PCR. Effect of estradiol (E2), tamoxifen, and trichostatin A (TSA) on gene expression was examined in ER + and ER- breast cancer cell lines. Chromatin immunoprecipitation and luciferase reporter assay were performed to identify binding and influencing of the ER to the promoters. Examination of both cancer tissues and cell lines revealed that the two genes showed an opposite expression pattern according to ER status; higher expression of MTO1 and MRPL41 in ER- and ER+ cancer types, respectively, and their expression levels were inversely correlated with promoter methylation. Tamoxifen, E2, and TSA upregulated MTO1 expression only in ER+ cells with no significant changes in ER- cells. However, these chemicals upregulated MRPL41 expression only in ER- cells without significant changes in ER+ cells, except for tamoxifen that induced downregulation. Chromatin immunoprecipitation and luciferase reporter assay identified binding and influencing of the ER to the promoters and the binding profiles were differentially regulated in ER+ and ER- cells. These results indicate that different epigenetic status including promoter methylation and different responses through the ER are involved in the differential expression of MTO1 and MRPL41 in breast cancer

  17. Regulation of AMPA Receptor Trafficking by Protein Ubiquitination

    Directory of Open Access Journals (Sweden)

    Jocelyn Widagdo

    2017-10-01

    Full Text Available The molecular mechanisms underlying plastic changes in the strength and connectivity of excitatory synapses have been studied extensively for the past few decades and remain the most attractive cellular models of learning and memory. One of the major mechanisms that regulate synaptic plasticity is the dynamic adjustment of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-type glutamate receptor content on the neuronal plasma membrane. The expression of surface AMPA receptors (AMPARs is controlled by the delicate balance between the biosynthesis, dendritic transport, exocytosis, endocytosis, recycling and degradation of the receptors. These processes are dynamically regulated by AMPAR interacting proteins as well as by various post-translational modifications that occur on their cytoplasmic domains. In the last few years, protein ubiquitination has emerged as a major regulator of AMPAR intracellular trafficking. Dysregulation of AMPAR ubiquitination has also been implicated in the pathophysiology of Alzheimer’s disease. Here we review recent advances in the field and provide insights into the role of protein ubiquitination in regulating AMPAR membrane trafficking and function. We also discuss how aberrant ubiquitination of AMPARs contributes to the pathogenesis of various neurological disorders, including Alzheimer’s disease, chronic stress and epilepsy.

  18. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Science.gov (United States)

    Savage, Linda J; Imre, Kathleen M; Hall, David A; Last, Robert L

    2013-01-01

    The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

  19. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    International Nuclear Information System (INIS)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III; Billheimer, J.T.

    1991-01-01

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP 2 ). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP 2 amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP 2 . The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A) + RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP 2 gene in the human genome or that the SCP 2 gene is very large. Coexpression of the SCP 2 cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP 2 plays a role in regulating steroidogenesis, among other possible functions

  20. Identification and Expression Profiles of Six Transcripts Encoding Carboxylesterase Protein in Vitis flexuosa Infected with Pathogens

    Directory of Open Access Journals (Sweden)

    Md. Zaherul Islam

    2016-08-01

    Full Text Available Plants protect themselves from pathogen attacks via several mechanisms, including hypersensitive cell death. Recognition of pathogen attack by the plant resistance gene triggers expression of carboxylesterase genes associated with hypersensitive response. We identified six transcripts of carboxylesterase genes, Vitis flexuosa carboxylesterase 5585 (VfCXE5585, VfCXE12827, VfCXE13132, VfCXE17159, VfCXE18231, and VfCXE47674, which showed different expression patterns upon transcriptome analysis of V. flexuosa inoculated with Elsinoe ampelina. The lengths of genes ranged from 1,098 to 1,629 bp, and their encoded proteins consisted of 309 to 335 amino acids. The predicted amino acid sequences showed hydrolase like domains in all six transcripts and contained two conserved motifs, GXSXG of serine hydrolase characteristics and HGGGF related to the carboxylesterase family. The deduced amino acid sequence also contained a potential catalytic triad consisted of serine, aspartic acid and histidine. Of the six transcripts, VfCXE12827 showed upregulated expression against E. ampelina at all time points. Three genes (VfCXE5585, VfCXE12827, and VfCXE13132 showed upregulation, while others (VfCXE17159, VfCXE18231, and VfCXE47674 were down regulated in grapevines infected with Botrytis cinerea. All transcripts showed upregulated expression against Rhizobium vitis at early and later time points except VfCXE12827, and were downregulated for up to 48 hours post inoculation (hpi after upregulation at 1 hpi in response to R. vitis infection. All tested genes showed high and differential expression in response to pathogens, indicating that they all may play a role in defense pathways during pathogen infection in grapevines.

  1. The Down regulated in Adenoma (dra) gene encodes an intestine-specific membrane sulfate transport protein.

    Science.gov (United States)

    Silberg, D G; Wang, W; Moseley, R H; Traber, P G

    1995-05-19

    A gene has been described, Down Regulated in Adenoma (dra), which is expressed in normal colon but is absent in the majority of colon adenomas and adenocarcinomas. However, the function of this protein is unknown. Because of sequence similarity to a recently cloned membrane sulfate transporter in rat liver, the transport function of Dra was examined. We established that dra encodes for a Na(+)-independent transporter for both sulfate and oxalate using microinjected Xenopus oocytes as an assay system. Sulfate transport was sensitive to the anion exchange inhibitor DIDS (4,4'-diisothiocyano-2,2' disulfonic acid stilbene). Using an RNase protection assay, we found that dra mRNA expression is limited to the small intestine and colon in mouse, therefore identifying Dra as an intestine-specific sulfate transporter. dra also had a unique pattern of expression during intestinal development. Northern blot analysis revealed a low level of expression in colon at birth with a marked increase in the first 2 postnatal weeks. In contrast, there was a lower, constant level of expression in small intestine in the postnatal period. Caco-2 cells, a colon carcinoma cell line that differentiates over time in culture, demonstrated a marked induction of dra mRNA as cells progressed from the preconfluent (undifferentiated) to the postconfluent (differentiated) state. These results show that Dra is an intestine-specific Na(+)-independent sulfate transporter that has differential expression during colonic development. This functional characterization provides the foundation for investigation of the role of Dra in intestinal sulfate transport and in the malignant phenotype.

  2. Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

    Science.gov (United States)

    Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

    2016-03-01

    The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

  3. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Directory of Open Access Journals (Sweden)

    Linda J Savage

    Full Text Available The Chloroplast 2010 Project (http://www.plastid.msu.edu/ identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/. Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles

  4. Expression of the gene encoding the ghrelin receptor in rats selected for differential alcohol preference.

    Science.gov (United States)

    Landgren, Sara; Engel, Jörgen A; Hyytiä, Petri; Zetterberg, Henrik; Blennow, Kaj; Jerlhag, Elisabet

    2011-08-01

    The mechanisms involved in alcohol use disorder, a chronic relapsing brain disorder, are complex and involve various signalling systems in the brain. Recently, the orexigenic peptide ghrelin was shown to be required for alcohol-induced reward, an effect mediated via ghrelin receptors, GHS-R1A, at the level of the cholinergic-dopaminergic reward link. Moreover, ghrelin increases and GHR-R1A antagonists reduce moderate alcohol consumption in mice, and a single nucleotide polymorphism in the GHS-R1A gene has been associated with high alcohol consumption in humans. Therefore, GHS-R1A gene expression and alcohol intake were investigated in high, AA (Alko, Alcohol), versus low, ANA (Alko, Non-Alcohol), alcohol consuming rats as well as in Wistar rats. In the AA and ANA rats plasma ghrelin levels were also measured. GHS-R1A gene expression was increased in AA compared to ANA rats in nucleus accumbens, ventral tegmental area, amygdala, prefrontal cortex and hippocampus. A similar trend was observed in the ventral tegmental area of Wistar rats consuming high amounts of alcohol. Furthermore, the AA rats had significantly smaller reduction of plasma ghrelin levels over time, after several weeks of alcohol exposure, than had the ANA rats. The present study provides further evidence for that the ghrelin signalling system, in particular at the level of the mesocortocolimbic dopamine system, is involved in alcohol consumption, and thus possibly contributes to alcohol use disorder. Therefore the GHS-R1A may constitute a novel candidate for development of new treatment strategies for alcohol dependence. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Virally encoded chemokines and chemokine receptors in the role of viral infections

    DEFF Research Database (Denmark)

    Holst, Peter J; Lüttichau, Hans R; Schwartz, Thue W

    2003-01-01

    of these or potent ways to alter an efficient antiviral response to a weak Th2-driven response. Examples here are the chemokine scavenging by US28, attractance of Th2 cells and regulatory cells by vMIP1-3 and the selective engaging of CCR8 by MC148. Important insights into viral pathology and possible targets...... for antiviral therapies have been provided by UL33, UL78 and in particular ORF74 and the chances are that many more will follow. In HHV8 vMIP-2 and the chemokine-binding proteins potent anti-inflammatory agents have been provided. These have already had their potential demonstrated in animal models and may...

  6. Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination

    International Nuclear Information System (INIS)

    Madiraju, M.V.; Templin, A.; Clark, A.J.

    1988-01-01

    A mutation partially suppressing the UV sensitivity caused by recF143 in a uvrA6 background was located at codon 37 of recA where GTG (valine) became ATG (methionine). This mutation, originally named srf-803, was renamed recA803. Little if any suppression of the recF143 defect in UV induction of a lexA regulon promoter was detected. This led to the hypothesis that a defect in recombination repair of UV damage was suppressed by recA803. The mutant RecA protein (RecA803) was purified and compared with wild-type protein (RecA+) as a catalyst of formation of joint molecules. Under suboptimal conditions, RecA803 produces both a higher rate of formation and a higher yield of joint molecules. The suboptimal conditions tested included addition of single-stranded DNA binding protein to single-stranded DNA prior to addition of RecA. We hypothesize that the ability of RecA803 to overcome interference by single-stranded DNA binding protein is the property that allows recA803 to suppress partially the deficiency in repair caused by recF mutations in the uvrA6 background. Implications of this hypothesis for the function of RecF protein in recombination are discussed

  7. Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

    International Nuclear Information System (INIS)

    Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.; Smith, W.C.; Talamantes, F.

    1990-01-01

    [ 125 I]Iodomouse GH [( 125 I]iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operated pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse

  8. Identification of G Protein-Coupled Receptors (GPCRs in Primary Cilia and Their Possible Involvement in Body Weight Control.

    Directory of Open Access Journals (Sweden)

    Yoshihiro Omori

    Full Text Available Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs. We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR, neuropeptide FF receptor 1 (NPFFR1, and neuromedin U receptor 1 (NMUR1, localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.

  9. Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control.

    Science.gov (United States)

    Omori, Yoshihiro; Chaya, Taro; Yoshida, Satoyo; Irie, Shoichi; Tsujii, Toshinori; Furukawa, Takahisa

    2015-01-01

    Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.

  10. Melanocortin receptor accessory proteins in adrenal gland physiology and beyond.

    Science.gov (United States)

    Novoselova, T V; Jackson, D; Campbell, D C; Clark, A J L; Chan, L F

    2013-04-01

    The melanocortin receptor (MCR) family consists of five G-protein-coupled receptors (MC1R-MC5R) with diverse physiological roles. MC1R controls pigmentation, MC2R is a critical component of the hypothalamic-pituitary-adrenal axis, MC3R and MC4R have a vital role in energy homeostasis and MC5R is involved in exocrine function. The melanocortin receptor accessory protein (MRAP) and its paralogue MRAP2 are small single-pass transmembrane proteins that have been shown to regulate MCR expression and function. In the adrenal gland, MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency, where inactivating MRAP mutations account for ∼20% of cases. MRAP is highly expressed in both the zona fasciculata and the undifferentiated zone. Expression in the undifferentiated zone suggests that MRAP could also be important in adrenal cell differentiation and/or maintenance. In contrast, the role of adrenal MRAP2, which is highly expressed in the foetal gland, is unclear. The expression of MRAPs outside the adrenal gland is suggestive of a wider physiological purpose, beyond MC2R-mediated adrenal steroidogenesis. In vitro, MRAPs have been shown to reduce surface expression and signalling of all the other MCRs (MC1,3,4,5R). MRAP2 is predominantly expressed in the hypothalamus, a site that also expresses a high level of MC3R and MC4R. This raises the intriguing possibility of a CNS role for the MRAPs.

  11. Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

    International Nuclear Information System (INIS)

    Parris, D.S.; Cross, A.; Orr, A.; Frame, M.C.; Murphy, M.; McGeoch, D.J.; Marsden, H.S.; Haarr, L.

    1988-01-01

    Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65K DBP ) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65K DBP . Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65K DBP , was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65K DBP . The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65K DBP , thus confirming the gene assignment

  12. Cloning of gene-encoded stem bromelain on system coming from Pichia pastoris as therapeutic protein candidate

    Science.gov (United States)

    Yusuf, Y.; Hidayati, W.

    2018-01-01

    The process of identifying bacterial recombination using PCR, and restriction, and then sequencing process was done after identifying the bacteria. This research aimed to get a yeast cell of Pichia pastoris which has an encoder gene of stem bromelain enzyme. The production of recombinant stem bromelain enzymes using yeast cells of P. pastoris can produce pure bromelain rod enzymes and have the same conformation with the enzyme’s conformation in pineapple plants. This recombinant stem bromelain enzyme can be used as a therapeutic protein in inflammatory, cancer and degenerative diseases. This study was an early stage of a step series to obtain bromelain rod protein derived from pineapple made with genetic engineering techniques. This research was started by isolating the RNA of pineapple stem which was continued with constructing cDNA using reserve transcriptase-PCR technique (RT-PCR), doing the amplification of bromelain enzyme encoder gene with PCR technique using a specific premiere couple which was designed. The process was continued by cloning into bacterium cells of Escherichia coli. A vector which brought the encoder gene of stem bromelain enzyme was inserted into the yeast cell of P. pastoris and was continued by identifying the yeast cell of P. pastoris which brought the encoder gene of stem bromelain enzyme. The research has not found enzyme gene of stem bromelain in yeast cell of P. pastoris yet. The next step is repeating the process by buying new reagent; RNase inhibitor, and buying liquid nitrogen.

  13. G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

    Science.gov (United States)

    Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

    2015-03-13

    Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop.

    Science.gov (United States)

    Ligerot, Yasmine; de Saint Germain, Alexandre; Waldie, Tanya; Troadec, Christelle; Citerne, Sylvie; Kadakia, Nikita; Pillot, Jean-Paul; Prigge, Michael; Aubert, Grégoire; Bendahmane, Abdelhafid; Leyser, Ottoline; Estelle, Mark; Debellé, Frédéric; Rameau, Catherine

    2017-12-01

    Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner.

  15. D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons.

    Directory of Open Access Journals (Sweden)

    Khursheed A Wani

    Full Text Available Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1 required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.

  16. Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

    International Nuclear Information System (INIS)

    Rodríguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Akerboom, Jasper; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.

    2008-01-01

    The genetically encoded fluorescent calcium-indicator protein GCaMP2 was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution and the structure was solved by molecular replacement. Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, β = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way

  17. The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

    Science.gov (United States)

    Delfosse, Verónica C; Agrofoglio, Yamila C; Casse, María F; Kresic, Iván Bonacic; Hopp, H Esteban; Ziegler-Graff, Véronique; Distéfano, Ana J

    2014-02-13

    Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Regulation of G Protein-Coupled Receptors by Ubiquitination

    Directory of Open Access Journals (Sweden)

    Kamila Skieterska

    2017-04-01

    Full Text Available G protein-coupled receptors (GPCRs comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

  19. Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics.

    Science.gov (United States)

    Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

    2014-01-01

    There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. This technique can now be extended to the study of other HERV genomes within the human chromosomes that may have contributed to

  20. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains...... in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...

  1. Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

    Science.gov (United States)

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

    2013-11-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons. © 2013 International Society for Neurochemistry.

  2. The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory

    NARCIS (Netherlands)

    Schmitz, Leanne J M; Klaassen, Remco V; Ruiperez-Alonso, Marta; Zamri, Azra Elia; Stroeder, Jasper; Rao-Ruiz, Priyanka; Lodder, Johannes C; van der Loo, Rolinka J; Mansvelder, Huib D; Smit, August B; Spijker, Sabine; Verhage, Matthijs

    2017-01-01

    Glutamatergic synapses rely on AMPA receptors (AMPARs) for fast synaptic transmission and plasticity. AMPAR auxiliary proteins regulate receptor trafficking, and modulate receptor mobility and its biophysical properties. The AMPAR auxiliary protein Shisa7 (CKAMP59) has been shown to interact with

  3. TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis

    NARCIS (Netherlands)

    van der Meer, Jonathan H. M.; van der Poll, Tom; van 't Veer, Cornelis

    2014-01-01

    TAM receptors (Tyro3, Axl, andMer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein

  4. Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

    Science.gov (United States)

    Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

  5. Characterization of dacC, which encodes a new low-molecular-weight penicillin-binding protein in Bacillus subtilis

    DEFF Research Database (Denmark)

    Pedersen, Lotte Bang; Murray, T; Popham, D L

    1998-01-01

    The pbp gene (renamed dacC), identified by the Bacillus subtilis genome sequencing project, encodes a putative 491-residue protein with sequence homology to low-molecular-weight penicillin-binding proteins. Use of a transcriptional dacC-lacZ fusion revealed that dacC expression (i) is initiated...... at the end of stationary phase; (ii) depends strongly on transcription factor sigmaH; and (iii) appears to be initiated from a promoter located immediately upstream of yoxA, a gene of unknown function located upstream of dacC on the B. subtilis chromosome. A B. subtilis dacC insertional mutant grew...

  6. Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

    Science.gov (United States)

    Otsuki, Tetsuji; Ota, Toshio; Nishikawa, Tetsuo; Hayashi, Koji; Suzuki, Yutaka; Yamamoto, Jun-ichi; Wakamatsu, Ai; Kimura, Kouichi; Sakamoto, Katsuhiko; Hatano, Naoto; Kawai, Yuri; Ishii, Shizuko; Saito, Kaoru; Kojima, Shin-ichi; Sugiyama, Tomoyasu; Ono, Tetsuyoshi; Okano, Kazunori; Yoshikawa, Yoko; Aotsuka, Satoshi; Sasaki, Naokazu; Hattori, Atsushi; Okumura, Koji; Nagai, Keiichi; Sugano, Sumio; Isogai, Takao

    2005-01-01

    We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

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

    Science.gov (United States)

    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).

  8. Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

    Science.gov (United States)

    Seo, H S; Kim, H Y; Jeong, J Y; Lee, S Y; Cho, M J; Bahk, J D

    1995-03-01

    A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

  9. Protein tyrosine phosphatase encoded in Cotesia plutellae bracovirus suppresses a larva-to-pupa metamorphosis of the diamondback moth, Plutella xylostella.

    Science.gov (United States)

    Kim, Jiwan; Hepat, Rahul; Lee, Daeweon; Kim, Yonggyun

    2013-09-01

    Parasitization by an endoparasitoid wasp, Cotesia plutellae, inhibits a larva-to-pupa metamorphosis of the diamondback moth, Plutella xylostella. This study tested an inhibitory effect of C. plutellae bracovirus (CpBV) on the metamorphosis of P. xylostella. Parasitized P. xylostella exhibited significantly reduced prothoracic gland (PTG) development at the last instar compared to nonparasitized larvae. Expression of the ecdysone receptor (EcR) was markedly suppressed during the last instar larvae parasitized by C. plutellae. By contrast, expression of the insulin receptor (InR) significantly increased in the parasitized larvae. Microinjection of CpBV significantly inhibited the larva-to-pupa metamorphosis of nonparasitized larvae in a dose-dependent manner. Injection of CpBV also inhibited the expression of the EcR and increased the expression of the InR. Individual CpBV segments were transiently expressed in its encoded genes in nonparasitized larvae and screened to determine antimetamorphic viral gene(s). Out of 21 CpBV segments, two viral segments (CpBV-S22 and CpBV-S27) were proved to inhibit larva-to-pupa metamorphosis by transient expression assay. RNA interference of each gene encoded in the viral segments was applied to determine antimetamorphic gene(s). Protein tyrosine phosphatase, early expressed gene, and four hypothetical genes were selected to be associated with the antimetamorphic activity of CpBV. These results suggest that antimetamorphosis of P. xylostella parasitized by C. plutellae is induced by inhibiting PTG development and subsequent ecdysteroid signaling with viral factors of CpBV. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Molecular Cloning and Characterization of Growth Factor Receptor Bound-Protein in Clonorchis sinensis

    Science.gov (United States)

    Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong

    2014-01-01

    Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892

  11. Molecular cloning and characterization of growth factor receptor bound-protein in Clonorchis sinensis.

    Directory of Open Access Journals (Sweden)

    Xuelian Bai

    Full Text Available BACKGROUND: Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2 is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2 from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. CONCLUSION: Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths.

  12. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  13. Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development

    Directory of Open Access Journals (Sweden)

    Hongyu Chen

    2018-02-01

    Full Text Available In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem, cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

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

    Science.gov (United States)

    2013-01-01

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

  15. Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.

    Science.gov (United States)

    Bubeck Wardenburg, J; Fu, C; Jackman, J K; Flotow, H; Wilkinson, S E; Williams, D H; Johnson, R; Kong, G; Chan, A C; Findell, P R

    1996-08-16

    Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

  16. The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

    Science.gov (United States)

    Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

    2009-10-01

    Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

  17. Regulation of neuronal communication by G protein-coupled receptors.

    Science.gov (United States)

    Huang, Yunhong; Thathiah, Amantha

    2015-06-22

    Neuronal communication plays an essential role in the propagation of information in the brain and requires a precisely orchestrated connectivity between neurons. Synaptic transmission is the mechanism through which neurons communicate with each other. It is a strictly regulated process which involves membrane depolarization, the cellular exocytosis machinery, neurotransmitter release from synaptic vesicles into the synaptic cleft, and the interaction between ion channels, G protein-coupled receptors (GPCRs), and downstream effector molecules. The focus of this review is to explore the role of GPCRs and G protein-signaling in neurotransmission, to highlight the function of GPCRs, which are localized in both presynaptic and postsynaptic membrane terminals, in regulation of intrasynaptic and intersynaptic communication, and to discuss the involvement of astrocytic GPCRs in the regulation of neuronal communication. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  18. GPCRdb: an information system for G protein-coupled receptors.

    Science.gov (United States)

    Isberg, Vignir; Mordalski, Stefan; Munk, Christian; Rataj, Krzysztof; Harpsøe, Kasper; Hauser, Alexander S; Vroling, Bas; Bojarski, Andrzej J; Vriend, Gert; Gloriam, David E

    2016-01-04

    Recent developments in G protein-coupled receptor (GPCR) structural biology and pharmacology have greatly enhanced our knowledge of receptor structure-function relations, and have helped improve the scientific foundation for drug design studies. The GPCR database, GPCRdb, serves a dual role in disseminating and enabling new scientific developments by providing reference data, analysis tools and interactive diagrams. This paper highlights new features in the fifth major GPCRdb release: (i) GPCR crystal structure browsing, superposition and display of ligand interactions; (ii) direct deposition by users of point mutations and their effects on ligand binding; (iii) refined snake and helix box residue diagram looks; and (iii) phylogenetic trees with receptor classification colour schemes. Under the hood, the entire GPCRdb front- and back-ends have been re-coded within one infrastructure, ensuring a smooth browsing experience and development. GPCRdb is available at http://www.gpcrdb.org/ and it's open source code at https://bitbucket.org/gpcr/protwis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

    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.

  20. Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.

    Directory of Open Access Journals (Sweden)

    Xavier Charest-Morin

    Full Text Available The bradykinin (BK B1 receptor (B1R is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP (enhanced green FP [EGFP] or mCherry prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively. The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy. Both assays indicated that the best design was FP-(Asn-Glyn-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology.

  1. Molecular Cloning and Characterization of Four Genes Encoding Ethylene Receptors Associated with Pineapple (Ananas comosus L.) Flowering.

    Science.gov (United States)

    Li, Yun-He; Wu, Qing-Song; Huang, Xia; Liu, Sheng-Hui; Zhang, Hong-Na; Zhang, Zhi; Sun, Guang-Ming

    2016-01-01

    Exogenous ethylene, or ethephon, has been widely used to induce pineapple flowering, but the molecular mechanism behind ethephon induction is still unclear. In this study, we cloned four genes encoding ethylene receptors (designated AcERS1a, AcERS1b, AcETR2a, and AcETR2b). The 5' flanking sequences of these four genes were also cloned by self-formed adaptor PCR and SiteFinding-PCR, and a group of putative cis-acting elements was identified. Phylogenetic tree analysis indicated that AcERS1a, AcERS1b, AcETR2a, and AcETR2b belonged to the plant ERS1s and ETR2/EIN4-like groups. Quantitative real-time PCR showed that AcETR2a and AcETR2b (subfamily 2) were more sensitive to ethylene treatment compared with AcERS1a and AcERS1b (subfamily 1). The relative expression of AcERS1b, AcETR2a, and AcETR2b was significantly increased during the earlier period of pineapple inflorescence formation, especially at 1-9 days after ethylene treatment (DAET), whereas AcERS1a expression changed less than these three genes. In situ hybridization results showed that bract primordia (BP) and flower primordia (FP) appeared at 9 and 21 DAET, respectively, and flowers were formed at 37 DAET. AcERS1a, AcERS1b, AcETR2a, and AcETR2b were mainly expressed in the shoot apex at 1-4 DAET; thereafter, with the appearance of BP and FP, higher expression of these genes was found in these new structures. Finally, at 37 DAET, the expression of these genes was mainly focused in the flower but was also low in other structures. These findings indicate that these four ethylene receptor genes, especially AcERS1b, AcETR2a, and AcETR2b, play important roles during pineapple flowering induced by exogenous ethephon.

  2. Nuclear Receptors, RXR, and the Big Bang.

    Science.gov (United States)

    Evans, Ronald M; Mangelsdorf, David J

    2014-03-27

    Isolation of genes encoding the receptors for steroids, retinoids, vitamin D, and thyroid hormone and their structural and functional analysis revealed an evolutionarily conserved template for nuclear hormone receptors. This discovery sparked identification of numerous genes encoding related proteins, termed orphan receptors. Characterization of these orphan receptors and, in particular, of the retinoid X receptor (RXR) positioned nuclear receptors at the epicenter of the "Big Bang" of molecular endocrinology. This Review provides a personal perspective on nuclear receptors and explores their integrated and coordinated signaling networks that are essential for multicellular life, highlighting the RXR heterodimer and its associated ligands and transcriptional mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Ocular toxoplasmosis: susceptibility in respect to the genes encoding the KIR receptors and their HLA class I ligands

    Science.gov (United States)

    Ayo, Christiane Maria; Frederico, Fábio Batista; Siqueira, Rubens Camargo; Brandão de Mattos, Cinara de Cássia; Previato, Mariana; Barbosa, Amanda Pires; Murata, Fernando Henrique Antunes; Silveira-Carvalho, Aparecida Perpétuo; de Mattos, Luiz Carlos

    2016-01-01

    The objective of this study was to investigate the influence of the genes encoding the KIR receptors and their HLA ligands in the susceptibility of ocular toxoplasmosis. A total of 297 patients serologically-diagnosed with toxoplasmosis were selected and stratified according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping was performed by PCR-SSOP. Statistical analyses were conducted using the Chi-square test, and odds ratio with a 95% confidence interval was also calculated to evaluate the risk association. The activating KIR3DS1 gene was associated with increased susceptibility for ocular toxoplasmosis. The activating KIR together with their HLA ligands (KIR3DS1-Bw4-80Ile and KIR2DS1+/C2++ KIR3DS1+/Bw4-80Ile+) were associated with increased susceptibility for ocular toxoplasmosis and its clinical manifestations. KIR-HLA inhibitory pairs -KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1- were associated with decreased susceptibility for ocular toxoplasmosis and its clinical forms, while the KIR3DS1−/KIR3DL1+/Bw4-80Ile+ combination was associated as a protective factor against the development of ocular toxoplasmosis and, in particular, against recurrent manifestations. Our data demonstrate that activating and inhibitory KIR genes may influence the development of ocular toxoplasmosis. PMID:27827450

  4. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  5. The endothelial protein C receptor and activated protein C play a limited role in host defense during experimental tuberculosis

    NARCIS (Netherlands)

    Kager, Liesbeth M.; Roelofs, Joris J. T. H.; de Vos, Alex F.; Wieland, Catharina W.; Schouten, Marcel; Meijers, Joost C. M.; Isermann, Berend; van't Veer, Cornelis; Esmon, Charles T.; van der Poll, Tom

    2013-01-01

    The protein C (PC) system is an important regulator of both coagulation and inflammation. Activated PC (APC), together with its receptor the endothelial protein C receptor (EPCR), has anticoagulant and anti-inflammatory properties. During tuberculosis (TB), a devastating chronic pulmonary disease

  6. Simvastatin enhances bone morphogenetic protein receptor type II expression

    International Nuclear Information System (INIS)

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N.

    2006-01-01

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function

  7. Scotopic vision in the monkey is modulated by the G protein-coupled receptor 55

    DEFF Research Database (Denmark)

    Bouskila, Joseph; Harrar, Vanessa; Javadi, Pasha

    2016-01-01

    The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors in the mon......The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors...

  8. A modeling strategy for G-protein coupled receptors

    Directory of Open Access Journals (Sweden)

    Anna Kahler

    2016-03-01

    Full Text Available Cell responses can be triggered via G-protein coupled receptors (GPCRs that interact with small molecules, peptides or proteins and transmit the signal over the membrane via structural changes to activate intracellular pathways. GPCRs are characterized by a rather low sequence similarity and exhibit structural differences even for functionally closely related GPCRs. An accurate structure prediction for GPCRs is therefore not straightforward. We propose a computational approach that relies on the generation of several independent models based on different template structures, which are subsequently refined by molecular dynamics simulations. A comparison of their conformational stability and the agreement with GPCR-typical structural features is then used to select a favorable model. This strategy was applied to predict the structure of the herpesviral chemokine receptor US28 by generating three independent models based on the known structures of the chemokine receptors CXCR1, CXCR4, and CCR5. Model refinement and evaluation suggested that the model based on CCR5 exhibits the most favorable structural properties. In particular, the GPCR-typical structural features, such as a conserved water cluster or conserved non-covalent contacts, are present to a larger extent in the model based on CCR5 compared to the other models. A final model validation based on the recently published US28 crystal structure confirms that the CCR5-based model is the most accurate and exhibits 80.8% correctly modeled residues within the transmembrane helices. The structural agreement between the selected model and the crystal structure suggests that our modeling strategy may also be more generally applicable to other GPCRs of unknown structure.

  9. Translational control and differential RNA decay are key elements regulating postsegregational expression of the killer protein encoded by the parB locus of plasmid R1

    DEFF Research Database (Denmark)

    Gerdes, K; Helin, K; Christensen, O W

    1988-01-01

    The parB locus of plasmid R1, which mediates plasmid stability via postsegregational killing of plasmid-free cells, encodes two genes, hok and sok. The hok gene product is a potent cell-killing protein. The hok gene is regulated at the translational level by the sok gene-encoded repressor, a small...

  10. Regulation of hematopoietic cell function by protein tyrosine kinase-encoding oncogenes, a review

    NARCIS (Netherlands)

    Punt, C. J.

    1992-01-01

    Tyrosine phosphorylation of proteins by protein tyrosine kinases (PTKs) is an important mechanism in the regulation of various cellular processes such as proliferation, differentiation, and transformation. Accumulating data implicate PTKs as essential intermediates in the transduction of

  11. The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

    Science.gov (United States)

    Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

    2017-08-11

    Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

  12. Mutations in DZIP1L, which encodes a ciliary-transition-zone protein, cause autosomal recessive polycystic kidney disease.

    Science.gov (United States)

    Lu, Hao; Galeano, Maria C Rondón; Ott, Elisabeth; Kaeslin, Geraldine; Kausalya, P Jaya; Kramer, Carina; Ortiz-Brüchle, Nadina; Hilger, Nadescha; Metzis, Vicki; Hiersche, Milan; Tay, Shang Yew; Tunningley, Robert; Vij, Shubha; Courtney, Andrew D; Whittle, Belinda; Wühl, Elke; Vester, Udo; Hartleben, Björn; Neuber, Steffen; Frank, Valeska; Little, Melissa H; Epting, Daniel; Papathanasiou, Peter; Perkins, Andrew C; Wright, Graham D; Hunziker, Walter; Gee, Heon Yung; Otto, Edgar A; Zerres, Klaus; Hildebrandt, Friedhelm; Roy, Sudipto; Wicking, Carol; Bergmann, Carsten

    2017-07-01

    Autosomal recessive polycystic kidney disease (ARPKD), usually considered to be a genetically homogeneous disease caused by mutations in PKHD1, has been associated with ciliary dysfunction. Here, we describe mutations in DZIP1L, which encodes DAZ interacting protein 1-like, in patients with ARPKD. We further validated these findings through loss-of-function studies in mice and zebrafish. DZIP1L localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone. In agreement with a defect in the diffusion barrier, we found that the ciliary-membrane translocation of the PKD proteins polycystin-1 and polycystin-2 is compromised in DZIP1L-mutant cells. Together, these data provide what is, to our knowledge, the first conclusive evidence that ARPKD is not a homogeneous disorder and further establish DZIP1L as a second gene involved in ARPKD pathogenesis.

  13. Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

    International Nuclear Information System (INIS)

    Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

    1988-01-01

    The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

  14. Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

    Science.gov (United States)

    Liu, Feng; Roth, Richard A.

    1995-10-01

    To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

  15. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Science.gov (United States)

    Zhang, Yang; Devries, Mark E; Skolnick, Jeffrey

    2006-02-01

    G protein-coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha) root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness

  16. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    2006-02-01

    Full Text Available G protein-coupled receptors (GPCRs, encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness

  17. Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease

    Science.gov (United States)

    Basmanav, F. Buket; Oprisoreanu, Ana-Maria; Pasternack, Sandra M.; Thiele, Holger; Fritz, Günter; Wenzel, Jörg; Größer, Leopold; Wehner, Maria; Wolf, Sabrina; Fagerberg, Christina; Bygum, Anette; Altmüller, Janine; Rütten, Arno; Parmentier, Laurent; El Shabrawi-Caelen, Laila; Hafner, Christian; Nürnberg, Peter; Kruse, Roland; Schoch, Susanne; Hanneken, Sandra; Betz, Regina C.

    2014-01-01

    Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4∗), c.652C>T (p.Arg218∗), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218∗) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology. PMID:24387993

  18. A Legionella pneumophila effector protein encoded in a region of genomic plasticity binds to Dot/Icm-modified vacuoles.

    Directory of Open Access Journals (Sweden)

    Shira Ninio

    2009-01-01

    Full Text Available Legionella pneumophila is an opportunistic pathogen that can cause a severe pneumonia called Legionnaires' disease. In the environment, L. pneumophila is found in fresh water reservoirs in a large spectrum of environmental conditions, where the bacteria are able to replicate within a variety of protozoan hosts. To survive within eukaryotic cells, L. pneumophila require a type IV secretion system, designated Dot/Icm, that delivers bacterial effector proteins into the host cell cytoplasm. In recent years, a number of Dot/Icm substrate proteins have been identified; however, the function of most of these proteins remains unknown, and it is unclear why the bacterium maintains such a large repertoire of effectors to promote its survival. Here we investigate a region of the L. pneumophila chromosome that displays a high degree of plasticity among four sequenced L. pneumophila strains. Analysis of GC content suggests that several genes encoded in this region were acquired through horizontal gene transfer. Protein translocation studies establish that this region of genomic plasticity encodes for multiple Dot/Icm effectors. Ectopic expression studies in mammalian cells indicate that one of these substrates, a protein called PieA, has unique effector activities. PieA is an effector that can alter lysosome morphology and associates specifically with vacuoles that support L. pneumophila replication. It was determined that the association of PieA with vacuoles containing L. pneumophila requires modifications to the vacuole mediated by other Dot/Icm effectors. Thus, the localization properties of PieA reveal that the Dot/Icm system has the ability to spatially and temporally control the association of an effector with vacuoles containing L. pneumophila through activities mediated by other effector proteins.

  19. G-protein-coupled receptors for free fatty acids

    DEFF Research Database (Denmark)

    Milligan, Graeme; Ulven, Trond; Murdoch, Hannah

    2014-01-01

    of these receptors. However, ongoing clinical trials of agonists of free fatty acid receptor 1 suggest that this receptor and other receptors for free fatty acids may provide a successful strategy for controlling hyperglycaemia and providing novel approaches to treat diabetes. Receptors responsive to free fatty acid...

  20. Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending.

    Science.gov (United States)

    Landel, C C; Potthoff, S J; Nardulli, A M; Kushner, P J; Greene, G L

    1997-01-01

    Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis

  1. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor

    DEFF Research Database (Denmark)

    Chen, Y; Grall, D; Salcini, A E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have......-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4...

  2. Characterization of Bombyx mori nucleopolyhedrovirus orf68 gene that encodes a novel structural protein of budded virus.

    Science.gov (United States)

    Iwanaga, Masashi; Kurihara, Masaaki; Kobayashi, Masahiko; Kang, WonKyung

    2002-05-25

    All lepidopteran baculovirus genomes sequenced to date encode a homolog of the Bombyx mori nucleopolyhedrovirus (BmNPV) orf68 gene, suggesting that it performs an important role in the virus life cycle. In this article we describe the characterization of BmNPV orf68 gene. Northern and Western analyses demonstrated that orf68 gene was expressed as a late gene and encoded a structural protein of budded virus (BV). Immunohistochemical analysis by confocal microscopy showed that ORF68 protein was localized mainly in the nucleus of infected cells. To examine the function of orf68 gene, we constructed orf68 deletion mutant (BmD68) and characterized it in BmN cells and larvae of B. mori. BV production was delayed in BmD68-infected cells. The larval bioassays also demonstrated that deletion of orf68 did not reduce the infectivity, but mutant virus took 70 h longer to kill the host than wild-type BmNPV. In addition, dot-blot analysis showed viral DNA accumulated more slowly in mutant infected cells. Further examination suggested that BmD68 was less efficient in entry and budding from cells, although it seemed to possess normal attachment ability. These results suggest that ORF68 is a BV-associated protein involved in secondary infection from cell-to-cell. (c) 2002 Elsevier Science (USA).

  3. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne

    2005-01-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...

  4. Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

    Science.gov (United States)

    Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

    2002-03-01

    Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

  5. Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction.

    Science.gov (United States)

    Beliaev, A S; Saffarini, D A

    1998-12-01

    Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.

  6. β2-Adrenergic receptors and G-protein-coupled receptor kinase 2 in rabbit pleural mesothelium.

    Science.gov (United States)

    Sironi, Chiara; Bodega, Francesca; Armilli, Marta; Porta, Cristina; Zocchi, Luciano; Agostoni, Emilio

    2010-09-30

    Former studies on net rate of liquid absorption from small Ringer or 1% albumin-Ringer hydrothoraces in rabbits indicated that Na+ transport and solute-coupled liquid absorption by mesothelium is increased by pleural liquid dilution, and stimulation of β2-adrenoreceptors (β2AR). In this research we tried to provide molecular evidence for β2AR in visceral and parietal mesothelium of rabbit pleura. Moreover, because prolonged stimulation of β2AR may lead to desensitization mediated by G-protein-coupled receptor kinase 2 (GRK2), we also checked whether GRK2 is expressed in pleural mesothelium. To this end we performed immunoblot assays on total protein extracts from scraped visceral and parietal mesothelium, and from cultured pleural mesothelial cells of rabbits. All three samples showed β2AR and GRK2 specific bands. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Polymorphisms in the gene encoding bovine interleukin-10 receptor alpha are associated with Mycobacterium avium ssp. paratuberculosis infection status

    Directory of Open Access Journals (Sweden)

    Kelton David F

    2010-04-01

    Full Text Available Abstract Background Johne's disease is a chronic inflammatory bowel disease (IBD of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP. Since this pathogen has been implicated in the pathogenesis of human IBDs, the goal of this study was to assess whether single nucleotide polymorphism (SNPs in several well-known candidate genes for human IBD are associated with susceptibility to MAP infection in dairy cattle. Methods The bovine candidate genes, interleukin-10 (IL10, IL10 receptor alpha/beta (IL10RA/B, transforming growth factor beta 1 (TGFB1, TGFB receptor class I/II (TGFBR1/2, and natural resistance-associated macrophage protein 1 (SLC11A1 were sequenced for SNP discovery using pooled DNA samples, and the identified SNPs were genotyped in a case-control association study comprised of 242 MAP negative and 204 MAP positive Holstein dairy cattle. Logistic regression was used to determine the association of SNPs and reconstructed haplotypes with MAP infection status. Results A total of 13 SNPs were identified. Four SNPs in IL10RA (984G > A, 1098C > T, 1269T > C, and 1302A > G were tightly linked, and showed a strong additive and dominance relationship with MAP infection status. Haplotypes AGC and AAT, containing the SNPs IL10RA 633C > A, 984G > A and 1185C > T, were associated with an elevated and reduced likelihood of positive diagnosis by serum ELISA, respectively. Conclusions SNPs in IL10RA are associated with MAP infection status in dairy cattle. The functional significance of these SNPs warrants further investigation.

  8. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  9. Arabidopsis VARIEGATED 3 encodes a chloroplasttargeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, A.; Jenkins, T.

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

  10. FRPR-4 Is a G-Protein Coupled Neuropeptide Receptor That Regulates Behavioral Quiescence and Posture in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Matthew D Nelson

    Full Text Available Neuropeptides signal through G-protein coupled receptors (GPCRs to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regulation of behavioral quiescence and locomotive posture. FRPR-4 is activated in cell culture by several neuropeptides with an amidated isoleucine-arginine-phenylalanine (IRF motif or an amidated valine-arginine-phenylalanine (VRF motif at their carboxy termini, including those encoded by the gene flp-13. Loss of frpr-4 function results in a minor feeding quiescence defect after heat-induced cellular stress. Overexpression of frpr-4 induces quiescence of locomotion and feeding as well as an exaggerated body bend posture. The exaggerated body bend posture requires the gene flp-13. While frpr-4 is expressed broadly, selective overexpression of frpr-4 in the proprioceptive DVA neurons results in exaggerated body bends that require flp-13 in the ALA neuron. Our results suggest that FLP-13 and other neuropeptides signal through FRPR-4 and other receptors to regulate locomotion posture and behavioral quiescence.

  11. Receptor activity modifying proteins (RAMPs) interact with the VPAC1 receptor: evidence for differential RAMP modulation of multiple signalling pathways

    International Nuclear Information System (INIS)

    Christopoulos, G.; Morfis, M.; Sexton, P.M.; Christopoulos, A.; Laburthe, M.; Couvineau, A.

    2001-01-01

    Full text: Receptor activity modifying proteins (RAMP) constitute a family of three accessory proteins that affect the expression and/or phenotype of the calcitonin receptor (CTR) or CTR-like receptor (CRLR). In this study we screened a range of class II G protein-coupled receptors (PTH1, PTH2, GHRH, VPAC1, VPAC2 receptors) for possible RAMP interactions by measurement of receptor-induced translocation of c-myc tagged RAMP1 or HA tagged RAMP3. Of these, only the VPAC1 receptor caused significant translocation of c-myc-RAMP1 or HA-RAMP3 to the cell surface. Co-transfection of VPAC1 and RAMPs did not alter 125 I-VIP binding and specificity. VPAC1 receptor function was subsequently analyzed through parallel determinations of cAMP accumulation and phosphoinositide (PI) hydrolysis in the presence and absence of each of the three RAMPs. In contrast to CTR-RAMP interaction, where there was an increase in cAMP Pharmacologisand a decrease in PI hydrolysis, VPAC1-RAMP interaction was characterized by a specific increase in agonist-mediated PI hydrolysis when co-transfected with RAMP2. This change was due to an enhancement of Emax with no change in EC 50 value for VIP. No significant change in cAMP accumulation was observed. This is the first demonstration of an interaction of RAMPs with a G protein-coupled receptor outside the CTR family and may suggest a more generalized role for RAMPs in modulating G protein-coupled receptor signaling. Copyright (2001) Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists

  12. Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

    DEFF Research Database (Denmark)

    Mahony, Jennifer; Kot, Witold Piotr; Murphy, James

    2013-01-01

    Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been...... implicated as a host receptor of (certain) 936-type phages, is specified by a large gene cluster, which, among different lactococcal strains, contains highly conserved regions as well as regions of diversity. The regions of diversity within this cluster on the genomes of lactococcal strains MG1363, SK11, IL......1403, KF147, CV56, and UC509.9 were used for the development of a multiplex PCR system to identify the pellicle genotype of lactococcal strains used in this study. The resulting comparative analysis revealed an apparent correlation between the pellicle genotype of a given host strain and the host range...

  13. The repertoire of trace amine G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gloriam, David E.; Bjarnadóttir, Thóra K; Yan, Yi-Lin

    2005-01-01

    eukaryotic species for receptors similar to the mammalian trace amine (TA) receptor subfamily. We identified 18 new receptors in rodents that are orthologous to the previously known TA-receptors. Remarkably, we found 57 receptors (and 40 pseudogenes) of this type in the zebrafish (Danio rerio), while fugu...... (Takifugu rubripes) had only eight receptors (and seven pseudogenes). We mapped 47 of the zebrafish TA-receptors on chromosomes using radiation hybrid panels and meiotic mapping. The results, together with the degree of conservation and phylogenetic relationships displayed among the zebrafish receptors...

  14. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng

    2003-01-01

    Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha...

  15. Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

    Science.gov (United States)

    Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

    2011-01-01

    P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

  16. The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids

    Directory of Open Access Journals (Sweden)

    Ngai John

    2006-12-01

    Full Text Available Abstract Background Vertebrate odorant receptors comprise at least three types of G protein-coupled receptors (GPCRs: the OR, V1R, and V2R/V2R-like receptors, the latter group belonging to the C family of GPCRs. These receptor families are thought to receive chemosensory information from a wide spectrum of odorant and pheromonal cues that influence critical animal behaviors such as feeding, reproduction and other social interactions. Results Using genome database mining and other informatics approaches, we identified and characterized the repertoire of 54 intact "V2R-like" olfactory C family GPCRs in the zebrafish. Phylogenetic analysis – which also included a set of 34 C family GPCRs from fugu – places the fish olfactory receptors in three major groups, which are related to but clearly distinct from other C family GPCRs, including the calcium sensing receptor, metabotropic glutamate receptors, GABA-B receptor, T1R taste receptors, and the major group of V2R vomeronasal receptor families. Interestingly, an analysis of sequence conservation and selective pressure in the zebrafish receptors revealed the retention of a conserved sequence motif previously shown to be required for ligand binding in other amino acid receptors. Conclusion Based on our findings, we propose that the repertoire of zebrafish olfactory C family GPCRs has evolved to allow the detection and discrimination of a spectrum of amino acid and/or amino acid-based compounds, which are potent olfactory cues in fish. Furthermore, as the major groups of fish receptors and mammalian V2R receptors appear to have diverged significantly from a common ancestral gene(s, these receptors likely mediate chemosensation of different classes of chemical structures by their respective organisms.

  17. Endothelial Protein C Receptor and Pediatric Arterial Stroke

    Directory of Open Access Journals (Sweden)

    Nejat Akar

    2013-03-01

    Full Text Available Objective: The aim of this study was to investigate the endothelial protein C receptor (EPCR gene A3 haplotype and plasma soluble EPCR (sEPCR levels in Turkish pediatric arterial stroke patients. Materials and Methods: We analyzed 44 pediatric arterial stroke patients and 75 healthy controls. Following DNA isolation, genotyping of the A3 haplotype was determined via PCR and RFLP. Additionally, fasting sEPCR levels were determined via ELISA. Results: There wasn’t a significant difference in the sEPCR level between the control and patient groups, although the sEPCR level was higher in the patient group. We didn’t observe a difference in the distribution of the CC and CG/GG genotypes between the control and patient groups. Conclusion: Further study on sEPCR levels at the onset of pediatric stroke is needed in order to reach a more definitive conclusion.

  18. Peptide drugs to target G protein-coupled receptors.

    Science.gov (United States)

    Bellmann-Sickert, Kathrin; Beck-Sickinger, Annette G

    2010-09-01

    Major indications for use of peptide-based therapeutics include endocrine functions (especially diabetes mellitus and obesity), infectious diseases, and cancer. Whereas some peptide pharmaceuticals are drugs, acting as agonists or antagonists to directly treat cancer, others (including peptide diagnostics and tumour-targeting pharmaceuticals) use peptides to 'shuttle' a chemotherapeutic agent or a tracer to the tumour and allow sensitive imaging or targeted therapy. Significant progress has been made in the last few years to overcome disadvantages in peptide design such as short half-life, fast proteolytic cleavage, and low oral bioavailability. These advances include peptide PEGylation, lipidisation or multimerisation; the introduction of peptidomimetic elements into the sequences; and innovative uptake strategies such as liposomal, capsule or subcutaneous formulations. This review focuses on peptides targeting G protein-coupled receptors that are promising drug candidates or that have recently entered the pharmaceutical market. Copyright 2010 Elsevier Ltd. All rights reserved.

  19. Protein-protein association and cellular localization of four essential gene products encoded by tellurite resistance-conferring cluster "ter" from pathogenic Escherichia coli.

    Science.gov (United States)

    Valkovicova, Lenka; Vavrova, Silvia Minarikova; Mravec, Jozef; Grones, Jozef; Turna, Jan

    2013-12-01

    Gene cluster "ter" conferring high tellurite resistance has been identified in various pathogenic bacteria including Escherichia coli O157:H7. However, the precise mechanism as well as the molecular function of the respective gene products is unclear. Here we describe protein-protein association and localization analyses of four essential Ter proteins encoded by minimal resistance-conferring fragment (terBCDE) by means of recombinant expression. By using a two-plasmid complementation system we show that the overproduced single Ter proteins are not able to mediate tellurite resistance, but all Ter members play an irreplaceable role within the cluster. We identified several types of homotypic and heterotypic protein-protein associations among the Ter proteins by in vitro and in vivo pull-down assays and determined their cellular localization by cytosol/membrane fractionation. Our results strongly suggest that Ter proteins function involves their mutual association, which probably happens at the interface of the inner plasma membrane and the cytosol.

  20. The prostaglandin EP1 receptor potentiates kainate receptor activation via a protein kinase C pathway and exacerbates status epilepticus

    Science.gov (United States)

    Rojas, Asheebo; Gueorguieva, Paoula; Lelutiu, Nadia; Quan, Yi; Shaw, Renee; Dingledine, Raymond

    2014-01-01

    Prostaglandin E2 (PGE2) regulates membrane excitability, synaptic transmission, plasticity, and neuronal survival. The consequences of PGE2 release following seizures has been the subject of much study. Here we demonstrate that the prostaglandin E2 receptor 1 (EP1, or Ptger1) modulates native kainate receptors, a family of ionotropic glutamate receptors widely expressed throughout the central nervous system. Global ablation of the EP1 gene in mice (EP1-KO) had no effect on seizure threshold after kainate injection but reduced the likelihood to enter status epilepticus. EP1-KO mice that did experience typical status epilepticus had reduced hippocampal neurodegeneration and a blunted inflammatory response. Further studies with native prostanoid and kainate receptors in cultured cortical neurons, as well as with recombinant prostanoid and kainate receptors expressed in Xenopus oocytes, demonstrated that EP1 receptor activation potentiates heteromeric but not homomeric kainate receptors via a second messenger cascade involving phospholipase C, calcium and protein kinase C. Three critical GluK5 C-terminal serines underlie the potentiation of the GluK2/GluK5 receptor by EP1 activation. Taken together, these results indicate that EP1 receptor activation during seizures, through a protein kinase C pathway, increases the probability of kainic acid induced status epilepticus, and independently promotes hippocampal neurodegeneration and a broad inflammatory response. PMID:24952362

  1. Hypomorphic mutations in PGAP2, encoding a GPI-anchor-remodeling protein, cause autosomal-recessive intellectual disability

    DEFF Research Database (Denmark)

    Hansen, Lars; Tawamie, Hasan; Murakami, Yoshiko

    2013-01-01

    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...

  2. DMBT1 encodes a protein involved in the immune defense and in epithelial differentiation and is highly unstable in cancer

    DEFF Research Database (Denmark)

    Mollenhauer, J; Herbertz, S; Holmskov, U

    2000-01-01

    in the respiratory immune defense. Immunohistochemical analyses revealed that DMBT1 is produced by both tumor-associated macrophages and tumor cells and that it is deregulated in glioblastoma multiforme in comparison to normal brain tissue. Our data further suggest that the proteins CRP-ductin and hensin, both...... of which have been implicated in epithelial differentiation, are the DMBT1 orthologs in mice and rabbits, respectively. These findings and the spatial and temporal distribution of DMBT1 in fetal and adult epithelia suggest that DMBT1 further plays a role in epithelial development. Rearrangements of DMBT1......, DMBT1 is a gene that is highly unstable in cancer and encodes for a protein with at least two different functions, one in the immune defense and a second one in epithelial differentiation....

  3. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    Science.gov (United States)

    Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

    2012-01-01

    Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  4. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    Directory of Open Access Journals (Sweden)

    Satoshi Sekiguchi

    Full Text Available Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV, is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis, liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25, which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-/MxCre((+/- mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor TNF-α and (interleukin IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  5. Conformational Fluctuations in G-Protein-Coupled Receptors

    Science.gov (United States)

    Brown, Michael F.

    2014-03-01

    G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets, where rhodopsin is an important prototype and occurs naturally in a lipid membrane. Rhodopsin photoactivation entails 11-cis to all-trans isomerization of the retinal cofactor, yielding an equilibrium between inactive Meta-I and active Meta-II states. Two important questions are: (1) Is rhodopsin is a simple two-state switch? Or (2) does isomerization of retinal unlock an activated conformational ensemble? For an ensemble-based activation mechanism (EAM) a role for conformational fluctuations is clearly indicated. Solid-state NMR data together with theoretical molecular dynamics (MD) simulations detect increased local mobility of retinal after light activation. Resultant changes in local dynamics of the cofactor initiate large-scale fluctuations of transmembrane helices that expose recognition sites for the signal-transducing G-protein. Time-resolved FTIR studies and electronic spectroscopy further show the conformational ensemble is strongly biased by the membrane lipid composition, as well as pH and osmotic pressure. A new flexible surface model (FSM) describes how the curvature stress field of the membrane governs the energetics of active rhodopsin, due to the spontaneous monolayer curvature of the lipids. Furthermore, influences of osmotic pressure dictate that a large number of bulk water molecules are implicated in rhodopsin activation. Around 60 bulk water molecules activate rhodopsin, which is much larger than the number of structural waters seen in X-ray crystallography, or inferred from studies of bulk hydrostatic pressure. Conformational selection and promoting vibrational motions of rhodopsin lead to activation of the G-protein (transducin). Our biophysical data give a paradigm shift in understanding GPCR activation. The new view is: dynamics and conformational fluctuations involve an ensemble of substates that activate the cognate G-protein in the amplified visual

  6. Unfolded Protein Response (UPR Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Martin Hampel

    Full Text Available The unfolded protein response (UPR, a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER, coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

  7. Identification and characterization of a novel Cut family cDNA that encodes human copper transporter protein CutC

    International Nuclear Information System (INIS)

    Li Jixi; Ji Chaoneng; Chen Jinzhong; Yang Zhenxing; Wang Yijing; Fei, Xiangwei; Zheng Mei; Gu Xing; Wen Ge; Xie Yi; Mao Yumin

    2005-01-01

    Copper is an essential heavy metal trace element that plays important roles in cell physiology. The Cut family was associated with the copper homeostasis and involved in several important metabolisms, such as uptake, storage, delivery, and efflux of copper. In this study, a novel Cut family cDNA was isolated from the human fetal brain library, which encodes a 273 amino acid protein with a molecular mass of about 29.3 kDa and a calculated pI of 8.17. It was named hCutC (human copper transporter protein CutC). The ORF of hCutC gene was cloned into pQE30 vector and expressed in Escherichia coli M15. The secreted hCutC protein was purified to a homogenicity of 95% by using the Ni-NTA affinity chromatography. RT-PCR analysis showed that the hCutC gene expressed extensively in human tissues. Subcellular location analysis of hCutC-EGFP fusion protein revealed that hCutC was distributed to cytoplasm of COS-7 cells, and both cytoplasm and nucleus of AD293 cells. The results suggest that hCutC may be one shuttle protein and play important roles in intracellular copper trafficking

  8. [Roles of G protein-coupled estrogen receptor in the male reproductive system].

    Science.gov (United States)

    Chen, Kai-hong; Zhang, Xian; Jiang, Xue-wu

    2016-02-01

    The G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30), was identified in the recent years as a functional membrane receptor different from the classical nuclear estrogen receptors. This receptor is widely expressed in the cortex, cerebellum, hippocampus, heart, lung, liver, skeletal muscle, and the urogenital system. It is responsible for the mediation of nongenomic effects associated with estrogen and its derivatives, participating in the physiological activities of the body. The present study reviews the molecular structure, subcellular localization, signaling pathways, distribution, and function of GPER in the male reproductive system.

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

    Science.gov (United States)

    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.

  10. Phylogenetic analysis of fungal heterotrimeric G protein-encoding genes and their expression during dimorphism in Mucor circinelloides.

    Science.gov (United States)

    Valle-Maldonado, Marco Iván; Jácome-Galarza, Irvin Eduardo; Díaz-Pérez, Alma Laura; Martínez-Cadena, Guadalupe; Campos-García, Jesús; Ramírez-Díaz, Martha Isela; Reyes-De la Cruz, Homero; Riveros-Rosas, Héctor; Díaz-Pérez, César; Meza-Carmen, Víctor

    2015-12-01

    In fungi, heterotrimeric G proteins are key regulators of biological processes such as mating, virulence, morphology, among others. Mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric G protein subunits in the fungal kingdom: twelve Gα (McGpa1-12), three Gβ (McGpb1-3), and three Gγ (McGpg1-3). Phylogenetic analysis of fungal Gα showed that they are divided into four distinct groups as reported previously. Fungal Gβ and Gγ are also divided into four phylogenetic groups, and to our understanding this is the first report of a phylogenetic classification for fungal Gβ and Gγ subunits. Almost all genes that encode putative heterotrimeric G subunits in M. circinelloides are differentially expressed during dimorphic growth, except for McGpg1 (Gγ) that showed very low mRNA levels at all developmental stages. Moreover, several of the subunits are expressed in a similar pattern and at the same level, suggesting that they constitute discrete complexes. For example, McGpb3 (Gβ), and McGpg2 (Gγ), are co-expressed during mycelium growth, and McGpa1, McGpb2, and McGpg2, are co-expressed during yeast development. These findings provide the conceptual framework to study the biological role of these genes during M. circinelloides morphogenesis. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  11. Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Klahre, U; Hemmings-Mieszczak, M; Filipowicz, W

    1995-06-01

    We have previously characterized nuclear cDNA clones encoding two RNA binding proteins, CP-RBP30 and CP-RBP-31, which are targeted to chloroplasts in Nicotiana plumbaginifolia. In this report we describe the analysis of the 3'-untranslated regions (3'-UTRs) in 22 CP-RBP30 and 8 CP-RBP31 clones which reveals that mRNAs encoding both proteins have a very complex polyadenylation pattern. Fourteen distinct poly(A) sites were identified among CP-RBP30 clones and four sites among the CP-RBP31 clones. The authenticity of the sites was confirmed by RNase A/T1 mapping of N. plumbaginifolia RNA. CP-RBP30 provides an extreme example of the heterogeneity known to be a feature of mRNA polyadenylation in higher plants. Using PCR we have demonstrated that CP-RBP genes in N. plumbaginifolia and N. sylvestris, in addition to the previously described introns interrupting the coding region, contain an intron located in the 3' non-coding part of the gene. In the case of the CP-RBP31, we have identified one polyadenylation event occurring in this intron.

  12. The number of genes encoding repeat domain-containing proteins positively correlates with genome size in amoebal giant viruses

    Science.gov (United States)

    Shukla, Avi; Chatterjee, Anirvan

    2018-01-01

    Abstract Curiously, in viruses, the virion volume appears to be predominantly driven by genome length rather than the number of proteins it encodes or geometric constraints. With their large genome and giant particle size, amoebal viruses (AVs) are ideally suited to study the relationship between genome and virion size and explore the role of genome plasticity in their evolutionary success. Different genomic regions of AVs exhibit distinct genealogies. Although the vertically transferred core genes and their functions are universally conserved across the nucleocytoplasmic large DNA virus (NCLDV) families and are essential for their replication, the horizontally acquired genes are variable across families and are lineage-specific. When compared with other giant virus families, we observed a near–linear increase in the number of genes encoding repeat domain-containing proteins (RDCPs) with the increase in the genome size of AVs. From what is known about the functions of RDCPs in bacteria and eukaryotes and their prevalence in the AV genomes, we envisage important roles for RDCPs in the life cycle of AVs, their genome expansion, and plasticity. This observation also supports the evolution of AVs from a smaller viral ancestor by the acquisition of diverse gene families from the environment including RDCPs that might have helped in host adaption. PMID:29308275

  13. Proteins Encoded in Genomic Regions Associated with Immune-Mediated Disease Physically Interact and Suggest Underlying Biology

    Science.gov (United States)

    Rossin, Elizabeth J.; Lage, Kasper; Raychaudhuri, Soumya; Xavier, Ramnik J.; Tatar, Diana; Benita, Yair

    2011-01-01

    Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein–protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in

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

    Science.gov (United States)

    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

  15. Ligand binding to G protein-coupled receptors in tethered cell membranes

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

    2003-01-01

    for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

  16. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  17. Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem

    Czech Academy of Sciences Publication Activity Database

    Ernst, A.; Jekat, S. B.; Zielonka, S.; Mueller, B.; Neumann, U.; Ruping, B.; Twyman, R. M.; Krzyžánek, Vladislav; Pruefer, D.; Noll, G. A.

    2012-01-01

    Roč. 109, č. 28 (2012), E1980-E1989 ISSN 0027-8424 Institutional support: RVO:68081731 Keywords : photoassimilate transport * wound response * exudation * phloem protein 1 Subject RIV: CE - Biochemistry Impact factor: 9.737, year: 2012

  18. Mutations in Three Genes Encoding Proteins Involved in Hair Shaft Formation Cause Uncombable Hair Syndrome

    DEFF Research Database (Denmark)

    Ü Basmanav, F Buket; Cau, Laura; Tafazzoli, Aylar

    2016-01-01

    Uncombable hair syndrome (UHS), also known as "spun glass hair syndrome," "pili trianguli et canaliculi," or "cheveux incoiffables" is a rare anomaly of the hair shaft that occurs in children and improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant...... in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments...... and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic...

  19. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    International Nuclear Information System (INIS)

    Gualdrón-López, Melisa; Michels, Paul A.M.

    2013-01-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M r of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M r of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and 35 S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed

  20. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: paul.michels@uclouvain.be [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)

    2013-02-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  1. Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA

    DEFF Research Database (Denmark)

    Nielsen, Morten S; Gustafsen, Camilla; Madsen, Peder

    2007-01-01

    -formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its...

  2. Computational studies of G protein-coupled receptor complexes : Structure and dynamics

    NARCIS (Netherlands)

    Sensoy, Ozge; Almeida, Jose G; Shabbir, Javeria; de Sousa Moreira, Irina; Morra, Giulia

    2017-01-01

    G protein-coupled receptors (GPCRs) are ubiquitously expressed transmembrane proteins associated with a wide range of diseases such as Alzheimer's, Parkinson, schizophrenia, and also implicated in in several abnormal heart conditions. As such, this family of receptors is regarded as excellent drug

  3. Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

    International Nuclear Information System (INIS)

    Senogles, S.E.; Caron, M.G.

    1986-01-01

    The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

  4. A molecular pharmacologist's guide to G protein-coupled receptor crystallography

    NARCIS (Netherlands)

    Piscitelli, Chayne L.; Kean, James; de Graaf, C.; Deupi, Xavier

    2015-01-01

    G protein-coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including class B, C, and Frizzled

  5. Protein complexes associated with the Kaposi's sarcoma-associated herpesvirus-encoded LANA

    International Nuclear Information System (INIS)

    Kaul, Rajeev; Verma, Subhash C.; Robertson, Erle S.

    2007-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA is known to modulate viral and cellular gene expression. We report here on some initial proteomic studies to identify cellular proteins associated with the amino and carboxy-terminal domains of LANA. The results of these studies show an association of known cellular proteins which support LANA functions and have identified additional LANA-associated proteins. These results provide new evidence for complexes involving LANA with a number of previously unreported functional classes of proteins including DNA polymerase, RNA helicase and cell cycle control proteins. The results also indicate that the amino terminus of LANA can interact with its carboxy-terminal domain. This interaction is potentially important for facilitating associations with other cell cycle regulatory proteins which include CENP-F identified in association with both the amino and carboxy-termini. These novel associations add to the diversity of LANA functions in relation to the maintenance of latency and subsequent transformation of KSHV infected cells

  6. Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer's disease: Identification as the microtubule-associated protein tau

    International Nuclear Information System (INIS)

    Goedert, M.; Wischik, C.M.; Crowther, R.A.; Walker, J.E.; Klug, A.

    1988-01-01

    Screening of cDNA libraries prepared from the frontal cortex of an Alzheimer's disease patient and from fetal human brain has led to isolation of the cDNA for a core protein of the paired helical filament of Alzheimer's disease. The partial amino acid sequence of this core protein was used to design synthetic oligonucleotide probes. The cDNA encodes a protein of 352 amino acids that contains a characteristic amino acid repeat in its carboxyl-terminal half. This protein is highly homologous to the sequence of the mouse microtubule-associated protein tau and thus constitutes the human equivalent of mouse tau. RNA blot analysis indicates the presence of two major transcripts, 6 and 2 kilobases long, with a wide distribution in normal human brain. Tau protein mRNAs were found in normal amounts in the frontal cortex from patients with Alzheimer's disease. The proof that at least part of tau protein forms a component of the paired helical filament core opens the way to understanding the mode of formation of paired helical filaments and thus, ultimately, the pathogenesis of Alzheimer's disease

  7. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  8. Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis.

    Science.gov (United States)

    Martin, Carol-Anne; Murray, Jennie E; Carroll, Paula; Leitch, Andrea; Mackenzie, Karen J; Halachev, Mihail; Fetit, Ahmed E; Keith, Charlotte; Bicknell, Louise S; Fluteau, Adeline; Gautier, Philippe; Hall, Emma A; Joss, Shelagh; Soares, Gabriela; Silva, João; Bober, Michael B; Duker, Angela; Wise, Carol A; Quigley, Alan J; Phadke, Shubha R; Wood, Andrew J; Vagnarelli, Paola; Jackson, Andrew P

    2016-10-01

    Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish "condensinopathies" as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size. © 2016 Martin et al.; Published by Cold Spring Harbor Laboratory Press.

  9. Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes

    DEFF Research Database (Denmark)

    Schubert, J.; Siekierska, A.; Langlois, M.

    2014-01-01

    Febrile seizures affect 2-4% of all children(1) and have a strong genetic component(2). Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2)(3-5) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding...... syntaxin-1B(6), that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees(7,8) identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations...... and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature...

  10. Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

    OpenAIRE

    Ping, P; Gelzer-Bell, R; Roth, D A; Kiel, D; Insel, P A; Hammond, H K

    1995-01-01

    To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling as...

  11. An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

    International Nuclear Information System (INIS)

    Meier, K.; Klein, C.

    1988-01-01

    The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

  12. The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

    Directory of Open Access Journals (Sweden)

    M. A. Prado

    2001-01-01

    Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

  13. Analysis of viral protein-2 encoding gene of avian encephalomyelitis virus from field specimens in Central Java region, Indonesia

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2016-01-01

    Full Text Available Aim: Avian encephalomyelitis (AE is a viral disease which can infect various types of poultry, especially chicken. In Indonesia, the incidence of AE infection in chicken has been reported since 2009, the AE incidence tends to increase from year to year. The objective of this study was to analyze viral protein 2 (VP-2 encoding gene of AE virus (AEV from various species of birds in field specimen by reverse transcription polymerase chain reaction (RT-PCR amplification using specific nucleotides primer for confirmation of AE diagnosis. Materials and Methods: A total of 13 AEV samples are isolated from various species of poultry which are serologically diagnosed infected by AEV from some areas in central Java, Indonesia. Research stage consists of virus samples collection from field specimens, extraction of AEV RNA, amplification of VP-2 protein encoding gene by RT-PCR, separation of RT-PCR product by agarose gel electrophoresis, DNA sequencing and data analysis. Results: Amplification products of the VP-2 encoding gene of AEV by RT-PCR methods of various types of poultry from field specimens showed a positive results on sample code 499/4/12 which generated DNA fragment in the size of 619 bp. Sensitivity test of RT-PCR amplification showed that the minimum concentration of RNA template is 127.75 ng/μl. The multiple alignments of DNA sequencing product indicated that positive sample with code 499/4/12 has 92% nucleotide homology compared with AEV with accession number AV1775/07 and 85% nucleotide homology with accession number ZCHP2/0912695 from Genbank database. Analysis of VP-2 gene sequence showed that it found 46 nucleotides difference between isolate 499/4/12 compared with accession number AV1775/07 and 93 nucleotides different with accession number ZCHP2/0912695. Conclusions: Analyses of the VP-2 encoding gene of AEV with RT-PCR method from 13 samples from field specimen generated the DNA fragment in the size of 619 bp from one sample with

  14. Proteins Encoded in Genomic Regions Associated with Immune-Mediated Disease Physically Interact and Suggest Underlying Biology

    DEFF Research Database (Denmark)

    Rossin, Elizabeth J.; Hansen, Kasper Lage; Raychaudhuri, Soumya

    2011-01-01

    Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these r......Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed...... in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein-protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more...... that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non...

  15. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

    Directory of Open Access Journals (Sweden)

    Yingying Cai

    Full Text Available Family B G protein-coupled receptors (GPCRs play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1 receptor (GLP1R, whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

  16. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

    Science.gov (United States)

    Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

    2017-01-01

    Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

  17. Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

    International Nuclear Information System (INIS)

    Pires, L.A.; Hegg, R.; Freitas, F.R.; Tavares, E.R.; Almeida, C.P.; Baracat, E.C.; Maranhão, R.C.

    2012-01-01

    Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy

  18. A novel gene encoding a TIG multiple domain protein is a positional candidate for autosomal recessive polycystic kidney disease.

    Science.gov (United States)

    Xiong, Huaqi; Chen, Yongxiong; Yi, Yajun; Tsuchiya, Karen; Moeckel, Gilbert; Cheung, Joseph; Liang, Dan; Tham, Kyi; Xu, Xiaohu; Chen, Xing-Zhen; Pei, York; Zhao, Zhizhuang Jeo; Wu, Guanqing

    2002-07-01

    Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-p12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. We recently identified a novel gene in this genetic interval from kidney cDNA, using cloning strategies. The gene PKHD1 (PKHD1-tentative) encodes a novel 3396-amino-acid protein with no apparent homology with any known proteins. We named its gene product "tigmin" because it contains multiple TIG domains, which usually are seen in proteins containing immunoglobulin-like folds. PKHD1 encodes an 11.6-kb transcript and is composed of 61 exons spanning an approximately 365-kb genomic region on chromosome 6p12-p11.2 adjacent to the marker D6S1714. Northern blot analyses demonstrated that the gene has discrete bands with one peak signal at approximately 11 kb, indicating that PKHD1 is likely to have multiple alternative transcripts. PKHD1 is highly expressed in adult and infant kidneys and weakly expressed in liver in northern blot analysis. This expression pattern parallels the tissue involvement observed in ARPKD. In situ hybridization analysis further revealed that the expression of PKHD1 in the kidney is mainly localized to the epithelial cells of the collecting duct, the specific tubular segment involved in cyst formation in ARPKD. These features of PKHD1 make it a strong positional candidate gene for ARPKD.

  19. Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation.

    Science.gov (United States)

    Kanekura, Kohsuke; Yagi, Takuya; Cammack, Alexander J; Mahadevan, Jana; Kuroda, Masahiko; Harms, Matthew B; Miller, Timothy M; Urano, Fumihiko

    2016-05-01

    The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the Chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This genetic alteration leads to the accumulation of five types of poly-dipeptides translated from the GGGGCC hexanucleotide repeat. Among these, poly-proline-arginine (poly-PR) and poly-glycine-arginine (poly-GR) peptides are known to be neurotoxic. However, the mechanisms of neurotoxicity associated with these poly-dipeptides are not clear. A proteomics approach identified a number of interacting proteins with poly-PR peptide, including mRNA-binding proteins, ribosomal proteins, translation initiation factors and translation elongation factors. Immunostaining of brain sections from patients with C9orf72 ALS showed that poly-GR was colocalized with a mRNA-binding protein, hnRNPA1. In vitro translation assays showed that poly-PR and poly-GR peptides made insoluble complexes with mRNA, restrained the access of translation factors to mRNA, and blocked protein translation. Our results demonstrate that impaired protein translation mediated by poly-PR and poly-GR peptides plays a role in neurotoxicity and reveal that the pathways altered by the poly-dipeptides-mRNA complexes are potential therapeutic targets for treatment of C9orf72 FTD/ALS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. The Compass-like locus, exclusive to the Ambulacrarians, encodes a chromatin insulator binding protein in the sea urchin embryo.

    Directory of Open Access Journals (Sweden)

    Vincenzo Cavalieri

    Full Text Available Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl, a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer

  1. Getting a Handle on Neuropharmacology by Targeting Receptor-Associated Proteins.

    Science.gov (United States)

    Maher, Michael P; Matta, Jose A; Gu, Shenyan; Seierstad, Mark; Bredt, David S

    2017-12-06

    Targeted therapy for neuropsychiatric disorders requires selective modulation of dysfunctional neuronal pathways. Receptors relevant to CNS disorders typically have associated proteins discretely expressed in specific neuronal pathways; these accessory proteins provide a new dimension for drug discovery. Recent studies show that targeting a TARP auxiliary subunit of AMPA receptors selectively modulates neuronal excitability in specific forebrain pathways relevant to epilepsy. Other medicinally important ion channels, gated by glutamate, γ-aminobutyric acid (GABA), and acetylcholine, also have associated proteins, which may be druggable. This emerging pharmacology of receptor-associated proteins provides a new approach for improving drug efficacy while mitigating side effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Identification of two novel genes encoding 97- to 99-kilodalton outer membrane proteins of Chlamydia pneumoniae.Infect Immun. 1999 Jan;67(1):375-83

    DEFF Research Database (Denmark)

    Knudsen, K; Madsen, AS; Mygind, P

    1999-01-01

    Two genes encoding 97- to 99-kDa Chlamydia pneumoniae VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the C. pneumoniae outer membrane complex, and the deduced amino acid sequences were similar to those...

  3. Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393T

    NARCIS (Netherlands)

    Martínez, B.; Sillanpää, J.; Smit, E.; Korhonen, T.K.; Pouwels, P.H.

    2000-01-01

    The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393T. The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting

  4. Local anesthetic inhibition of G protein-coupled receptor signaling by interference with Galpha(q) protein function

    NARCIS (Netherlands)

    Hollmann, M. W.; Wieczorek, K. S.; Berger, A.; Durieux, M. E.

    2001-01-01

    Although local anesthetics are considered primarily Na(+) channel blockers, previous studies suggest a common intracellular site of action on different G protein-coupled receptors. In the present study, we characterized this site for the LPA, m1 muscarinic, and trypsin receptor. Xenopus laevis

  5. Comparative genomic analyses of transport proteins encoded within the genomes of Leptospira species.

    Science.gov (United States)

    Buyuktimkin, Bora; Saier, Milton H

    2015-11-01

    Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity was accompanied by progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Comparative analyses of transport proteins encoded within the genomes of Leptospira species.

    Science.gov (United States)

    Buyuktimkin, Bora; Saier, Milton H

    2016-09-01

    Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they all have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity arose in Leptospira correlating to progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. Copyright © 2016. Published by Elsevier Ltd.

  7. rad-Dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint

    NARCIS (Netherlands)

    Walworth, N.C.; Bernards, R.A.

    1996-01-01

    Exposure of eukaryotic cells to agents that generate DNA damage results in transient arrest of progression through the cell cycle. In fission yeast, the DNA damage checkpoint associated with cell cycle arrest before mitosis requires the protein kinase p56chk1. DNA damage induced by ultraviolet

  8. Characterization of the hormone-binding domain of the chicken c-erbA/thyroid hormone receptor protein

    DEFF Research Database (Denmark)

    Muñoz, A; Zenke, M; Gehring, U

    1988-01-01

    mutations present in the carboxy-terminal half of P75gag-v-erbA co-operate in abolishing hormone binding, and that the ligand-binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand-binding domains of P75......To identify and characterize the hormone-binding domain of the thyroid hormone receptor, we analyzed the ligand-binding capacities of proteins representing chimeras between the normal receptor and P75gag-v-erbA, the retrovirus-encoded form deficient in binding ligand. Our results show that several......gag-v-erbA and that renders it biologically inactive fails to affect hormone binding by the c-erbA protein. These results suggest that the mutation changed the ability of P75gag-v-erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone...

  9. A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

    Science.gov (United States)

    Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

    2008-12-01

    A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

  10. Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

    Science.gov (United States)

    Bell, Nicholas A. W.; Keyser, Ulrich F.

    2016-07-01

    The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.

  11. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling...... regarding total protein tyrosine phosphorylation, TCR down-regulation, mobilization of intracellular free calcium, or induction of the activation markers CD69 and CD25....

  12. The Scaffolding Protein Synapse-Associated Protein 97 is Required for Enhanced Signaling Through Isotype-Switched IgG Memory B Cell Receptors

    Science.gov (United States)

    Liu, Wanli; Chen, Elizabeth; Zhao, Xing Wang; Wan, Zheng Peng; Gao, Yi Ren; Davey, Angel; Huang, Eric; Zhang, Lijia; Crocetti, Jillian; Sandoval, Gabriel; Joyce, M. Gordon; Miceli, Carrie; Lukszo, Jan; Aravind, L.; Swat, Wojciech; Brzostowski, Joseph; Pierce, Susan K.

    2012-01-01

    Memory B cells are generated during an individual's first encounter with a foreign antigen and respond to re-encounter with the same antigen through cell surface immunoglobulin G (IgG) B cell receptors (BCRs) resulting in rapid, high-titered IgG antibody responses. Despite a central role for IgG BCRs in B cell memory, our understanding of the molecular mechanism by which IgG BCRs enhance antibody responses is incomplete. Here, we showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ-binding motif, associated with synapse-associated protein 97 (SAP97), a member of the PDZ domain–containing, membrane-associated guanylate-kinase family of scaffolding molecules that play key roles in controlling receptor density and signal strength at neuronal synapses. We showed that SAP97 accumulated and bound to IgG BCRs in the immune synapses that formed in response to engagement of the B cell with antigen. Knocking down SAP97 in IgG-expressing B cells or mutating the putative PDZ-binding motif in the tail impaired immune synapse formation, the initiation of IgG BCR signaling, and downstream activation of p38 mitogen-activated protein kinase. Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immune synapse. PMID:22855505

  13. A ligand channel through the G protein coupled receptor opsin.

    Directory of Open Access Journals (Sweden)

    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  14. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  15. BDNF downregulates 5-HT(2A) receptor protein levels in hippocampal cultures

    DEFF Research Database (Denmark)

    Trajkovska, V; Santini, M A; Marcussen, Anders Bue

    2009-01-01

    Both brain-derived neurotrophic factor (BDNF) and the serotonin receptor 2A (5-HT(2A)) have been related to depression pathology. Specific 5-HT(2A) receptor changes seen in BDNF conditional mutant mice suggest that BDNF regulates the 5-HT(2A) receptor level. Here we show a direct effect of BDNF...... on 5-HT(2A) receptor protein levels in primary hippocampal neuronal and mature hippocampal organotypic cultures exposed to different BDNF concentrations for either 1, 3, 5 or 7 days. In vivo effects of BDNF on hippocampal 5-HT(2A) receptor levels were further corroborated in (BDNF +/-) mice...... with reduced BDNF levels. In primary neuronal cultures, 7 days exposure to 25 and 50ng/mL BDNF resulted in downregulation of 5-HT(2A), but not of 5-HT(1A), receptor protein levels. The BDNF-associated downregulation of 5-HT(2A) receptor levels was also observed in mature hippocampal organotypic cultures...

  16. Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

    Directory of Open Access Journals (Sweden)

    Michael S. Parker

    2014-03-01

    Full Text Available The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY receptors and could apply to many receptors that use large peptidic agonists.

  17. Modulation of expression of genes encoding nuclear proteins following exposure to JANUS neutrons or γ-rays

    International Nuclear Information System (INIS)

    Woloschak, G.E.; Chang-Liu, Chin-Mei

    1994-01-01

    Previous work has shown that exposure of cells to ionizing radiations causes modulation of a variety of genes, including those encoding c-fos, interleukin-1, tumor necrosis factor, cytoskeletal elements, and many more. The experiments reported herein were designed to examine the effects of either JANUS neutron or γ-ray exposure on expression of genes encoding nucleus-associated proteins (H4-histone, c-jun, c-myc, Rb, and p53). Cycling Syrian hamster embryo cells were irradiated with varying doses and dose rates of either JANUS fission-spectrum neutrons or γ-rays; after incubation of the cell cultures for 1 h following radiation exposure, mRNA was harvested and analyzed by Northern blot. Results revealed induction of transcripts for c-jun, H4-histone, and Rb following γ-ray but not following neutron exposure. Interestingly, expression of c-myc was repressed following γ-ray but not following neutron exposure. Radiations at different doses and dose rates were compared for each of the genes studied

  18. Recent Advances on the Role of G Protein-Coupled Receptors in Hypoxia-Mediated Signaling

    OpenAIRE

    Lappano, Rosamaria; Rigiracciolo, Damiano; De Marco, Paola; Avino, Silvia; Cappello, Anna Rita; Rosano, Camillo; Maggiolini, Marcello; De Francesco, Ernestina Marianna

    2016-01-01

    G protein-coupled receptors (GPCRs) are cell surface proteins mainly involved in signal transmission; however, they play a role also in several pathophysiological conditions. Chemically heterogeneous molecules like peptides, hormones, lipids, and neurotransmitters activate second messengers and induce several biological responses by binding to these seven transmembrane receptors, which are coupled to heterotrimeric G proteins. Recently, additional molecular mechanisms have been involved in GP...

  19. Identification and Characterization of Pheromone Receptors and Interplay between Receptors and Pheromone Binding Proteins in the Diamondback Moth, Plutella xyllostella

    OpenAIRE

    Sun, Mengjing; Liu, Yang; Walker, William B.; Liu, Chengcheng; Lin, Kejian; Gu, Shaohua; Zhang, Yongjun; Zhou, Jingjiang; Wang, Guirong

    2013-01-01

    Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic...

  20. Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers

    OpenAIRE

    Levoye, Angélique; Dam, Julie; Ayoub, Mohammed A; Guillaume, Jean-Luc; Jockers, Ralf

    2006-01-01

    G-protein-coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand-independent functions for these receptors. This emerging concept is connected with the observ...

  1. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans.

    Directory of Open Access Journals (Sweden)

    Su Yang

    Full Text Available The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2 treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998, Lon proteases (dr0349 and dr1974, NADH-quinone oxidoreductase (dr1506, thiosulfate sulfurtransferase (dr2531, the DNA repair protein UvsE (dr1819, PprA (dra0346, and RecN (dr1447, are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.

  2. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

    International Nuclear Information System (INIS)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-01

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

  3. Bcmimp1, a Botrytis cinerea gene transiently expressed in planta, encodes a mitochondrial protein

    Directory of Open Access Journals (Sweden)

    David eBenito-Pescador

    2016-02-01

    Full Text Available Botrytis cinerea is a widespread necrotrophic fungus which infects more than 200 plant species. In an attempt to characterize the physiological status of the fungus in planta and to identify genetic factors contributing to its ability to infect the host cells, a differential gene expression analysis during the interaction B. cinerea-tomato was carried out. Gene Bcmimp1 codes for a mRNA detected by differential display in the course of this analysis. During the interaction with the host, it shows a transient expression pattern with maximal expression levels during the colonization and maceration of the infected tissues. Bioinformatic analysis suggested that BCMIMP1 is an integral membrane protein located in the mitochondrial inner membrane. Co-localization experiments with a BCMIMP1-GFP fusion protein confirmed that the protein is targeted to the mitochondria. ΔBcmimp1 mutants do not show obvious phenotypic differences during saprophytic growth and their infection ability was unaltered as compared to the wild-type. Interestingly, the mutants produced increased levels of ROS, likely as a consequence of disturbed mitochondrial function. Although Bcmimp1 expression is enhanced in planta it cannot be considered a pathogenicity factor.

  4. Deleted in malignant brain tumors-1 protein (DMBT1): a pattern recognition receptor with multiple binding sites.

    Science.gov (United States)

    Ligtenberg, Antoon J M; Karlsson, Niclas G; Veerman, Enno C I

    2010-01-01

    Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1(SAG)), and lung glycoprotein-340 (DMBT1(GP340)) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

  5. Deleted in Malignant Brain Tumors-1 Protein (DMBT1: A Pattern Recognition Receptor with Multiple Binding Sites

    Directory of Open Access Journals (Sweden)

    Enno C. I. Veerman

    2010-12-01

    Full Text Available Deleted in Malignant Brain Tumors-1 protein (DMBT1, salivary agglutinin (DMBT1SAG, and lung glycoprotein-340 (DMBT1GP340 are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW. Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

  6. Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2.

    Science.gov (United States)

    Li, Qun; Laumonnier, Yves; Syrovets, Tatiana; Simmet, Thomas

    2011-11-01

    To identify proteins that interact with the C-terminal fragment of annexin A2 (A2IC), generated by plasmin cleavage of the plasmin receptor, a heterotetramer (AA2t) containing annexin A2. The gene that encodes the A2IC fragment was obtained from PCR-amplified cDNA isolated from human monocytes, and was ligated into the pBTM116 vector using a DNA ligation kit. The resultant plasmid (pBTM116-A2IC) was sequenced with an ABI PRISM 310 Genetic Analyzer. The expression of an A2IC bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis. The identification of proteins that interact with A2IC and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening. The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit. Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov). Confirmation of the interaction between the protein LexA-A2IC and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay. The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach. After screening 1×10(7) clones, 23 independent β-Gal-positive clones were identified. Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17, ProCathepsin S, RPS2, ZBTB4, OGDH, CCDC32, PAPD4, and actin which was already known to interact with annexin A2). A2IC A2IC interacts with various proteins to form protein complexes, which may contribute to the molecular mechanism of monocyte activation induced by plasmin. The yeast two-hybrid system is an efficient approach for investigating protein interactions.

  7. Identification of cDNA encoding an additional α subunit of a human GTP-binding protein: Expression of three αi subtypes in human tissues and cell lines

    International Nuclear Information System (INIS)

    Kim, S.; Ang, S.L.; Bloch, D.B.; Bloch, K.D.; Kawahara, Y.; Tolman, C.; Lee, R.; Seidman, J.G.; Neer, E.J.

    1988-01-01

    The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of α, β, and γ subunits. The authors have cloned and characterized cDNA from a human T-cell library encoding a form of α i that is different from the human α i subtypes previously reported. α i is the α subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the α i-3 subtype of G proteins on the basis of its similarity to other α i -like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. They have determined the expression of mRNA for this and two other subtypes of human α i (α i-1 and α i-2 ) in a variety of human fetal tissues and in human cell lines. All three α i subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of α i-1 expression. mRNA for α i-1 is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of α i-1 genes may permit characterization of distinct physiological roles for this α i subunit. mRNA for α i-2 and α i-3 was found in all the primary and transformed cell lines tested. Thus, some cells contain all three α i subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar α proteins

  8. The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors.

    Science.gov (United States)

    Bondar, Alexey; Lazar, Josef

    2017-06-09

    The G i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G i/o -GPCR coupling and the high rate of G i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G i/o proteins and their cognate GPCRs in the inactive state (G i/o -GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα i1 subunits labeled with fluorescent proteins and four GPCRs: the α 2A -adrenergic receptor, GABA B , cannabinoid receptor type 1 (CB 1 R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα i1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα i1 subunits and α 2A -adrenergic receptor, GABA B , or dopamine receptor type 2 receptors did not reveal any interaction between the G i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB 1 R exhibited an interaction with the G i1 protein. Further experiments revealed that this interaction is caused solely by CB 1 R basal activity; no preassembly between CB 1 R and the G i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G i1 (basal coupling). These findings suggest that G i1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-01-01

    taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

  10. Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene

    International Nuclear Information System (INIS)

    Li, G.C.; Li, Ligeng; Liu, Yunkang; Mak, J.Y.; Chen, Lili; Lee, W.M.F.

    1991-01-01

    The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. The authors have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, low-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45C and their thermal survivals were determined by colony-formation assay, they found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress

  11. Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

    Science.gov (United States)

    Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

    2010-07-01

    Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

  12. Stress tolerances of nullmutants of function-unknown genes encoding menadione stress-responsive proteins in Aspergillus nidulans.

    Science.gov (United States)

    Leiter, Éva; Bálint, Mihály; Miskei, Márton; Orosz, Erzsébet; Szabó, Zsuzsa; Pócsi, István

    2016-07-01

    A group of menadione stress-responsive function-unkown genes of Aspergillus nidulans (Locus IDs ANID_03987.1, ANID_06058.1, ANID_10219.1, and ANID_10260.1) was deleted and phenotypically characterized. Importantly, comparative and phylogenetic analyses of the tested A. nidulans genes and their orthologs shed light only on the presence of a TANGO2 domain with NRDE protein motif in the translated ANID_06058.1 gene but did not reveal any recognizable protein-encoding domains in other protein sequences. The gene deletion strains were subjected to oxidative, osmotic, and metal ion stress and, surprisingly, only the ΔANID_10219.1 mutant showed an increased sensitivity to 0.12 mmol l(-1) menadione sodium bisulfite. The gene deletions affected the stress sensitivities (tolerances) irregularly, for example, some strains grew more slowly when exposed to various oxidants and/or osmotic stress generating agents, meanwhile the ΔANID_10260.1 mutant possessed a wild-type tolerance to all stressors tested. Our results are in line with earlier studies demonstrating that the deletions of stress-responsive genes do not confer necessarily any stress-sensitivity phenotypes, which can be attributed to compensatory mechanisms based on other elements of the stress response system with overlapping functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants.

    Science.gov (United States)

    Rosales-Mendoza, Sergio; Soria-Guerra, Ruth E; Moreno-Fierros, Leticia; Govea-Alonso, Dania O; Herrera-Díaz, Areli; Korban, Schuyler S; Alpuche-Solís, Ángel G

    2011-06-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.

  14. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine

    2002-01-01

    into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the beta2-adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where...... changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the beta2-adrenergic receptor in Xenopus laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co...

  15. Comparative analysis of the repertoire of G protein-coupled receptors of three species of the fungal genus Trichoderma

    Science.gov (United States)

    2013-01-01

    Background Eukaryotic organisms employ cell surface receptors such as the seven-transmembrane G protein-coupled receptors (GPCRs) as sensors to connect to the environment. GPCRs react to a variety of extracellular cues and are considered to play central roles in the signal transduction in fungi. Several species of the filamentous ascomycete Trichoderma are potent mycoparasites, i.e. can attack and parasitize other fungi, which turns them into successful bio-fungicides for the protection of plants against fungal phytopathogens. The identification and characterization of GPCRs will provide insights into how Trichoderma communicates with its environment and senses the presence of host fungi. Results We mined the recently published genomes of the two mycoparasitic biocontrol agents Trichoderma atroviride and Trichoderma virens and compared the identified GPCR-like proteins to those of the saprophyte Trichoderma reesei. Phylogenetic analyses resulted in 14 classes and revealed differences not only among the three Trichoderma species but also between Trichoderma and other fungi. The class comprising proteins of the PAQR family was significantly expanded both in Trichoderma compared to other fungi as well as in the two mycoparasites compared to T. reesei. Expression analysis of the PAQR-encoding genes of the three Trichoderma species revealed that all except one were actually transcribed. Furthermore, the class of receptors with a DUF300 domain was expanded in T. atroviride, and T. virens showed an expansion of PTH11-like receptors compared to T. atroviride and T. reesei. Conclusions Comparative genome analyses of three Trichoderma species revealed a great diversity of putative GPCRs with genus- and species- specific differences. The expansion of certain classes in the mycoparasites T. atroviride and T. virens is likely to reflect the capability of these fungi to establish various ecological niches and interactions with other organisms such as fungi and plants. These

  16. Comparative analysis of the repertoire of G protein-coupled receptors of three species of the fungal genus Trichoderma.

    Science.gov (United States)

    Gruber, Sabine; Omann, Markus; Zeilinger, Susanne

    2013-05-16

    Eukaryotic organisms employ cell surface receptors such as the seven-transmembrane G protein-coupled receptors (GPCRs) as sensors to connect to the environment. GPCRs react to a variety of extracellular cues and are considered to play central roles in the signal transduction in fungi. Several species of the filamentous ascomycete Trichoderma are potent mycoparasites, i.e. can attack and parasitize other fungi, which turns them into successful bio-fungicides for the protection of plants against fungal phytopathogens. The identification and characterization of GPCRs will provide insights into how Trichoderma communicates with its environment and senses the presence of host fungi. We mined the recently published genomes of the two mycoparasitic biocontrol agents Trichoderma atroviride and Trichoderma virens and compared the identified GPCR-like proteins to those of the saprophyte Trichoderma reesei. Phylogenetic analyses resulted in 14 classes and revealed differences not only among the three Trichoderma species but also between Trichoderma and other fungi. The class comprising proteins of the PAQR family was significantly expanded both in Trichoderma compared to other fungi as well as in the two mycoparasites compared to T. reesei. Expression analysis of the PAQR-encoding genes of the three Trichoderma species revealed that all except one were actually transcribed. Furthermore, the class of receptors with a DUF300 domain was expanded in T. atroviride, and T. virens showed an expansion of PTH11-like receptors compared to T. atroviride and T. reesei. Comparative genome analyses of three Trichoderma species revealed a great diversity of putative GPCRs with genus- and species- specific differences. The expansion of certain classes in the mycoparasites T. atroviride and T. virens is likely to reflect the capability of these fungi to establish various ecological niches and interactions with other organisms such as fungi and plants. These GPCRs consequently represent

  17. Cloning and functional analysis of human mTERFL encoding a novel mitochondrial transcription termination factor-like protein

    International Nuclear Information System (INIS)

    Chen Yao; Zhou Guangjin; Yu Min; He Yungang; Tang Wei; Lai Jianhua; He Jie; Liu Wanguo; Tan Deyong

    2005-01-01

    Serum plays an important role in the regulation of cell cycle and cell growth. To identify novel serum-inhibitory factors and study their roles in cell cycle regulation, we performed mRNA differential display analysis of U251 cells in the presence or absence of serum and cloned a novel gene encoding the human mitochondrial transcription termination factor-like protein (mTERFL). The full-length mTERFL cDNA has been isolated and the genomic structure determined. The mTERFL gene consists of three exons and encodes 385 amino acids with 52% sequence similarity to the human mitochondrial transcription termination factor (mTERF). However, mTERFL and mTERF have an opposite expression pattern in response to serum. The expression of mTERFL is dramatically inhibited by the addition of serum in serum-starved cells while the mTERF is rather induced. Northern blot analysis detected three mTERFL transcripts of 1.7, 3.2, and 3.5 kb. Besides the 3.2 kb transcript that is unique to skeletal muscle, other two transcripts express predominant in heart, liver, pancreas, and skeletal muscle. Expression of the GFP-mTERFL fusion protein in HeLa cells localized it to the mitochondria. Furthermore, ectopic expression of mTERFL suppresses cell growth and arrests cells in the G1 stage demonstrated by MTT and flow cytometry analysis. Collectively, our data suggest that mTERFL is a novel mTERF family member and a serum-inhibitory factor probably participating in the regulation of cell growth through the modulation of mitochondrial transcription

  18. Heterogeneous genetic diversity pattern in Plasmodium vivax genes encoding merozoite surface proteins (MSP) -7E, -7F and -7L.

    Science.gov (United States)

    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.

  19. Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data

    DEFF Research Database (Denmark)

    Hauser, Frank; Williamson, Michael; Cazzamali, Giuseppe

    2006-01-01

    insect genome, that of the fruitfly Drosophila melanogaster, was sequenced in 2000, and about 200 GPCRs have been annnotated in this model insect. About 50 of these receptors were predicted to have neuropeptides or protein hormones as their ligands. Since 2000, the cDNAs of most of these candidate...... receptors have been cloned and for many receptors the endogenous ligand has been identified. In this review, we will give an update about the current knowledge of all Drosophila neuropeptide and protein hormone receptors, and discuss their phylogenetic relationships. Udgivelsesdato: 2006-Feb...

  20. Evaluation of 10 genes encoding cardiac proteins in Doberman Pinschers with dilated cardiomyopathy.

    Science.gov (United States)

    O'Sullivan, M Lynne; O'Grady, Michael R; Pyle, W Glen; Dawson, John F

    2011-07-01

    To identify a causative mutation for dilated cardiomyopathy (DCM) in Doberman Pinschers by sequencing the coding regions of 10 cardiac genes known to be associated with familial DCM in humans. 5 Doberman Pinschers with DCM and congestive heart failure and 5 control mixed-breed dogs that were euthanized or died. RNA was extracted from frozen ventricular myocardial samples from each dog, and first-strand cDNA was synthesized via reverse transcription, followed by PCR amplification with gene-specific primers. Ten cardiac genes were analyzed: cardiac actin, α-actinin, α-tropomyosin, β-myosin heavy chain, metavinculin, muscle LIM protein, myosinbinding protein C, tafazzin, titin-cap (telethonin), and troponin T. Sequences for DCM-affected and control dogs and the published canine genome were compared. None of the coding sequences yielded a common causative mutation among all Doberman Pinscher samples. However, 3 variants were identified in the α-actinin gene in the DCM-affected Doberman Pinschers. One of these variants, identified in 2 of the 5 Doberman Pinschers, resulted in an amino acid change in the rod-forming triple coiled-coil domain. Mutations in the coding regions of several genes associated with DCM in humans did not appear to consistently account for DCM in Doberman Pinschers. However, an α-actinin variant was detected in some Doberman Pinschers that may contribute to the development of DCM given its potential effect on the structure of this protein. Investigation of additional candidate gene coding and noncoding regions and further evaluation of the role of α-actinin in development of DCM in Doberman Pinschers are warranted.

  1. DMPD: G-protein-coupled receptor expression, function, and signaling in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17456803 G-protein-coupled receptor expression, function, and signaling in macropha...2007 Apr 24. (.png) (.svg) (.html) (.csml) Show G-protein-coupled receptor expression, function, and signali...ng in macrophages. PubmedID 17456803 Title G-protein-coupled receptor expression, function

  2. DMPD: The role of Toll-like receptors and Nod proteins in bacterial infection. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15476921 The role of Toll-like receptors and Nod proteins in bacterial infection. P...of Toll-like receptors and Nod proteins in bacterial infection. PubmedID 15476921 Title The role of Toll-like receptors and Nod prote...ins in bacterial infection. Authors Philpott DJ, Girardi

  3. DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:...le Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflam

  4. Sex- and age-related differences in ribosomal proteins L17 and L37, as well as androgen receptor protein, in the song control system of zebra finches.

    Science.gov (United States)

    Tang, Y P; Wade, J

    2010-12-29

    The zebra finch song system is sexually dimorphic--only males sing, and the morphology of forebrain regions controlling the learning and production of this song is greatly enhanced in males compared to females. Masculinization appears to involve effects of steroid hormones as well as other factors, perhaps including the expression of sex chromosome genes (males: ZZ, females: ZW). The present study investigated three proteins--two encoded by Z-linked genes, ribosomal proteins L17 and L37 (RPL17 and RPL37), including their co-localization with androgen receptor (AR), from post-hatching day 25 to adulthood. Extensive co-expression of AR with the ribosomal proteins was detected in the three song nuclei investigated (HVC, robust nucleus of the arcopallium (RA), and Area X) across these ages. In general, more cells expressed each of these proteins in males compared to females, and the sex differences increased as animals matured. Specific patterns differed across regions and between RPL17 and RPL37, which suggest potential roles of one or both of these proteins in the incorporation and/or differentiation of song system cells. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  5. The human herpes virus 8-encoded chemokine receptor is required for angioproliferation in a murine model of Kaposi's sarcoma

    DEFF Research Database (Denmark)

    Jensen, Kristian K; Manfra, Denise J; Grisotto, Marcos G

    2005-01-01

    Kaposi's sarcoma (KS)-associated herpesvirus or human herpes virus 8 is considered the etiological agent of KS, a highly vascularized neoplasm that is the most common tumor affecting HIV/AIDS patients. The KS-associated herpesvirus/human herpes virus 8 open reading frame 74 encodes a constitutively...

  6. Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120

    DEFF Research Database (Denmark)

    Prihandoko, Rudi; Alvarez-Curto, Elisa; Hudson, Brian D

    2016-01-01

    of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment...... activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode...

  7. Does protein binding modulate the effect of angiotensin II receptor antagonists?

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  8. MBA1 encodes a mitochondrial membrane-associated protein required for biogenesis of the respiratory chain.

    Science.gov (United States)

    Rep, M; Grivell, L A

    1996-06-17

    The yeast MBA 1 gene (Multi-copy Bypass of AFG3) is one of three genes whose overexpression suppresses afg3-null and rca1-null mutations. Bypass of AFG3 and RCA1, whose products are essential for assembly of mitochondrial inner membrane enzyme complexes, suggests a related role for MBA1. The predicted translation product is a 30 kDa hydrophilic protein with a putative mitochondrial targeting sequence and no homology to any sequence in protein or EST databases. Gene disruption leads to a partial respiratory growth defect, which is more pronounced at temperatures above 30 degrees C. Concomitantly, amounts of cytochromes b and aa3 are reduced. A C-terminal c-myc-tagged MBA1 gene product is functional and is found associated with the mitochondrial inner membrane, from which it can he extracted by carbonate, but not by high salt. These observations give further support to a role of MBA1 in assembly of the respiratory chain.

  9. Selection of unadapted, pathogenic SHIVs encoding newly transmitted HIV-1 envelope proteins.

    Science.gov (United States)

    Del Prete, Gregory Q; Ailers, Braiden; Moldt, Brian; Keele, Brandon F; Estes, Jacob D; Rodriguez, Anthony; Sampias, Marissa; Oswald, Kelli; Fast, Randy; Trubey, Charles M; Chertova, Elena; Smedley, Jeremy; LaBranche, Celia C; Montefiori, David C; Burton, Dennis R; Shaw, George M; Markowitz, Marty; Piatak, Michael; KewalRamani, Vineet N; Bieniasz, Paul D; Lifson, Jeffrey D; Hatziioannou, Theodora

    2014-09-10

    Infection of macaques with chimeric viruses based on SIVMAC but expressing the HIV-1 envelope (Env) glycoproteins (SHIVs) remains the most powerful model for evaluating prevention and therapeutic strategies against AIDS. Unfortunately, only a few SHIVs are currently available. Furthermore, their generation has required extensive adaptation of the HIV-1 Env sequences in macaques so they may not accurately represent HIV-1 Env proteins circulating in humans, potentially limiting their translational utility. We developed a strategy for generating large numbers of SHIV constructs expressing Env proteins from newly transmitted HIV-1 strains. By inoculating macaques with cocktails of multiple SHIV variants, we selected SHIVs that can replicate and cause AIDS-like disease in immunologically intact rhesus macaques without requiring animal-to-animal passage. One of these SHIVs could be transmitted mucosally. We demonstrate the utility of the SHIVs generated by this method for evaluating neutralizing antibody administration as a protection against mucosal SHIV challenge. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available Membrane proteins, particularly G-protein coupled receptors (GPCRs, are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

  11. Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

    Science.gov (United States)

    Mnpotra, Jagjeet S; Qiao, Zhuanhong; Cai, Jian; Lynch, Diane L; Grossfield, Alan; Leioatts, Nicholas; Hurst, Dow P; Pitman, Michael C; Song, Zhao-Hui; Reggio, Patricia H

    2014-07-18

    In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

    Directory of Open Access Journals (Sweden)

    Gerd Krause

    2017-04-01

    Full Text Available The thyroid-stimulating hormone receptor (TSHR is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs. TSHR and its endogenous ligand thyrotropin (TSH are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016 concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other

  13. Structural-Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work.

    Science.gov (United States)

    Kleinau, Gunnar; Worth, Catherine L; Kreuchwig, Annika; Biebermann, Heike; Marcinkowski, Patrick; Scheerer, Patrick; Krause, Gerd

    2017-01-01

    The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to

  14. Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

    Science.gov (United States)

    Kleinau, Gunnar; Worth, Catherine L.; Kreuchwig, Annika; Biebermann, Heike; Marcinkowski, Patrick; Scheerer, Patrick; Krause, Gerd

    2017-01-01

    The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to

  15. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    Science.gov (United States)

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-09-01

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. A structural study for the optimisation of functional motifs encoded in protein sequences

    Directory of Open Access Journals (Sweden)

    Helmer-Citterich Manuela

    2004-04-01

    Full Text Available Abstract Background A large number of PROSITE patterns select false positives and/or miss known true positives. It is possible that – at least in some cases – the weak specificity and/or sensitivity of a pattern is due to the fact that one, or maybe more, functional and/or structural key residues are not represented in the pattern. Multiple sequence alignments are commonly used to build functional sequence patterns. If residues structurally conserved in proteins sharing a function cannot be aligned in a multiple sequence alignment, they are likely to be missed in a standard pattern construction procedure. Results Here we present a new procedure aimed at improving the sensitivity and/ or specificity of poorly-performing patterns. The procedure can be summarised as follows: 1. residues structurally conserved in different proteins, that are true positives for a pattern, are identified by means of a computational technique and by visual inspection. 2. the sequence positions of the structurally conserved residues falling outside the pattern are used to build extended sequence patterns. 3. the extended patterns are optimised on the SWISS-PROT database for their sensitivity and specificity. The method was applied to eight PROSITE patterns. Whenever structurally conserved residues are found in the surface region close to the pattern (seven out of eight cases, the addition of information inferred from structural analysis is shown to improve pattern selectivity and in some cases selectivity and sensitivity as well. In some of the cases considered the procedure allowed the identification of functionally interesting residues, whose biological role is also discussed. Conclusion Our method can be applied to any type of functional motif or pattern (not only PROSITE ones which is not able to select all and only the true positive hits and for which at least two true positive structures are available. The computational technique for the identification of

  17. TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis.

    Science.gov (United States)

    van der Meer, Jonathan H M; van der Poll, Tom; van 't Veer, Cornelis

    2014-04-17

    TAM receptors (Tyro3, Axl, and Mer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein S. Protein S is more commonly known as an important cofactor for protein C as well as a direct inhibitor of multiple coagulation factors. To our knowledge, the functions of Gas6 are limited to TAM receptor activation. When activated, the TAM receptors have effects on primary hemostasis and coagulation and display an anti-inflammatory or a proinflammatory effect, depending on cell type. To comprehend the effects that the TAM receptors and their ligands have on hemostasis and inflammation, we compare studies that report the different phenotypes displayed by mice with deficiencies in the genes of this receptor family and its ligands (protein S(+/-), Gas6(-/-), TAM(-/-), and variations of these). In this manner, we aim to display which features are attributable to the different ligands. Because of the effects TAM receptors have on hemostasis, inflammation, and cancer growth, their modulation could make interesting therapeutic targets in thromboembolic disease, atherosclerosis, sepsis, autoimmune disease, and cancer.

  18. A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER

    Directory of Open Access Journals (Sweden)

    Haga Christopher L

    2007-09-01

    Full Text Available Abstract Background In mouse the cytokine interleukin-7 (IL-7 is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER. The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR, a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules.

  19. Cloning and expression of gene encoding P23 protein from Cryptosporidium parvum

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    Dinh Thi Bich Lan

    2014-12-01

    Full Text Available We cloned the cp23 gene coding P23 (glycoprotein from Cryptosporidium parvum isolated from Thua Thien Hue province, Vietnam. The coding region of cp23 gene from C. parvum is 99% similar with cp23 gene deposited in NCBI (accession number: U34390. SDS-PAGE and Western blot analysis showed that the cp23 gene in E. coli BL21 StarTM (DE3 produced polypeptides with molecular weights of approximately 37, 40 and 49 kDa. These molecules may be non-glycosylated or glycosylated P23 fusion polypeptides. Recombinant P23 protein purified by GST (glutathione S-transferase affinity chromatography can be used as an antigen for C. parvum antibody production as well as to develop diagnostic kit for C. parvum.

  20. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

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    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  1. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

    Science.gov (United States)

    Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J; Mobarec, Juan Carlos; Woodlock, David A; Reynolds, Christopher A; Poyner, David R; Watkins, Harriet A; Ladds, Graham

    2016-10-14

    The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα s -mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα s and Gα q but also identify a Gα i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα s , Gα i , and Gα q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Present perspectives on the automated classification of the G-protein coupled receptors (GPCRs) at the protein sequence level

    DEFF Research Database (Denmark)

    Davies, Matthew N; Gloriam, David E; Secker, Andrew

    2011-01-01

    The G-protein coupled receptors--or GPCRs--comprise simultaneously one of the largest and one of the most multi-functional protein families known to modern-day molecular bioscience. From a drug discovery and pharmaceutical industry perspective, the GPCRs constitute one of the most commercially an...

  3. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins

    DEFF Research Database (Denmark)

    Bruun, A W; Svendsen, I; Sørensen, S O

    1998-01-01

    signals for transport into the endoplasmic reticulum. Surprisingly, Ic is encoded by TFS1, which has previously been isolated as a high-copy suppressor of cdc25-1. CDC25 encodes the putative GTP exchange factor for Ras1p/Ras2p in yeast. In an attempt to rationalize this finding, we looked...... degree of specificity, showing a 200-fold higher Ki toward a carboxypeptidase from Candida albicans which is highly homologous to carboxypeptidase Y. The TFS1 gene product shows extensive similarity to a class of proteins termed "21-23-kDa lipid binding proteins", members of which are found in several...

  4. Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor

    International Nuclear Information System (INIS)

    Pohlmann, R.; Nagel, G.; Schmidt, B.

    1987-01-01

    Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in λgt11. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids, and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential N-glycosylation sites from a C-terminal region lacking N-glycosylation sites. Thus the N-terminal (M/sub r/ = 18,299) and C-terminal (M/sub r/ ≤ 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12

  5. Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors.

    Science.gov (United States)

    Pardon, Els; Betti, Cecilia; Laeremans, Toon; Chevillard, Florent; Guillemyn, Karel; Kolb, Peter; Ballet, Steven; Steyaert, Jan

    2018-05-04

    The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β 2 -adrenoreceptor-nanobody fusion locked in its active-state conformation by a G-protein-mimicking nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody-enabled reverse pharmacology. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

    International Nuclear Information System (INIS)

    Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

    1988-01-01

    In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [ 3 H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating

  7. Improved in Vitro Folding of the Y2 G Protein-Coupled Receptor into Bicelles

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    Peter Schmidt

    2018-01-01

    Full Text Available Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

  8. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    DEFF Research Database (Denmark)

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae......, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While...... demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease....

  9. E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

    Energy Technology Data Exchange (ETDEWEB)

    Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

    1988-12-01

    In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with (/sup 3/H)DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating.

  10. The effects of Epstein-Barr virus-encoded latent membrane protein-1 in the irradiated nasopharyngeal carcinoma cells

    International Nuclear Information System (INIS)

    Hsu, H.-Y.; Hsu, W.-L.

    2003-01-01

    Full text: Nasopharyngeal carcinoma (NPC) is a common cancer in southern China and Taiwan. NPC is closely associated with Epstein-Barr virus (EBV) and the EBV encoded latent membrane protein-1 (LMP-1)is commonly found in the tumor cells. Radiotherapy is the effective choice for most of the NPC patients with different classification and stages. The previous studies showed that EBV-associated NPC tend to be more sensitive to radiotherapy and have been shown the better prognosis than that of EBV-negative NPC. It suggests that LMP-1 may be able to modulate the cellular sensitivity of apoptosis induced by radiation in some kinds of NPC cells. In our study, LMP-1-expressing HONE-1 NPC cells were taken to treat with radiation to investigate whether the LMP-1 can prevent the cells from apoptosis induced by irradiation. The growth of LMP-1-expressing HONE-1 NPC cells were not significantly different from that without expressing LMP-1 at a giving dose of 2, 4 or 6Gy. However, the arrested cellular growth was found in LMP-1-expressing cells irradiated with a dose higher than 8Gy. In addition to the DNA fragmentation, the different levels of several related proteins of the apoptotic pathway and the mRNA of cell cycle arrest in these transfected cells were also investigated after various treatments

  11. ClRTL1 Encodes a Chinese Fir RNase III–Like Protein Involved in Regulating Shoot Branching

    Directory of Open Access Journals (Sweden)

    Xia Li

    2015-10-01

    Full Text Available Identification of genes controlling shoot branching is crucial for improving plant architecture and increasing crop yield or biomass. A branching mutant of Chinese fir named “Dugansha” (Cunninghamia lanceolata var. dugan. has been isolated in our laboratory. We chose the cDNA-AFLP technique and an effective strategy to screen genes that potentially regulate shoot branching in Chinese fir using this mutant. An RNase III-like1 cDNA fragment named ClRTL1 was identified as a potential positive regulator. To investigate the function of ClRTL1 in regulating shoot branching, we cloned the full-length cDNA sequence from C. lanceolata (Lamb. Hook, deduced its secondary structure and function, and overexpressed the coding sequence in Arabidopsis. The ClRTL1 cDNA is 1045 bp and comprises an open reading frame of 705 bp. It encodes a protein of 235 amino acids. The deduced secondary structure of the ClRTL1 indicates that it is a mini-RNase III-like protein. The expression analysis and phenotypes of 35S: ClRTL1 in A. thaliana implies that ClRTL1 plays a role in promoting shoot branching in Chinese fir.

  12. The Arabidopsis GASA10 gene encodes a cell wall protein strongly expressed in developing anthers and seeds.

    Science.gov (United States)

    Trapalis, Menelaos; Li, Song Feng; Parish, Roger W

    2017-07-01

    The Arabidopsis GASA10 gene encodes a GAST1-like (Gibberellic Acid-Stimulated) protein. Reporter gene analysis identified consistent expression in anthers and seeds. In anthers expression was developmentally regulated, first appearing at stage 7 of anther development and reaching a maximum at stage 11. Strongest expression was in the tapetum and developing microspores. GASA10 expression also occurred throughout the seed and in root vasculature. GASA10 was shown to be transported to the cell wall. Using GASA1 and GASA6 as positive controls, gibberellic acid was found not to induce GASA10 expression in Arabidopsis suspension cells. Overexpression of GASA10 (35S promoter-driven) resulted in a reduction in silique elongation. GASA10 shares structural similarities to the antimicrobial peptide snakin1, however, purified GASA10 failed to influence the growth of a variety of bacterial and fungal species tested. We propose cell wall associated GASA proteins are involved in regulating the hydroxyl radical levels at specific sites in the cell wall to facilitate wall growth (regulating cell wall elongation). Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    International Nuclear Information System (INIS)

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-01-01

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  14. Brain transcriptional stability upon prion protein-encoding gene invalidation in zygotic or adult mouse

    Directory of Open Access Journals (Sweden)

    Béringue Vincent

    2010-07-01

    Full Text Available Abstract Background The physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages. Results Only subtle transcriptomic differences resulting from the Prnp knockout could be evidenced, beside Prnp itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal Prnp disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences. Conclusions These results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.

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

    Science.gov (United States)

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

    1993-01-01

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

  16. Differential Influence of Inositol Hexaphosphate on the Expression of Genes Encoding TGF-β Isoforms and Their Receptors in Intestinal Epithelial Cells Stimulated with Proinflammatory Agents

    Directory of Open Access Journals (Sweden)

    Małgorzata Kapral

    2013-01-01

    Full Text Available Transforming growth factor β (TGF-β is a multifunctional cytokine recognized as an important regulator of inflammatory responses. The effect of inositol hexaphosphate (IP6, a naturally occurring phytochemical, on the mRNA expression of TGF-β1, TGF-β2, TGF-β3 and TβRI, TβRII, and TβRIII receptors stimulated with bacterial lipopolysaccharides (Escherichia coli and Salmonella typhimurium and IL-1β in intestinal cells Caco-2 for 3 and 12 h was investigated. Real-time qRT-PCR was used to validate mRNAs level of examined genes. Bacterial endotoxin promoted differential expression of TGF-βs and their receptors in a time-dependent manner. IL-1β upregulated mRNA levels of all TGF-βs and receptors at both 3 h and 12 h. IP6 elicited the opposed to LPS effect by increasing downregulated transcription of the examined genes and suppressing the expression of TGF-β1 at 12 h. IP6 counteracted the stimulatory effect of IL-1β on TGF-β1 and receptors expression by decreasing their mRNA levels. IP6 enhanced LPS- and IL-1β-stimulated mRNA expression of TGF-β2 and -β3. Based on these studies it may be concluded that IP6 present in the intestinal milieu may exert immunoregulatory effects and chemopreventive activity on colonic epithelium under inflammatory conditions or during microbe-induced infection/inflammation by modulating the expression of genes encoding TGF-βs and their receptors at transcriptional level.

  17. Cloning, expression and characterization of a gene from earthworm Eisenia fetida encoding a blood-clot dissolving protein.

    Directory of Open Access Journals (Sweden)

    GangQiang Li

    Full Text Available A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida by RT-PCR amplification. The gene designated as CST1 (GenBank No. AY840996 was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA, urokinase (uPA, and vampire bat (DSPAα1 plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3. The molecular mass of recombinant CST1 (rCST was 25 kDa as estimated by SDS-PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7% of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.

  18. The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

    Science.gov (United States)

    Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

    2014-01-31

    The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

  19. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    DEFF Research Database (Denmark)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we...... in normal human intestinal epithelia and could play a role in cholera....

  20. Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

    Directory of Open Access Journals (Sweden)

    M.R. Hespanhol

    2002-03-01

    Full Text Available Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18 and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA by mouse peritoneal macrophages. We observed that a macrophages are able to recognize (bind to these red cells, b this interaction can be inhibited by denatured BSA in the fluid phase, c there is no phagocytosis of these particles by normal macrophages, d phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A.

  1. A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

    DEFF Research Database (Denmark)

    Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D

    2016-01-01

    Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand...

  2. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; DeVree, Brian T; Zou, Yaozhong

    2011-01-01

    -occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs...

  3. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

    Directory of Open Access Journals (Sweden)

    Cecilia Bucci

    2014-10-01

    Full Text Available Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC and p75NTR, a member of the tumor necrosis factor (TNF receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways.

  4. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

    Science.gov (United States)

    Bucci, Cecilia; Alifano, Pietro; Cogli, Laura

    2014-01-01

    Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways. PMID:25295627

  5. Interaction between the p21ras GTPase activating protein and the insulin receptor

    NARCIS (Netherlands)

    Pronk, G.J.; Medema, R.H.; Burgering, B.M.T.; Clark, R.; McCormick, F.; Bos, J.L.

    1992-01-01

    We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine

  6. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    LENUS (Irish Health Repository)

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  7. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    International Nuclear Information System (INIS)

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H.

    1990-01-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[ 3 H]ethylcarboxamidoadenosine [( 3 H]NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the [ 3 H]NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors

  8. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites....... In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines...... the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands....

  9. Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

    DEFF Research Database (Denmark)

    Cazzamali, Giuseppe; Hauser, Frank; Kobberup, Sune

    2003-01-01

    The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also...... part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila...... and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head...

  10. Cloning of a protein arginine methyltransferase PRMT1 homologue from Schistosoma mansoni: Evidence for roles in nuclear receptor signaling and RNA metabolism

    International Nuclear Information System (INIS)

    Mansure, Jose Joao; Furtado, Daniel Rodrigues; Bastos de Oliveira, Francisco Meirelles; Rumjanek, Franklin David; Franco, Gloria Regina; Fantappie, Marcelo Rosado

    2005-01-01

    The most studied arginine methyltransferase is the type I enzyme, which catalyzes the transfer of an S-adenosyl-L-methionine to a broad spectrum of substrates, including histones, RNA-transporting proteins, and nuclear hormone receptor coactivators. We cloned a cDNA encoding a protein arginine methyltransferase in Schistosoma mansoni (SmPRMT1). SmPRMT1 is highly homologous to the vertebrate PRMT1 enzyme. In vitro methylation assays showed that SmPRMT1 recombinant protein was able to specifically methylate histone H4. Two schistosome proteins likely to be involved in RNA metabolism, SMYB1 and SmSmD3, that display a number of RGG motifs, were strongly methylated by SmPRMT1. In vitro GST pull-down assays showed that SMYB1 and SmSmD3 physically interacted with SmPRMT1. Additional GST pull-down assay suggested the occurrence of a ternary complex including SmPRMT1, SmRXR1 nuclear receptor, and the p160 (SRC-1) nuclear receptor coactivator. Together, these data suggest a mechanism by which SmPRMT1 plays a role in nuclear receptor-mediated chromatin remodeling and RNA transactions

  11. Bombyx neuropeptide G protein-coupled receptor A7 is the third cognate receptor for short neuropeptide F from silkworm.

    Science.gov (United States)

    Ma, Qiang; Cao, Zheng; Yu, Yena; Yan, Lili; Zhang, Wenjuan; Shi, Ying; Zhou, Naiming; Huang, Haishan

    2017-12-15

    The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm ( Bombyx mori ) two orphan G protein-coupled receptors, Bombyx neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in B. mori remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of B. mori larvae and identified it as a receptor for Bombyx sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic Bombyx sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca 2+ mobilization via a G i/o -dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as Bommo -sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three Bombyx sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the Bombyx sNPF/sNPFR system in the regulation of physiological activities. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  13. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...

  14. Structure-Function Analysis of Cf-9, a Receptor-Like Protein with Extracytoplasmic Leucine-Rich Repeats

    NARCIS (Netherlands)

    Hoorn, van der R.A.L.; Wulff, B.B.H.; Rivas, S.; Durrant, M.C.; Ploeg, van der A.; Wit, de P.J.G.M.; Jones, J.D.G.

    2005-01-01

    The tomato (Lycopersicon pimpinellifolium) resistance protein Cf-9 belongs to a large class of plant proteins with extracytoplasmic Leu-rich repeats (eLRRs). eLRR proteins play key roles in plant defense and development, mainly as receptor-like proteins or receptor-like kinases, conferring

  15. G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.

    Science.gov (United States)

    Bouchard, Caroline; Pagé, Julie; Bédard, Andréanne; Tremblay, Pierrot; Vallières, Luc

    2007-06-01

    G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.

  16. Many Saccharomyces cerevisiae Cell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent.

    Science.gov (United States)

    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.

  17. Genome-wide identification and characterization of stress-associated protein (SAP gene family encoding A20/AN1 zinc-finger proteins in Medicago truncatula

    Directory of Open Access Journals (Sweden)

    Zhou Yong

    2018-01-01

    Full Text Available Stress associated proteins (SAPs play important roles in developmental processes, responses to various stresses and hormone stimulation in plants. However, little is known about the SAP gene family in Medicago truncatula. In this study, a total of 17 MtSAP genes encoding A20/AN1 zinc-finger proteins were characterized. Out of these 17 genes, 15 were distributed over all 8 chromosomes at different densities, and two segmental duplication events were detected. The phylogenetic analysis of these proteins and their orthologs from Arabidopsis and rice suggested that they could be classified into five out of the seven groups of SAP family genes, with genes in the same group showing similar structures and conserved domains. The cis-elements of the MtSAP promoters were studied, and many cis-elements related to stress and plant hormone responses were identified. We also investigated the stress-responsive expression patterns of the MtSAP genes under various stresses, including drought, exposure to NaCl and cold. The qRT-PCR results showed that numerous MtSAP genes exhibited transcriptional responses to multiple abiotic stresses. These results lay the foundation for further functional characterization of SAP genes. To the best of our knowledge, this is the first report of a genome-wide analysis of the SAP gene family in M. truncatula.

  18. Identification of human microRNA-like sequences embedded within the protein-encoding genes of the human immunodeficiency virus.

    Directory of Open Access Journals (Sweden)

    Bryan Holland

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are highly conserved, short (18-22 nts, non-coding RNA molecules that regulate gene expression by binding to the 3' untranslated regions (3'UTRs of mRNAs. While numerous cellular microRNAs have been associated with the progression of various diseases including cancer, miRNAs associated with retroviruses have not been well characterized. Herein we report identification of microRNA-like sequences in coding regions of several HIV-1 genomes. RESULTS: Based on our earlier proteomics and bioinformatics studies, we have identified 8 cellular miRNAs that are predicted to bind to the mRNAs of multiple proteins that are dysregulated during HIV-infection of CD4+ T-cells in vitro. In silico analysis of the full length and mature sequences of these 8 miRNAs and comparisons with all the genomic and subgenomic sequences of HIV-1 strains in global databases revealed that the first 18/18 sequences of the mature hsa-miR-195 sequence (including the short seed sequence, matched perfectly (100%, or with one nucleotide mismatch, within the envelope (env genes of five HIV-1 genomes from Africa. In addition, we have identified 4 other miRNA-like sequences (hsa-miR-30d, hsa-miR-30e, hsa-miR-374a and hsa-miR-424 within the env and the gag-pol encoding regions of several HIV-1 strains, albeit with reduced homology. Mapping of the miRNA-homologues of env within HIV-1 genomes localized these sequence to the functionally significant variable regions of the env glycoprotein gp120 designated V1, V2, V4 and V5. CONCLUSIONS: We conclude that microRNA-like sequences are embedded within the protein-encoding regions of several HIV-1 genomes. Given that the V1 to V5 regions of HIV-1 envelopes contain specific, well-characterized domains that are critical for immune responses, virus neutralization and disease progression, we propose that the newly discovered miRNA-like sequences within the HIV-1 genomes may have evolved to self-regulate survival of the

  19. Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

    Science.gov (United States)

    Navarro, Gemma; Cordomí, Arnau; Zelman-Femiak, Monika; Brugarolas, Marc; Moreno, Estefania; Aguinaga, David; Perez-Benito, Laura; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; García-Sáez, Ana J; McCormick, Peter J; Franco, Rafael

    2016-04-05

    G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

  20. Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

    Science.gov (United States)

    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.

  1. Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

    Science.gov (United States)

    Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf

    2002-07-01

    Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G

  2. Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

    Directory of Open Access Journals (Sweden)

    Bharat Bhusan Patnaik

    Full Text Available Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4, at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC. SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC. The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp. In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR. The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps involved in plant development and defense.The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit

  3. Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

    Science.gov (United States)

    Patnaik, Bharat Bhusan; Kim, Dong Hyun; Oh, Seung Han; Song, Yong-Su; Chanh, Nguyen Dang Minh; Kim, Jong Sun; Jung, Woo-jin; Saha, Atul Kumar; Bindroo, Bharat Bhushan; Han, Yeon Soo

    2012-01-01

    Background Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. Methodology/Principal Findings Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. Conclusions/Significance The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found

  4. Crystallization and preliminary X-ray crystallographic studies on the parD-encoded protein Kid from Escherichia coli plasmid R1

    NARCIS (Netherlands)

    Hargreaves, D.; Giraldo, R.; Santos-Sierra, S.; Boelens, R.; Rice, D.W.; Díaz Orejas, R.; Rafferty, J.B.

    2002-01-01

    DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficient functioning of the DnaB helicase. The toxin protein Kid from the plasmid-stability system parD encoded on plasmid R1 of E. coli is thought to target and block DnaB-dependent DNA replication. The

  5. A Gene Encoding a DUF247 Domain Protein Cosegregates with the S Self-Incompatibility Locus in Perennial Ryegrass

    DEFF Research Database (Denmark)

    Manzanares, Chloe; Barth, Susanne; Thorogood, Daniel

    2016-01-01

    genes cosegregating with the S-locus, a highly polymorphic gene encoding for a protein containing a DUF247 was fully predictive of known S-locus genotypes at the amino acid level in the seven mapping populations. Strikingly, this gene showed a frameshift mutation in self-compatible darnel (Lolium...

  6. The protein encoded by the proto-oncogene DEK changes the topology of chromatin and reduces the efficiency of DNA replication in a chromatin-specific manner

    DEFF Research Database (Denmark)

    Alexiadis, V; Waldmann, T; Andersen, Jens S.

    2000-01-01

    The structure of chromatin regulates the genetic activity of the underlying DNA sequence. We report here that the protein encoded by the proto-oncogene DEK, which is involved in acute myelogenous leukemia, induces alterations of the superhelical density of DNA in chromatin. The change in topology...

  7. WHITE PANICLE3, a Novel Nucleus-Encoded Mitochondrial Protein, Is Essential for Proper Development and Maintenance of Chloroplasts and Mitochondria in Rice

    Directory of Open Access Journals (Sweden)

    Hongchang Li

    2018-06-01

    Full Text Available Mitochondria and chloroplasts are interacting organelles that play important roles in plant development. In addition to a small number proteins encoded by their own genomes, the majority of mitochondrial and chloroplast proteins are encoded in the cell nucleus and imported into the organelle. As a consequence, coordination between mitochondria, chloroplasts, and the nucleus is of crucial importance to plant cells. Variegated mutants are chloroplast-defective mutants and are considered to be ideal models for studying the intercommunication between these organelles. Here, we report the isolation of WHITE PANICLE3 (WP3, a nuclear gene involved in variegation, from a naturally occurring white panicle rice mutant. Disrupted expression of WP3 in the mutant leads to severe developmental defects in both chloroplasts and mitochondria, and consequently causes the appearance of white-striped leaves and white panicles in the mutant plants. Further investigation showed that WP3 encodes a protein most likely targeted to mitochondria and is specifically expressed in rice panicles. Interestingly, we demonstrate that the recessive white-panicle phenotype in the wp3 mutant is inherited in a typical Mendelian manner, while the white-striped leaf phenotype in wp3 is maternally inherited. Our data collectively suggest that the nucleus-encoded mitochondrial protein, WP3, plays an essential role in the regulation of chloroplast development in rice panicles by maintaining functional mitochondria. Therefore, the wp3 mutant is an excellent model in which to explore the communication between the nucleus, mitochondria, and chloroplasts in plant cells.

  8. Detection of a Bacteriophage Gene Encoding a Mu-like Portal Protein in Haemophilus parasuis Reference Strains and Field Isolates by Nested Polymerase Chain Reaction

    Science.gov (United States)

    A nested PCR assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene’s sequence, were utilized. A majority of the virulent reference strai...

  9. Detection, Characterization, and In Vitro and In Vivo Expression of Genes Encoding S-Proteins in Lactobacillus gallinarum Strains Isolated from Chicken Crops

    Science.gov (United States)

    Hagen, Karen E.; Guan, Le Luo; Tannock, Gerald W.; Korver, Doug R.; Allison, Gwen E.

    2005-01-01

    Thirty-eight isolates of Lactobacillus gallinarum cultured from the crops of broiler chickens were screened for the presence of genes encoding S-layer proteins. All of the isolates had two S-protein genes, which were designated Lactobacillus gallinarum S-protein (lgs) genes. One gene in each isolate was either lgsA or lgsB. The Lactobacillus isolates were further characterized by pulsed-field gel electrophoresis of DNA digests, which grouped the isolates into 17 genotypes (strains). The second gene in each of eight representative strains was sequenced and shown to differ among strains (lgsC, lgsD, lgsE, lgsF, lgsG, lgsH, and lgsI). The genome of each strain thus encoded a common S-protein (encoded by either lgsA or lgsB) and a strain-specific S-protein. The extraction of cell surface proteins from cultures of the eight strains showed that each strain produced a single S-protein that was always encoded by the strain-specific lgs gene. Two of the strains were used to inoculate chickens maintained in a protected environment which were Lactobacillus-free prior to inoculation. DNAs and RNAs extracted from the digesta of the chickens were used for PCR and reverse transcription-PCR, respectively, to demonstrate the presence and transcription of lgs genes in vivo. In both cases, only the strain-specific gene was transcribed. Both of the strains adhered to the crop epithelium, consistent with published data predicting that S-proteins of lactobacilli are adhesins. The results of this study provide a basis for the investigation of gene duplication and sequence variation as mechanisms by which bacterial strains of the same species can share the same habitat. PMID:16269691

  10. α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

    Science.gov (United States)

    Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  11. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

    DEFF Research Database (Denmark)

    Whorton, Matthew R; Bokoch, Michael P; Rasmussen, Søren Gøgsig Faarup

    2007-01-01

    G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet...... the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However......, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein...

  12. Receptor-G Protein Interaction Studied by Bioluminescence Resonance Energy Transfer: Lessons From Protease-Activated Receptor 1

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAYOUB

    2012-06-01

    Full Text Available Since its development, the bioluminescence resonance energy transfer (BRET approach has been extensively applied to study G protein-coupled receptors (GPCRs in real time and in live cells. One of the major aspects of GPCRs investigated in considerable details is their physical coupling to the heterotrimeric G proteins. As a result, new concepts have emerged, but few questions are still a matter of debate illustrating the complexity of GPCR-G protein interactions and coupling. Here, we summarized the recent advances on our understanding of GPCR-G protein coupling based on BRET approaches and supported by other FRET-based studies. We essentially focused on our recent studies in which we addressed the concept of preassembly versus the agonist-dependent interaction between the protease-activated receptor 1 (PAR1 and its cognate G proteins. We discussed the concept of agonist-induced conformational changes within the preassembled PAR1-G protein complexes as well as the critical question how the multiple coupling of PAR1 with two different G proteins, Gi1 and G12, but also -arrestin 1, can be regulated.

  13. The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development

    Science.gov (United States)

    Reissmann, Eva; Jörnvall, Henrik; Blokzijl, Andries; Andersson, Olov; Chang, Chenbei; Minchiotti, Gabriella; Persico, M. Graziella; Ibáñez, Carlos F.; Brivanlou, Ali H.

    2001-01-01

    Nodal proteins have crucial roles in mesendoderm formation and left–right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling. PMID:11485994

  14. Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

    Science.gov (United States)

    Cook, Brian L.; Steuerwald, Dirk; Kaiser, Liselotte; Graveland-Bikker, Johanna; Vanberghem, Melanie; Berke, Allison P.; Herlihy, Kara; Pick, Horst; Vogel, Horst; Zhang, Shuguang

    2009-01-01

    Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be ≈50% α-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices. PMID:19581598

  15. Biallelic mutation of UNC50, encoding a protein involved in AChR trafficking, is responsible for arthrogryposis.

    Science.gov (United States)

    Abiusi, Emanuela; D'Alessandro, Manuela; Dieterich, Klaus; Quevarec, Loic; Turczynski, Sandrina; Valfort, Aurore-Cecile; Mezin, Paulette; Jouk, Pierre Simon; Gut, Marta; Gut, Ivo; Bessereau, Jean Louis; Melki, Judith

    2017-10-15

    Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. 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.

    Science.gov (United States)

    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.

  17. Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

    Science.gov (United States)

    Kemenesi, Gábor; Kurucz, Kornélia; Zana, Brigitta; Földes, Fanni; Urbán, Péter; Vlaschenko, Anton; Kravchenko, Kseniia; Budinski, Ivana; Szodoray-Parádi, Farkas; Bücs, Szilárd; Jére, Csaba; Csősz, István; Szodoray-Parádi, Abigél; Estók, Péter; Görföl, Tamás; Boldogh, Sándor; Jakab, Ferenc

    2018-03-01

    Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

  18. Molecular adaptation within the coat protein-encoding gene of Tunisian almond isolates of Prunus necrotic ringspot virus.

    Science.gov (United States)

    Boulila, Moncef; Ben Tiba, Sawssen; Jilani, Saoussen

    2013-04-01

    The sequence alignments of five Tunisian isolates of Prunus necrotic ringspot virus (PNRSV) were searched for evidence of recombination and diversifying selection. Since failing to account for recombination can elevate the false positive error rate in positive selection inference, a genetic algorithm (GARD) was used first and led to the detection of potential recombination events in the coat protein-encoding gene of that virus. The Recco algorithm confirmed these results by identifying, additionally, the potential recombinants. For neutrality testing and evaluation of nucleotide polymorphism in PNRSV CP gene, Tajima's D, and Fu and Li's D and F statistical tests were used. About selection inference, eight algorithms (SLAC, FEL, IFEL, REL, FUBAR, MEME, PARRIS, and GA branch) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of PNRSV capsid. Inferred phylogenies pointed out, in addition to the three classical groups (PE-5, PV-32, and PV-96), the delineation of a fourth cluster having the new proposed designation SW6, and a fifth clade comprising four Tunisian PNRSV isolates which underwent recombination and selective pressure and to which the name Tunisian outgroup was allocated.

  19. Domestication process of the goat revealed by an analysis of the nearly complete mitochondrial protein-encoding genes.

    Directory of Open Access Journals (Sweden)

    Koh Nomura

    Full Text Available Goats (Capra hircus are one of the oldest domesticated species, and they are kept all over the world as an essential resource for meat, milk, and fiber. Although recent archeological and molecular biological studies suggested that they originated in West Asia, their domestication processes such as the timing of population expansion and the dynamics of their selection pressures are little known. With the aim of addressing these issues, the nearly complete mitochondrial protein-encoding genes were determined from East, Southeast, and South Asian populations. Our coalescent time estimations suggest that the timing of their major population expansions was in the Late Pleistocene and significantly predates the beginning of their domestication in the Neolithic era (≈10,000 years ago. The ω (ratio of non-synonymous rate/synonymous substitution rate for each lineage was also estimated. We found that the ω of the globally distributed haplogroup A which is inherited by more than 90% of goats examined, turned out to be extremely low, suggesting that they are under severe selection pressure probably due to their large population size. Conversely, the ω of the Asian-specific haplogroup B inherited by about 5% of goats was relatively high. Although recent molecular studies suggest that domestication of animals may tend to relax selective constraints, the opposite pattern observed in our goat mitochondrial genome data indicates the process of domestication is more complex than may be presently appreciated and cannot be explained only by a simple relaxation model.

  20. Analysis of the CYP51 gene and encoded protein in propiconazole-resistant isolates of Mycosphaerella fijiensis.

    Science.gov (United States)

    Cañas-Gutiérrez, Gloria P; Angarita-Velásquez, Mónica J; Restrepo-Flórez, Juan M; Rodríguez, Paola; Moreno, Claudia X; Arango, Rafael

    2009-08-01

    Mycosphaerella fijiensis Morelet causes black sigatoka, the most important disease in bananas and plantains. Disease control is mainly through the application of systemic fungicides, including sterol demethylation inhibitors (DMIs). Their intensive use has favoured the appearance of resistant strains. However, no studies have been published on the possible resistance mechanisms. In this work, the CYP51 gene was isolated and sequenced in 11 M. fijiensis strains that had shown different degrees of in vitro sensitivity to propiconazole, one of the most widely used DMI fungicides. Six mutations that could be related to the loss in sensitivity to this fungicide were found: Y136F, A313G, Y461D, Y463D, Y463H and Y463N. The mutations were analysed using a homology model of the protein that was constructed from the crystallographic structure of Mycobacterium tuberculosis (Zoff.) Lehmann & Neumann. Additionally, gene expression was determined in 13 M. fijiensis strains through quantitative analysis of products obtained by RT-PCR. Several changes in the sequence of the gene encoding sterol 14alpha-demethylase were found that have been described in other fungi as being correlated with resistance to azole fungicides. No correlation was found between gene expression and propiconazole resistance.

  1. Interferon-induced transcription of a gene encoding a 15-kDA protein depends on an upstream enhancer element

    International Nuclear Information System (INIS)

    Reich, N.; Evans, B.; Levy, D.; Fahey, D.; Knight, E. Jr.; Darnell, J.E. Jr.

    1987-01-01

    A human gene encoding an interferon-induced 15-kDa protein has been isolated from a genomic library. The gene appears to be single-copy and is composed of two exons, the first of which contains the ATG translation initiation codon. In vitro nuclear run-on assays showed that the transcription rate of the gene is stimulated after interferon treatment. To analyze transcriptional regulatory sequences, the authors constructed recombinant plasmids for use in transient transfection assays of HeLa cells. Constructs containing 115 nucleotides 5' to the transcription initiation site were found to be fully inducible by interferon. Assays of deletion mutants identified a critical element for interferon induction located between -115 and -96, just upstream of the CCAAT box. Moreover, a DNA fragment including this region can confer interferon inducibility on a heterologous promoter (thymidine kinase) when cloned in either orientation upstream of the gene or downstream of the gene. These are properties characteristic of an enhancer element that is active only after treatment with interferon. This regulatory sequence may be shared by a group of interferon-induced genes, since a very similar sequence is present within the functional region near the RNA start site of another interferon-induced gene

  2. Immunogenicity in African Green Monkeys of M Protein Mutant Vesicular Stomatitis Virus Vectors and Contribution of Vector-Encoded Flagellin

    Directory of Open Access Journals (Sweden)

    Marlena M. Westcott

    2018-03-01

    Full Text Available Recombinant vesicular stomatitis virus (VSV is a promising platform for vaccine development. M51R VSV, an attenuated, M protein mutant strain, is an effective inducer of Type I interferon and dendritic cell (DC maturation, which are desirable properties to exploit for vaccine design. We have previously evaluated M51R VSV (M51R and M51R VSV that produces flagellin (M51R-F as vaccine vectors using murine models, and found that flagellin enhanced DC activation and VSV-specific antibody production after low-dose vaccination. In this report, the immunogenicity of M51R vectors and the adjuvant effect of virus-produced flagellin were evaluated in nonhuman primates following high-dose (108 pfu and low-dose (105 pfu vaccination. A single intramuscular vaccination of African green monkeys with M51R or M51R-F induced VSV-specific, dose-dependent humoral immune responses. Flagellin induced a significant increase in antibody production (IgM, IgG and neutralizing antibody at the low vaccination dose. A VSV-specific cellular response was detected at 6 weeks post-vaccination, but was neither dose-dependent nor enhanced by flagellin; similar numbers of VSV-specific, IFNγ-producing cells were detected in lymph node and spleen of all animals. These results indicate that virus-directed, intracellular flagellin production may improve VSV-based vaccines encoding heterologous antigens by lowering the dose required to achieve humoral immunity.

  3. The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities.

    Directory of Open Access Journals (Sweden)

    Madeleine Zerbato

    Full Text Available Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

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

    Science.gov (United States)

    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.

  5. Mutation in the novel nuclear-encoded mitochondrial protein CHCHD10 in a family with autosomal dominant mitochondrial myopathy.

    Science.gov (United States)

    Ajroud-Driss, Senda; Fecto, Faisal; Ajroud, Kaouther; Lalani, Irfan; Calvo, Sarah E; Mootha, Vamsi K; Deng, Han-Xiang; Siddique, Nailah; Tahmoush, Albert J; Heiman-Patterson, Terry D; Siddique, Teepu

    2015-01-01

    Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutations in either the mitochondrial or nuclear genome. Only 5% of all mitochondrial disorders are autosomal dominant. We analyzed DNA from members of the previously reported Puerto Rican kindred with an autosomal dominant mitochondrial myopathy (Heimann-Patterson et al. 1997). Linkage analysis suggested a putative locus on the pericentric region of the long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we established chromosome 22 open reading frame 16 (C22orf16) (later designated as CHCHD10) as the only high-scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double-missense mutation (R15S and G58R) in cis in CHCHD10 which encodes a coiled coil-helix-coiled coil-helix protein of unknown function. These two mutations completely co-segregated with the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic (including 32 Puerto Rican) controls. Expression profiling showed that CHCHD10 is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10 protein was confirmed using immunofluorescence in cells expressing either wild-type or mutant CHCHD10. We found that the expression of the G58R, but not the R15S, mutation induced mitochondrial fragmentation. Our findings identify a novel gene causing mitochondrial myopathy, thereby expanding the spectrum of mitochondrial myopathies caused by nuclear genes. Our findings also suggest a role for CHCHD10 in the morphologic remodeling of the mitochondria.

  6. Scavenger Receptor Class B, Type I, a CD36 Related Protein in Macrobrachium nipponense: Characterization, RNA Interference, and Expression Analysis with Different Dietary Lipid Sources

    Directory of Open Access Journals (Sweden)

    Zhili Ding

    2016-01-01

    Full Text Available The scavenger receptor class B, type I (SR-BI, is a member of the CD36 superfamily comprising transmembrane proteins involved in mammalian and fish lipid homeostasis regulation. We hypothesize that this receptor plays an important role in Macrobrachium nipponense lipid metabolism. However, little attention has been paid to SR-BI in commercial crustaceans. In the present study, we report a cDNA encoding M. nipponense scavenger receptor class B, type I (designated as MnSR-BI, obtained from a hepatopancreas cDNA library. The complete MnSR-BI coding sequence was 1545 bp, encoding 514 amino acid peptides. The MnSR-BI primary structure consisted of a CD36 domain that contained two transmembrane regions at the N- and C-terminals of the protein. SR-BI mRNA expression was specifically detected in muscle, gill, ovum, intestine, hepatopancreas, stomach, and ovary tissues. Furthermore, its expression in the hepatopancreas was regulated by dietary lipid sources, with prawns fed soybean and linseed oils exhibiting higher expression levels. RNAi-based SR-BI silencing resulted in the suppression of its expression in the hepatopancreas and variation in th