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Sample records for conserved protein required

  1. Ana3 is a conserved protein required for the structural integrity of centrioles and basal bodies.

    Science.gov (United States)

    Stevens, Naomi R; Dobbelaere, Jeroen; Wainman, Alan; Gergely, Fanni; Raff, Jordan W

    2009-11-02

    Recent studies have identified a conserved "core" of proteins that are required for centriole duplication. A small number of additional proteins have recently been identified as potential duplication factors, but it is unclear whether any of these proteins are components of the core duplication machinery. In this study, we investigate the function of one of these proteins, Drosophila melanogaster Ana3. We show that Ana3 is present in centrioles and basal bodies, but its behavior is distinct from that of the core duplication proteins. Most importantly, we find that Ana3 is required for the structural integrity of both centrioles and basal bodies and for centriole cohesion, but it is not essential for centriole duplication. We show that Ana3 has a mammalian homologue, Rotatin, that also localizes to centrioles and basal bodies and appears to be essential for cilia function. Thus, Ana3 defines a conserved family of centriolar proteins and plays an important part in ensuring the structural integrity of centrioles and basal bodies.

  2. Conserved RNA-Binding Proteins Required for Dendrite Morphogenesis in Caenorhabditis elegans Sensory Neurons

    Science.gov (United States)

    Antonacci, Simona; Forand, Daniel; Wolf, Margaret; Tyus, Courtney; Barney, Julia; Kellogg, Leah; Simon, Margo A.; Kerr, Genevieve; Wells, Kristen L.; Younes, Serena; Mortimer, Nathan T.; Olesnicky, Eugenia C.; Killian, Darrell J.

    2015-01-01

    The regulation of dendritic branching is critical for sensory reception, cell−cell communication within the nervous system, learning, memory, and behavior. Defects in dendrite morphology are associated with several neurologic disorders; thus, an understanding of the molecular mechanisms that govern dendrite morphogenesis is important. Recent investigations of dendrite morphogenesis have highlighted the importance of gene regulation at the posttranscriptional level. Because RNA-binding proteins mediate many posttranscriptional mechanisms, we decided to investigate the extent to which conserved RNA-binding proteins contribute to dendrite morphogenesis across phyla. Here we identify a core set of RNA-binding proteins that are important for dendrite morphogenesis in the PVD multidendritic sensory neuron in Caenorhabditis elegans. Homologs of each of these genes were previously identified as important in the Drosophila melanogaster dendritic arborization sensory neurons. Our results suggest that RNA processing, mRNA localization, mRNA stability, and translational control are all important mechanisms that contribute to dendrite morphogenesis, and we present a conserved set of RNA-binding proteins that regulate these processes in diverse animal species. Furthermore, homologs of these genes are expressed in the human brain, suggesting that these RNA-binding proteins are candidate regulators of dendrite development in humans. PMID:25673135

  3. A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion

    Science.gov (United States)

    Toret, Christopher P.; D’Ambrosio, Michael V.; Vale, Ronald D.; Simon, Michael A.

    2014-01-01

    Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (∼14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca2+-dependent adhesion in DE-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling. PMID:24446484

  4. The evolutionarily conserved protein PHOTOSYNTHESIS AFFECTED MUTANT71 is required for efficient manganese uptake at the thylakoid membrane in Arabidopsis

    DEFF Research Database (Denmark)

    Schneider, Anja; Steinberger, Iris; Herdean, Andrei

    2016-01-01

    In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylako...... was restored by supplementation with Mn2+, but not Ca2+. Furthermore, PAM71 suppressed the Mn2+-sensitive phenotype of the yeast mutant Δpmr1. Therefore, PAM71 presumably functions in Mn2+ uptake into thylakoids to ensure optimal PSII performance....

  5. Idas, a Novel Phylogenetically Conserved Geminin-related Protein, Binds to Geminin and Is Required for Cell Cycle Progression*

    Science.gov (United States)

    Pefani, Dafni-Eleutheria; Dimaki, Maria; Spella, Magda; Karantzelis, Nickolas; Mitsiki, Eirini; Kyrousi, Christina; Symeonidou, Ioanna-Eleni; Perrakis, Anastassis; Taraviras, Stavros; Lygerou, Zoi

    2011-01-01

    Development and homeostasis of multicellular organisms relies on an intricate balance between cell proliferation and differentiation. Geminin regulates the cell cycle by directly binding and inhibiting the DNA replication licensing factor Cdt1. Geminin also interacts with transcriptional regulators of differentiation and chromatin remodelling factors, and its balanced interactions are implicated in proliferation-differentiation decisions during development. Here, we describe Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology), a previously uncharacterised coiled-coil protein related to Geminin. We show that human Idas localizes to the nucleus, forms a complex with Geminin both in cells and in vitro through coiled-coil mediated interactions, and can change Geminin subcellular localization. Idas does not associate with Cdt1 and prevents Geminin from binding to Cdt1 in vitro. Idas depletion from cells affects cell cycle progression; cells accumulate in S phase and are unable to efficiently progress to mitosis. Idas protein levels decrease in anaphase, whereas its overexpression causes mitotic defects. During development, we show that Idas exhibits high level expression in the choroid plexus and the cortical hem of the mouse telencephalon. Our data highlight Idas as a novel Geminin binding partner, implicated in cell cycle progression, and a putative regulator of proliferation-differentiation decisions during development. PMID:21543332

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

    International Nuclear Information System (INIS)

    Liao Maofu; Kielian, Margaret

    2005-01-01

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

  7. Ubiquitin--conserved protein or selfish gene?

    Science.gov (United States)

    Catic, André; Ploegh, Hidde L

    2005-11-01

    The posttranslational modifier ubiquitin is encoded by a multigene family containing three primary members, which yield the precursor protein polyubiquitin and two ubiquitin moieties, Ub(L40) and Ub(S27), that are fused to the ribosomal proteins L40 and S27, respectively. The gene encoding polyubiquitin is highly conserved and, until now, those encoding Ub(L40) and Ub(S27) have been generally considered to be equally invariant. The evolution of the ribosomal ubiquitin moieties is, however, proving to be more dynamic. It seems that the genes encoding Ub(L40) and Ub(S27) are actively maintained by homologous recombination with the invariant polyubiquitin locus. Failure to recombine leads to deterioration of the sequence of the ribosomal ubiquitin moieties in several phyla, although this deterioration is evidently constrained by the structural requirements of the ubiquitin fold. Only a few amino acids in ubiquitin are vital for its function, and we propose that conservation of all three ubiquitin genes is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as a 'selfish gene'.

  8. Bug22p, a Conserved Centrosomal/Ciliary Protein Also Present in Higher Plants, Is Required for an Effective Ciliary Stroke in Paramecium ▿ †

    Science.gov (United States)

    Laligné, C.; Klotz, C.; Garreau de Loubresse, N.; Lemullois, M.; Hori, M.; Laurent, F. X.; Papon, J. F.; Louis, B.; Cohen, J.; Koll, F.

    2010-01-01

    Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate. PMID:20118210

  9. Bug22p, a conserved centrosomal/ciliary protein also present in higher plants, is required for an effective ciliary stroke in Paramecium.

    Science.gov (United States)

    Laligné, C; Klotz, C; de Loubresse, N Garreau; Lemullois, M; Hori, M; Laurent, F X; Papon, J F; Louis, B; Cohen, J; Koll, F

    2010-04-01

    Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.

  10. Identification of conserved amino acids in the herpes simplex virus type 1 UL8 protein required for DNA synthesis and UL52 primase interaction in the virus replisome.

    Science.gov (United States)

    Muylaert, Isabella; Zhao, Zhiyuan; Andersson, Torbjörn; Elias, Per

    2012-09-28

    We have used oriS-dependent transient replication assays to search for species-specific interactions within the herpes simplex virus replisome. Hybrid replisomes derived from herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1) failed to support DNA replication in cells. Moreover, the replisomes showed a preference for their cognate origin of replication. The results demonstrate that the herpesvirus replisome behaves as a molecular machine relying on functionally important interactions. We then searched for functional interactions in the replisome context by subjecting HSV-1 UL8 protein to extensive mutagenesis. 52 mutants were made by replacing single or clustered charged amino acids with alanines. Four mutants showed severe replication defects. Mutant A23 exhibited a lethal phenotype, and mutants A49, A52 and A53 had temperature-sensitive phenotypes. Mutants A49 and A53 did not interact with UL52 primase as determined by co-immunoprecipitation experiments. Using GFP-tagged UL8, we demonstrate that all mutants were unable to support formation of ICP8-containing nuclear replication foci. Extended mutagenesis suggested that a highly conserved motif corresponding to mutant A49 serves an important role for establishing a physical contact between UL8 and UL52. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8.

  11. A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions

    International Nuclear Information System (INIS)

    Szajner, Patricia; Jaffe, Howard; Weisberg, Andrea S.; Moss, Bernard

    2004-01-01

    Early events in vaccinia virus (VAC) morphogenesis, particularly the formation of viral membranes and their association with viroplasm, are poorly understood. Recently, we showed that repression of A30 or G7 expression results in the accumulation of normal viral membranes that form empty-looking immature virions (IV), which are separated from large masses of electron-dense viroplasm. In addition, A30 and G7 physically and functionally interact with each other and with the F10 protein kinase. To identify other proteins involved in early morphogenesis, proteins from cells that had been infected with vaccinia virus expressing an epitope-tagged copy of F10 were purified by immunoaffinity chromatography and analyzed by gel electrophoresis. In addition to F10, A30, and G7, viral proteins A15, D2, D3, and J1 were identified by mass spectrometry of tryptic peptides. Further evidence for the complex was obtained by immunopurification of proteins associated with epitope-tagged A15, D2, and D3. The previously unstudied A15, like other proteins in the complex, was expressed late in infection, associated with virus cores, and required for the stability and kinase activity of F10. Biochemical and electron microscopic analyses indicated that mutants in which A15 or D2 expression was regulated by the Escherichia coli lac operator system exhibited phenotypes characterized by the presence of large numbers of empty immature virions, similar to the results obtained with inducible A30 and G7 mutants. Empty immature virions were also seen by electron microscopy of cells infected with temperature-sensitive mutants of D2 or D3, though the numbers of membrane forms were reduced perhaps due to additional effects of high temperature

  12. 18 CFR 430.15 - Conservation requirements.

    Science.gov (United States)

    2010-04-01

    ... area of the municipal or public water supply. Such program shall include a program for leakage control... shall be adopted and implemented to provide for the detection and expeditious correction of leakage. (3... 18 Conservation of Power and Water Resources 2 2010-04-01 2010-04-01 false Conservation...

  13. Topology-function conservation in protein-protein interaction networks.

    Science.gov (United States)

    Davis, Darren; Yaveroğlu, Ömer Nebil; Malod-Dognin, Noël; Stojmirovic, Aleksandar; Pržulj, Nataša

    2015-05-15

    Proteins underlay the functioning of a cell and the wiring of proteins in protein-protein interaction network (PIN) relates to their biological functions. Proteins with similar wiring in the PIN (topology around them) have been shown to have similar functions. This property has been successfully exploited for predicting protein functions. Topological similarity is also used to guide network alignment algorithms that find similarly wired proteins between PINs of different species; these similarities are used to transfer annotation across PINs, e.g. from model organisms to human. To refine these functional predictions and annotation transfers, we need to gain insight into the variability of the topology-function relationships. For example, a function may be significantly associated with specific topologies, while another function may be weakly associated with several different topologies. Also, the topology-function relationships may differ between different species. To improve our understanding of topology-function relationships and of their conservation among species, we develop a statistical framework that is built upon canonical correlation analysis. Using the graphlet degrees to represent the wiring around proteins in PINs and gene ontology (GO) annotations to describe their functions, our framework: (i) characterizes statistically significant topology-function relationships in a given species, and (ii) uncovers the functions that have conserved topology in PINs of different species, which we term topologically orthologous functions. We apply our framework to PINs of yeast and human, identifying seven biological process and two cellular component GO terms to be topologically orthologous for the two organisms. © The Author 2015. Published by Oxford University Press.

  14. The relationship of protein conservation and sequence length

    Directory of Open Access Journals (Sweden)

    Panchenko Anna R

    2002-11-01

    Full Text Available Abstract Background In general, the length of a protein sequence is determined by its function and the wide variance in the lengths of an organism's proteins reflects the diversity of specific functional roles for these proteins. However, additional evolutionary forces that affect the length of a protein may be revealed by studying the length distributions of proteins evolving under weaker functional constraints. Results We performed sequence comparisons to distinguish highly conserved and poorly conserved proteins from the bacterium Escherichia coli, the archaeon Archaeoglobus fulgidus, and the eukaryotes Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens. For all organisms studied, the conserved and nonconserved proteins have strikingly different length distributions. The conserved proteins are, on average, longer than the poorly conserved ones, and the length distributions for the poorly conserved proteins have a relatively narrow peak, in contrast to the conserved proteins whose lengths spread over a wider range of values. For the two prokaryotes studied, the poorly conserved proteins approximate the minimal length distribution expected for a diverse range of structural folds. Conclusions There is a relationship between protein conservation and sequence length. For all the organisms studied, there seems to be a significant evolutionary trend favoring shorter proteins in the absence of other, more specific functional constraints.

  15. Optimal conservation outcomes require both restoration and protection.

    Science.gov (United States)

    Possingham, Hugh P; Bode, Michael; Klein, Carissa J

    2015-01-01

    Conservation outcomes are principally achieved through the protection of intact habitat or the restoration of degraded habitat. Restoration is generally considered a lower priority action than protection because protection is thought to provide superior outcomes, at lower costs, without the time delay required for restoration. Yet while it is broadly accepted that protected intact habitat safeguards more biodiversity and generates greater ecosystem services per unit area than restored habitat, conservation lacks a theory that can coherently compare the relative outcomes of the two actions. We use a dynamic landscape model to integrate these two actions into a unified conservation theory of protection and restoration. Using nonlinear benefit functions, we show that both actions are crucial components of a conservation strategy that seeks to optimise either biodiversity conservation or ecosystem services provision. In contrast to conservation orthodoxy, in some circumstances, restoration should be strongly preferred to protection. The relative priority of protection and restoration depends on their costs and also on the different time lags that are inherent to both protection and restoration. We derive a simple and easy-to-interpret heuristic that integrates these factors into a single equation that applies equally to biodiversity conservation and ecosystem service objectives. We use two examples to illustrate the theory: bird conservation in tropical rainforests and coastal defence provided by mangrove forests.

  16. Optimal conservation outcomes require both restoration and protection.

    Directory of Open Access Journals (Sweden)

    Hugh P Possingham

    2015-01-01

    Full Text Available Conservation outcomes are principally achieved through the protection of intact habitat or the restoration of degraded habitat. Restoration is generally considered a lower priority action than protection because protection is thought to provide superior outcomes, at lower costs, without the time delay required for restoration. Yet while it is broadly accepted that protected intact habitat safeguards more biodiversity and generates greater ecosystem services per unit area than restored habitat, conservation lacks a theory that can coherently compare the relative outcomes of the two actions. We use a dynamic landscape model to integrate these two actions into a unified conservation theory of protection and restoration. Using nonlinear benefit functions, we show that both actions are crucial components of a conservation strategy that seeks to optimise either biodiversity conservation or ecosystem services provision. In contrast to conservation orthodoxy, in some circumstances, restoration should be strongly preferred to protection. The relative priority of protection and restoration depends on their costs and also on the different time lags that are inherent to both protection and restoration. We derive a simple and easy-to-interpret heuristic that integrates these factors into a single equation that applies equally to biodiversity conservation and ecosystem service objectives. We use two examples to illustrate the theory: bird conservation in tropical rainforests and coastal defence provided by mangrove forests.

  17. Conservation and diversification of Msx protein in metazoan evolution.

    Science.gov (United States)

    Takahashi, Hirokazu; Kamiya, Akiko; Ishiguro, Akira; Suzuki, Atsushi C; Saitou, Naruya; Toyoda, Atsushi; Aruga, Jun

    2008-01-01

    Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family

  18. A conserved mammalian protein interaction network.

    Directory of Open Access Journals (Sweden)

    Åsa Pérez-Bercoff

    Full Text Available Physical interactions between proteins mediate a variety of biological functions, including signal transduction, physical structuring of the cell and regulation. While extensive catalogs of such interactions are known from model organisms, their evolutionary histories are difficult to study given the lack of interaction data from phylogenetic outgroups. Using phylogenomic approaches, we infer a upper bound on the time of origin for a large set of human protein-protein interactions, showing that most such interactions appear relatively ancient, dating no later than the radiation of placental mammals. By analyzing paired alignments of orthologous and putatively interacting protein-coding genes from eight mammals, we find evidence for weak but significant co-evolution, as measured by relative selective constraint, between pairs of genes with interacting proteins. However, we find no strong evidence for shared instances of directional selection within an interacting pair. Finally, we use a network approach to show that the distribution of selective constraint across the protein interaction network is non-random, with a clear tendency for interacting proteins to share similar selective constraints. Collectively, the results suggest that, on the whole, protein interactions in mammals are under selective constraint, presumably due to their functional roles.

  19. Demographic and habitat requirements for conservation of bull trout

    Science.gov (United States)

    Bruce E. Rieman; John D. Mclntyre

    1993-01-01

    Elements in bull trout biology, population dynamics, habitat, and biotic interactions important to conservation of the species are identified. Bull trout appear to have more specific habitat requirements than other salmonids, but no critical thresholds of acceptable habitat condition were found. Size, temporal variation, and spatial distribution are likely to influence...

  20. Biodiversity conservation in agriculture requires a multi-scale approach.

    Science.gov (United States)

    Gonthier, David J; Ennis, Katherine K; Farinas, Serge; Hsieh, Hsun-Yi; Iverson, Aaron L; Batáry, Péter; Rudolphi, Jörgen; Tscharntke, Teja; Cardinale, Bradley J; Perfecto, Ivette

    2014-09-22

    Biodiversity loss--one of the most prominent forms of modern environmental change--has been heavily driven by terrestrial habitat loss and, in particular, the spread and intensification of agriculture. Expanding agricultural land-use has led to the search for strong conservation strategies, with some suggesting that biodiversity conservation in agriculture is best maximized by reducing local management intensity, such as fertilizer and pesticide application. Others highlight the importance of landscape-level approaches that incorporate natural or semi-natural areas in landscapes surrounding farms. Here, we show that both of these practices are valuable to the conservation of biodiversity, and that either local or landscape factors can be most crucial to conservation planning depending on which types of organisms one wishes to save. We performed a quantitative review of 266 observations taken from 31 studies that compared the impacts of localized (within farm) management strategies and landscape complexity (around farms) on the richness and abundance of plant, invertebrate and vertebrate species in agro-ecosystems. While both factors significantly impacted species richness, the richness of sessile plants increased with less-intensive local management, but did not significantly respond to landscape complexity. By contrast, the richness of mobile vertebrates increased with landscape complexity, but did not significantly increase with less-intensive local management. Invertebrate richness and abundance responded to both factors. Our analyses point to clear differences in how various groups of organisms respond to differing scales of management, and suggest that preservation of multiple taxonomic groups will require multiple scales of conservation. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  1. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  2. 18 CFR 401.36 - Water supply projects-Conservation requirements.

    Science.gov (United States)

    2010-04-01

    ... 18 Conservation of Power and Water Resources 2 2010-04-01 2010-04-01 false Water supply projects-Conservation requirements. 401.36 Section 401.36 Conservation of Power and Water Resources DELAWARE RIVER BASIN... Compact § 401.36 Water supply projects—Conservation requirements. Maximum feasible efficiency in the use...

  3. Relationships between residue Voronoi volume and sequence conservation in proteins.

    Science.gov (United States)

    Liu, Jen-Wei; Cheng, Chih-Wen; Lin, Yu-Feng; Chen, Shao-Yu; Hwang, Jenn-Kang; Yen, Shih-Chung

    2018-02-01

    Functional and biophysical constraints can cause different levels of sequence conservation in proteins. Previously, structural properties, e.g., relative solvent accessibility (RSA) and packing density of the weighted contact number (WCN), have been found to be related to protein sequence conservation (CS). The Voronoi volume has recently been recognized as a new structural property of the local protein structural environment reflecting CS. However, for surface residues, it is sensitive to water molecules surrounding the protein structure. Herein, we present a simple structural determinant termed the relative space of Voronoi volume (RSV); it uses the Voronoi volume and the van der Waals volume of particular residues to quantify the local structural environment. RSV (range, 0-1) is defined as (Voronoi volume-van der Waals volume)/Voronoi volume of the target residue. The concept of RSV describes the extent of available space for every protein residue. RSV and Voronoi profiles with and without water molecules (RSVw, RSV, VOw, and VO) were compared for 554 non-homologous proteins. RSV (without water) showed better Pearson's correlations with CS than did RSVw, VO, or VOw values. The mean correlation coefficient between RSV and CS was 0.51, which is comparable to the correlation between RSA and CS (0.49) and that between WCN and CS (0.56). RSV is a robust structural descriptor with and without water molecules and can quantitatively reflect evolutionary information in a single protein structure. Therefore, it may represent a practical structural determinant to study protein sequence, structure, and function relationships. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Conserved chemosensory proteins in the proboscis and eyes of Lepidoptera.

    Science.gov (United States)

    Zhu, Jiao; Iovinella, Immacolata; Dani, Francesca Romana; Liu, Yu-Ling; Huang, Ling-Qiao; Liu, Yang; Wang, Chen-Zhu; Pelosi, Paolo; Wang, Guirong

    2016-01-01

    Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are endowed with several different functions besides being carriers for pheromones and odorants. Based on a previous report of a CSP acting as surfactant in the proboscis of the moth Helicoverpa armigera , we revealed the presence of orthologue proteins in two other moths Plutella xylostella and Chilo suppressalis , as well as two butterflies Papilio machaon and Pieris rapae , using immunodetection and proteomic analysis. The unusual conservation of these proteins across large phylogenetic distances indicated a common specific function for these CSPs. This fact prompted us to search for other functions of these proteins and discovered that CSPs are abundantly expressed in the eyes of H. armigera and possibly involved as carriers for carotenoids and visual pigments. This hypothesis is supported by ligand-binding experiments and docking simulations with retinol and β-carotene. This last orange pigment, occurring in many fruits and vegetables, is an antioxidant and the precursor of visual pigments. We propose that structurally related CSPs solubilise nutritionally important carotenoids in the proboscis, while they act as carriers of both β-carotene and its derived products 3-hydroxyretinol and 3-hydroxyretinal in the eye. The use of soluble olfactory proteins, such as CSPs, as carriers for visual pigments in insects, here reported for the first time, parallels the function of retinol-binding protein in vertebrates, a lipocalin structurally related to vertebrate odorant-binding proteins.

  5. [Family of ribosomal proteins S1 contains unique conservative domain].

    Science.gov (United States)

    Deriusheva, E I; Machulin, A V; Selivanova, O M; Serdiuk, I N

    2010-01-01

    Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

  6. Maximizing conservation benefit for grassland species with contrasting management requirements

    NARCIS (Netherlands)

    Teeffelen, van A.J.A.; Cabeza, M.; Poyry, J.; Raatikainen, K.; Kuussaari, M.

    2008-01-01

    Conservation management often encompasses multiple, alternative management actions on a given site, involving habitat restoration and maintenance for example. Which actions are preferable depends on the conservation goals, the expected outcomes of actions, and their associated costs. When actions

  7. The generation of recombinant influenza A viruses expressing a PB2 fusion protein requires the conservation of a packaging signal overlapping the coding and noncoding regions at the 5' end of the PB2 segment

    International Nuclear Information System (INIS)

    Dos Santos Afonso, Emmanuel; Escriou, Nicolas; Leclercq, India; Werf, Sylvie van der; Naffakh, Nadia

    2005-01-01

    We generated recombinant A/WSN/33 influenza A viruses expressing a PB2 protein fused to a Flag epitope at the N- (Flag-PB2) or C-terminus (PB2-Flag), which replicated efficiently and proved to be stable upon serial passage in vitro on MDCK cells. Rescue of PB2-Flag viruses required that the 5' end of the PB2 segment was kept identical to the wild-type beyond the 34 noncoding terminal nucleotides. This feature was achieved by a duplication of the 109 last nucleotides encoding PB2 between the Flag sequence and the 5'NCR. In PB2 minigenomes rescue experiments, both the 5' and 3' coding ends of the PB2 segment were found to promote the incorporation of minigenomes into virions. However, the presence of the Flag sequence at the junction between the 3'NCR and the coding sequence did not prevent the rescue of Flag-PB2 viruses. Our observations define requirements that may be useful for the purpose of engineering influenza RNAs

  8. [Interconnection between architecture of protein globule and disposition of conformational conservative oligopeptides in proteins from one protein family].

    Science.gov (United States)

    Batianovskiĭ, A V; Filatov, I V; Namiot, V A; Esipova, N G; Volotovskiĭ, I D

    2012-01-01

    It was shown that selective interactions between helical segments of macromolecules can realize in globular proteins in the segments characterized by the same periodicities of charge distribution i.e. between conformationally conservative oligopeptides. It was found that in the macromolecules of alpha-helical proteins conformationally conservative oligopeptides are disposed at a distance being characteristic of direct interactions. For representatives of many structural families of alpha-type proteins specific disposition of conformationally conservative segments is observed. This disposition is inherent to a particular structural family. Disposition of conformationally conservative segments is not related to homology of the amino acid sequence but reflects peculiarities of native 3D-architectures of protein globules.

  9. Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life.

    Science.gov (United States)

    Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

    2016-11-01

    Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  10. Protein requirements of finishing paca (Cuniculus paca).

    Science.gov (United States)

    Nogueira-Filho, Sérgio Luiz Gama; Bastos, Ivanise da Hora; Mendes, Alcester; Nogueira, Selene Siqueira da Cunha

    2016-06-01

    We conducted a nitrogen balance digestion trial to determine the crude protein requirements of paca (Cuniculus paca) during the last growth phase. In a 4 × 4 Latin square design, four young captive male pacas, aged 5 months, were fed four isoenergetic diets containing four different levels of nitrogen (N) (11.3, 16.6, 21.4, and 26.6 g N/kg of dry matter). After 15 days of adaptation, we collected all feces and urine for five consecutive days. By regression analysis between N intake and N in feces and urine, the metabolic fecal nitrogen (MFN = 4.2 g/kg of dry matter intake) and daily endogenous urinary N (EUN = 91.6 mg/kg(0.75)) were determined. Likewise, by regression analyses between nitrogen intake and nitrogen retention [NR = N intake-(fecal N + urine N)], we estimated the daily requirement of 280.5 mg N/kg(0.75). Therefore, a minimum of 55 g crude protein per kilogram dry matter and 13 MJ/kg of digestible energy are required by finishing paca on unrestricted diets. Such values are similar to those of other wild frugivores and below those of growing rabbits. The data confirm that farmers overfeed protein, and similar growth can be more economically achieved on lower protein diets.

  11. Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding

    DEFF Research Database (Denmark)

    Rossi, Marie L; Ghosh, Avik K; Kulikowicz, Tomasz

    2010-01-01

    Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer...... provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N...... activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases....

  12. Functionality of system components: Conservation of protein function in protein feature space

    DEFF Research Database (Denmark)

    Jensen, Lars Juhl; Ussery, David; Brunak, Søren

    2003-01-01

    well on organisms other than the one on which it was trained. We evaluate the performance of such a method, ProtFun, which relies on protein features as its sole input, and show that the method gives similar performance for most eukaryotes and performs much better than anticipated on archaea......Many protein features useful for prediction of protein function can be predicted from sequence, including posttranslational modifications, subcellular localization, and physical/chemical properties. We show here that such protein features are more conserved among orthologs than paralogs, indicating...... they are crucial for protein function and thus subject to selective pressure. This means that a function prediction method based on sequence-derived features may be able to discriminate between proteins with different function even when they have highly similar structure. Also, such a method is likely to perform...

  13. Energy conservation: a requirement of the present time

    International Nuclear Information System (INIS)

    Alcoforado, Fernando Antonio Goncalves

    1993-01-01

    The origins of the financial crisis that affects the Brazilian electrical sector is presented. The options available to overcome this situation and the role played by energy conservation are described. 7 refs

  14. A conserved regulatory mechanism in bifunctional biotin protein ligases.

    Science.gov (United States)

    Wang, Jingheng; Beckett, Dorothy

    2017-08-01

    Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization. Like E. coli BirA (EcBirA), Staphylococcus aureus, and Bacillus subtilis BirA (Sa and BsBirA) repress transcription in vivo in a biotin-dependent manner. In this work, sedimentation equilibrium measurements were performed to investigate the molecular basis of this biotin-responsive transcription regulation. The results reveal that, as observed for EcBirA, Sa, and BsBirA dimerization reactions are significantly enhanced by bio-5'-AMP binding. Thus, the molecular mechanism of the Biotin Regulatory System is conserved in the biotin repressors from these three organisms. © 2017 The Protein Society.

  15. Bound water at protein-protein interfaces: partners, roles and hydrophobic bubbles as a conserved motif.

    Directory of Open Access Journals (Sweden)

    Mostafa H Ahmed

    Full Text Available There is a great interest in understanding and exploiting protein-protein associations as new routes for treating human disease. However, these associations are difficult to structurally characterize or model although the number of X-ray structures for protein-protein complexes is expanding. One feature of these complexes that has received little attention is the role of water molecules in the interfacial region.A data set of 4741 water molecules abstracted from 179 high-resolution (≤ 2.30 Å X-ray crystal structures of protein-protein complexes was analyzed with a suite of modeling tools based on the HINT forcefield and hydrogen-bonding geometry. A metric termed Relevance was used to classify the general roles of the water molecules.The water molecules were found to be involved in: a (bridging interactions with both proteins (21%, b favorable interactions with only one protein (53%, and c no interactions with either protein (26%. This trend is shown to be independent of the crystallographic resolution. Interactions with residue backbones are consistent for all classes and account for 21.5% of all interactions. Interactions with polar residues are significantly more common for the first group and interactions with non-polar residues dominate the last group. Waters interacting with both proteins stabilize on average the proteins' interaction (-0.46 kcal mol(-1, but the overall average contribution of a single water to the protein-protein interaction energy is unfavorable (+0.03 kcal mol(-1. Analysis of the waters without favorable interactions with either protein suggests that this is a conserved phenomenon: 42% of these waters have SASA ≤ 10 Å(2 and are thus largely buried, and 69% of these are within predominantly hydrophobic environments or "hydrophobic bubbles". Such water molecules may have an important biological purpose in mediating protein-protein interactions.

  16. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  17. A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation.

    Science.gov (United States)

    Chilton, Scott S; Falbel, Tanya G; Hromada, Susan; Burton, Briana M

    2017-08-01

    Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation. IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA. Copyright © 2017 American Society for Microbiology.

  18. 77 FR 27411 - Sea Turtle Conservation; Shrimp Trawling Requirements

    Science.gov (United States)

    2012-05-10

    ... imbricata) turtles are listed as endangered. The loggerhead (Caretta caretta; Northwest Atlantic distinct... populations of green turtles in Florida and on the Pacific coast of Mexico, which are listed as endangered... regulations (50 CFR 223.206) are followed. The same conservation measures also apply to endangered sea turtles...

  19. Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea

    OpenAIRE

    Guo, Li; Feng, Yingang; Zhang, Zhenfeng; Yao, Hongwei; Luo, Yuanming; Wang, Jinfeng; Huang, Li

    2007-01-01

    Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a high...

  20. Evolutionarily conserved regions of the human c-myc protein can be uncoupled from transforming activity

    International Nuclear Information System (INIS)

    Sarid, J.; Halazonetis, T.D.; Murphy, W.; Leder, P.

    1987-01-01

    The myc family of oncogenes contains coding sequences that have been preserved in different species for over 400 million years. This conservation (which implies functional selection) is broadly represented throughout the C-terminal portion of the human c-myc protein but is largely restricted to three cluster of amino acid sequences in the N-terminal region. The authors have examined the role that the latter three regions of the c-myc protein might play in the transforming function of the c-myc gene. Several mutations, deletions and frameshifts, were introduced into the c-myc gene, and these mutant genes were tested for their ability to collaborate with the EJ-ras oncogene to transform rat embryo fibroblasts. Complete elimination of the first two N-terminal conserved segments abolished transforming activity. In contrast, genes altered in a portion of the second or the entire third conserved segment retained their transforming activity. Thus, the latter two segments are not required for the transformation process, suggesting that they serve another function related only to the normal expression of the c-myc gene

  1. A conserved endoplasmic reticulum membrane protein complex (EMC facilitates phospholipid transfer from the ER to mitochondria.

    Directory of Open Access Journals (Sweden)

    Sujoy Lahiri

    2014-10-01

    Full Text Available Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC has decreased phosphatidylserine (PS transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE. Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

  2. Combining modularity, conservation, and interactions of proteins significantly increases precision and coverage of protein function prediction

    Directory of Open Access Journals (Sweden)

    Sers Christine T

    2010-12-01

    Full Text Available Abstract Background While the number of newly sequenced genomes and genes is constantly increasing, elucidation of their function still is a laborious and time-consuming task. This has led to the development of a wide range of methods for predicting protein functions in silico. We report on a new method that predicts function based on a combination of information about protein interactions, orthology, and the conservation of protein networks in different species. Results We show that aggregation of these independent sources of evidence leads to a drastic increase in number and quality of predictions when compared to baselines and other methods reported in the literature. For instance, our method generates more than 12,000 novel protein functions for human with an estimated precision of ~76%, among which are 7,500 new functional annotations for 1,973 human proteins that previously had zero or only one function annotated. We also verified our predictions on a set of genes that play an important role in colorectal cancer (MLH1, PMS2, EPHB4 and could confirm more than 73% of them based on evidence in the literature. Conclusions The combination of different methods into a single, comprehensive prediction method infers thousands of protein functions for every species included in the analysis at varying, yet always high levels of precision and very good coverage.

  3. On the relationship between residue structural environment and sequence conservation in proteins.

    Science.gov (United States)

    Liu, Jen-Wei; Lin, Jau-Ji; Cheng, Chih-Wen; Lin, Yu-Feng; Hwang, Jenn-Kang; Huang, Tsun-Tsao

    2017-09-01

    Residues that are crucial to protein function or structure are usually evolutionarily conserved. To identify the important residues in protein, sequence conservation is estimated, and current methods rely upon the unbiased collection of homologous sequences. Surprisingly, our previous studies have shown that the sequence conservation is closely correlated with the weighted contact number (WCN), a measure of packing density for residue's structural environment, calculated only based on the C α positions of a protein structure. Moreover, studies have shown that sequence conservation is correlated with environment-related structural properties calculated based on different protein substructures, such as a protein's all atoms, backbone atoms, side-chain atoms, or side-chain centroid. To know whether the C α atomic positions are adequate to show the relationship between residue environment and sequence conservation or not, here we compared C α atoms with other substructures in their contributions to the sequence conservation. Our results show that C α positions are substantially equivalent to the other substructures in calculations of various measures of residue environment. As a result, the overlapping contributions between C α atoms and the other substructures are high, yielding similar structure-conservation relationship. Take the WCN as an example, the average overlapping contribution to sequence conservation is 87% between C α and all-atom substructures. These results indicate that only C α atoms of a protein structure could reflect sequence conservation at the residue level. © 2017 Wiley Periodicals, Inc.

  4. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

    Directory of Open Access Journals (Sweden)

    Deirdre R Ducken

    Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

  5. Hierarchical partitioning of metazoan protein conservation profiles provides new functional insights.

    Directory of Open Access Journals (Sweden)

    Jonathan Witztum

    Full Text Available The availability of many complete, annotated proteomes enables the systematic study of the relationships between protein conservation and functionality. We explore this question based solely on the presence or absence of protein homologues (a.k.a. conservation profiles. We study 18 metazoans, from two distinct points of view: the human's and the fly's. Using the GOrilla gene ontology (GO analysis tool, we explore functional enrichment of the "universal proteins", those with homologues in all 17 other species, and of the "non-universal proteins". A large number of GO terms are strongly enriched in both human and fly universal proteins. Most of these functions are known to be essential. A smaller number of GO terms, exhibiting markedly different properties, are enriched in both human and fly non-universal proteins. We further explore the non-universal proteins, whose conservation profiles are consistent with the "tree of life" (TOL consistent, as well as the TOL inconsistent proteins. Finally, we applied Quantum Clustering to the conservation profiles of the TOL consistent proteins. Each cluster is strongly associated with one or a small number of specific monophyletic clades in the tree of life. The proteins in many of these clusters exhibit strong functional enrichment associated with the "life style" of the related clades. Most previous approaches for studying function and conservation are "bottom up", studying protein families one by one, and separately assessing the conservation of each. By way of contrast, our approach is "top down". We globally partition the set of all proteins hierarchically, as described above, and then identify protein families enriched within different subdivisions. While supporting previous findings, our approach also provides a tool for discovering novel relations between protein conservation profiles, functionality, and evolutionary history as represented by the tree of life.

  6. 78 FR 9024 - Sea Turtle Conservation; Shrimp Trawling Requirements

    Science.gov (United States)

    2013-02-07

    ... restriction and require all skimmer trawls, pusher-head trawls, and wing nets (butterfly trawls) rigged for... purposes of this notice, skimmer trawls, pusher-head trawls, and wing nets (butterfly trawls) will be... trawls, pusher-head trawls, and wing nets (butterfly trawls) in the Gulf of Mexico to use TEDs in their...

  7. Intragenic suppressor of Osiaa23 revealed a conserved tryptophan residue crucial for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Jun Ni

    Full Text Available The Auxin/Indole-3-Acetic Acid (Aux/IAA and Auxin Response Factor (ARF are two important families that play key roles in auxin signal transduction. Both of the families contain a similar carboxyl-terminal domain (Domain III/IV that facilitates interactions between these two families. In spite of the importance of protein-protein interactions among these transcription factors, the mechanisms involved in these interactions are largely unknown. In this study, we isolated six intragenic suppressors of an auxin insensitive mutant, Osiaa23. Among these suppressors, Osiaa23-R5 successfully rescued all the defects of the mutant. Sequence analysis revealed that an amino acid substitution occurred in the Tryptophan (W residue in Domain IV of Osiaa23. Yeast two-hybrid experiments showed that the mutation in Domain IV prevents the protein-protein interactions between Osiaa23 and OsARFs. Phylogenetic analysis revealed that the W residue is conserved in both OsIAAs and OsARFs. Next, we performed site-specific amino acid substitutions within Domain IV of OsARFs, and the conserved W in Domain IV was exchanged by Serine (S. The mutated OsARF(WSs can be released from the inhibition of Osiaa23 and maintain the transcriptional activities. Expression of OsARF(WSs in Osiaa23 mutant rescued different defects of the mutant. Our results suggest a previously unknown importance of Domain IV in both families and provide an indirect way to investigate functions of OsARFs.

  8. Protein conservation and variation suggest mechanisms of cell type-specific modulation of signaling pathways.

    Directory of Open Access Journals (Sweden)

    Martin H Schaefer

    2014-06-01

    Full Text Available Many proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors and the output (transcription factors layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types.

  9. Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

  10. Structure-sequence based analysis for identification of conserved regions in proteins

    Science.gov (United States)

    Zemla, Adam T; Zhou, Carol E; Lam, Marisa W; Smith, Jason R; Pardes, Elizabeth

    2013-05-28

    Disclosed are computational methods, and associated hardware and software products for scoring conservation in a protein structure based on a computationally identified family or cluster of protein structures. A method of computationally identifying a family or cluster of protein structures in also disclosed herein.

  11. Quantitative and functional characterization of the hyper-conserved protein of Prochlorococcus and marine Synechococcus.

    Directory of Open Access Journals (Sweden)

    Caroline E Whidden

    Full Text Available A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs. While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.

  12. Prediction of functional sites in proteins using conserved functional group analysis.

    Science.gov (United States)

    Innis, C Axel; Anand, A Prem; Sowdhamini, R

    2004-04-02

    A detailed knowledge of a protein's functional site is an absolute prerequisite for understanding its mode of action at the molecular level. However, the rapid pace at which sequence and structural information is being accumulated for proteins greatly exceeds our ability to determine their biochemical roles experimentally. As a result, computational methods are required which allow for the efficient processing of the evolutionary information contained in this wealth of data, in particular that related to the nature and location of functionally important sites and residues. The method presented here, referred to as conserved functional group (CFG) analysis, relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologues. We show that CFG analysis can fully or partially predict the location of functional sites in approximately 96% of the 470 cases tested and that, unlike other methods available, it is able to tolerate wide variations in sequence identity. In addition, we discuss its potential in a structural genomics context, where automation, scalability and efficiency are critical, and an increasing number of protein structures are determined with no prior knowledge of function. This is exemplified by our analysis of the hypothetical protein Ydde_Ecoli, whose structure was recently solved by members of the North East Structural Genomics consortium. Although the proposed active site for this protein needs to be validated experimentally, this example illustrates the scope of CFG analysis as a general tool for the identification of residues likely to play an important role in a protein's biochemical function. Thus, our method offers a convenient solution to rapidly and automatically process the vast amounts of data that are beginning to emerge from structural genomics projects.

  13. Conservation

    NARCIS (Netherlands)

    Noteboom, H.P.

    1985-01-01

    The IUCN/WWF Plants Conservation Programme 1984 — 1985. World Wildlife Fund chose plants to be the subject of their fund-raising campaign in the period 1984 — 1985. The objectives were to: 1. Use information techniques to achieve the conservation objectives of the Plants Programme – to save plants;

  14. Conservation.

    Science.gov (United States)

    National Audubon Society, New York, NY.

    This set of teaching aids consists of seven Audubon Nature Bulletins, providing the teacher and student with informational reading on various topics in conservation. The bulletins have these titles: Plants as Makers of Soil, Water Pollution Control, The Ground Water Table, Conservation--To Keep This Earth Habitable, Our Threatened Air Supply,…

  15. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    International Nuclear Information System (INIS)

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong

    2012-01-01

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  16. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong, E-mail: yerong24@fudan.edu.cn

    2012-06-05

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  17. A Conserved GPG-Motif in the HIV-1 Nef Core Is Required for Principal Nef-Activities.

    Directory of Open Access Journals (Sweden)

    Marta Martínez-Bonet

    Full Text Available To find out new determinants required for Nef activity we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified the GPG-motif, located at the 121-137 region of HIV-1 NL4.3 Nef, as a novel protein signature strictly required for the p56Lck dependent Nef-induced CD4-downregulation in T-cells. Since the Nef-GPG motif was dispensable for CD4-downregulation in HeLa-CD4 cells, Nef/AP-1 interaction and Nef-dependent effects on Tf-R trafficking, the observed effects on CD4 downregulation cannot be attributed to structure constraints or to alterations on general protein trafficking. Besides, we found that the GPG-motif was also required for Nef-dependent inhibition of ring actin re-organization upon TCR triggering and MHCI downregulation, suggesting that the GPG-motif could actively cooperate with the Nef PxxP motif for these HIV-1 Nef-related effects. Finally, we observed that the Nef-GPG motif was required for optimal infectivity of those viruses produced in T-cells. According to these findings, we propose the conserved GPG-motif in HIV-1 Nef as functional region required for HIV-1 infectivity and therefore with a potential interest for the interference of Nef activity during HIV-1 infection.

  18. Mitonuclear protein imbalance as a conserved longevity mechanism

    NARCIS (Netherlands)

    Houtkooper, Riekelt H.; Mouchiroud, Laurent; Ryu, Dongryeol; Moullan, Norman; Katsyuba, Elena; Knott, Graham; Williams, Robert W.; Auwerx, Johan

    2013-01-01

    Longevity is regulated by a network of closely linked metabolic systems. We used a combination of mouse population genetics and RNA interference in Caenorhabditis elegans to identify mitochondrial ribosomal protein S5 (Mrps5) and other mitochondrial ribosomal proteins as metabolic and longevity

  19. When Heterotrimeric G Proteins Are Not Activated by G Protein-Coupled Receptors: Structural Insights and Evolutionary Conservation.

    Science.gov (United States)

    DiGiacomo, Vincent; Marivin, Arthur; Garcia-Marcos, Mikel

    2018-01-23

    Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the "Gα-binding and -activating (GBA) motif". So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subunits in vitro and activates G protein signaling in cells. The importance of this mechanism of signal transduction is highlighted by the fact that its dysregulation underlies human diseases, such as cancer, which has made the proteins attractive new candidates for therapeutic intervention. Here we discuss recent discoveries on the structural basis of GBA-mediated activation of G proteins and its evolutionary conservation and compare them with the better-studied mechanism mediated by GPCRs.

  20. TOPDOM: database of conservatively located domains and motifs in proteins.

    Science.gov (United States)

    Varga, Julia; Dobson, László; Tusnády, Gábor E

    2016-09-01

    The TOPDOM database-originally created as a collection of domains and motifs located consistently on the same side of the membranes in α-helical transmembrane proteins-has been updated and extended by taking into consideration consistently localized domains and motifs in globular proteins, too. By taking advantage of the recently developed CCTOP algorithm to determine the type of a protein and predict topology in case of transmembrane proteins, and by applying a thorough search for domains and motifs as well as utilizing the most up-to-date version of all source databases, we managed to reach a 6-fold increase in the size of the whole database and a 2-fold increase in the number of transmembrane proteins. TOPDOM database is available at http://topdom.enzim.hu The webpage utilizes the common Apache, PHP5 and MySQL software to provide the user interface for accessing and searching the database. The database itself is generated on a high performance computer. tusnady.gabor@ttk.mta.hu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  1. Regulation of G-protein coupled receptor traffic by an evolutionary conserved hydrophobic signal.

    Science.gov (United States)

    Angelotti, Tim; Daunt, David; Shcherbakova, Olga G; Kobilka, Brian; Hurt, Carl M

    2010-04-01

    Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

  2. Structural Conservation of the Myoviridae Phage Tail Sheath Protein Fold

    Energy Technology Data Exchange (ETDEWEB)

    Aksyuk, Anastasia A.; Kurochkina, Lidia P.; Fokine, Andrei; Forouhar, Farhad; Mesyanzhinov, Vadim V.; Tong, Liang; Rossmann, Michael G. (SOIBC); (Purdue); (Columbia)

    2012-02-21

    Bacteriophage phiKZ is a giant phage that infects Pseudomonas aeruginosa, a human pathogen. The phiKZ virion consists of a 1450 {angstrom} diameter icosahedral head and a 2000 {angstrom}-long contractile tail. The structure of the whole virus was previously reported, showing that its tail organization in the extended state is similar to the well-studied Myovirus bacteriophage T4 tail. The crystal structure of a tail sheath protein fragment of phiKZ was determined to 2.4 {angstrom} resolution. Furthermore, crystal structures of two prophage tail sheath proteins were determined to 1.9 and 3.3 {angstrom} resolution. Despite low sequence identity between these proteins, all of these structures have a similar fold. The crystal structure of the phiKZ tail sheath protein has been fitted into cryo-electron-microscopy reconstructions of the extended tail sheath and of a polysheath. The structural rearrangement of the phiKZ tail sheath contraction was found to be similar to that of phage T4.

  3. Sequence, structure and function relationships in flaviviruses as assessed by evolutive aspects of its conserved non-structural protein domains.

    Science.gov (United States)

    da Fonseca, Néli José; Lima Afonso, Marcelo Querino; Pedersolli, Natan Gonçalves; de Oliveira, Lucas Carrijo; Andrade, Dhiego Souto; Bleicher, Lucas

    2017-10-28

    Flaviviruses are responsible for serious diseases such as dengue, yellow fever, and zika fever. Their genomes encode a polyprotein which, after cleavage, results in three structural and seven non-structural proteins. Homologous proteins can be studied by conservation and coevolution analysis as detected in multiple sequence alignments, usually reporting positions which are strictly necessary for the structure and/or function of all members in a protein family or which are involved in a specific sub-class feature requiring the coevolution of residue sets. This study provides a complete conservation and coevolution analysis on all flaviviruses non-structural proteins, with results mapped on all well-annotated available sequences. A literature review on the residues found in the analysis enabled us to compile available information on their roles and distribution among different flaviviruses. Also, we provide the mapping of conserved and coevolved residues for all sequences currently in SwissProt as a supplementary material, so that particularities in different viruses can be easily analyzed. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation.

    Science.gov (United States)

    Ribis, John W; Ravichandran, Priyanka; Putnam, Emily E; Pishdadian, Keyan; Shen, Aimee

    2017-01-01

    The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis , only two of these morphogenetic proteins have homologs in the Clostridia : SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis . Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis , C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia , but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and

  5. Identification of the conserved hypothetical protein BPSL0317 in Burkholderia pseudomallei K96243

    Science.gov (United States)

    Yusoff, Nur Syamimi; Damiri, Nadzirah; Firdaus-Raih, Mohd

    2014-09-01

    Burkholderia pseudomallei K96243 is the causative agent of melioidosis, a disease which is endemic in Northern Australia and Southeastern Asia. The genome encodes several essential proteins including those currently annotated as hypothetical proteins. We studied the conservation and the essentiality of expressed hypothetical proteins in normal and different stress conditions. Based on the comparative genomics, we identified a hypothetical protein, BPSL0317, a potential essential gene that is being expressed in all normal and stress conditions. BPSL0317 is also phylogenetically conserved in the Burkholderiales order suggesting that this protein is crucial for survival among the order's members. BPSL0317 therefore has a potential to be a candidate antimicrobial drug target for this group of bacteria.

  6. Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

    Directory of Open Access Journals (Sweden)

    Jackson Champer

    2016-01-01

    Full Text Available We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy. Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4, Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

  7. Pleiotropic Regulation of Virulence Genes in Streptococcus mutans by the Conserved Small Protein SprV.

    Science.gov (United States)

    Shankar, Manoharan; Hossain, Mohammad S; Biswas, Indranil

    2017-04-15

    critical for successful disease establishment. Sometimes these regulators, which are potential targets for antimicrobials, are lost in the genomic context due to the lack of annotated homologs. This work outlines the regulatory impact of a small, highly conserved hypothetical protein, SprV, encoded by S. mutans We show that SprV affects the transcript levels of various virulence factors required for normal growth, biofilm formation, stress tolerance, genetic competence, and bacteriocin production. Copyright © 2017 American Society for Microbiology.

  8. A computational tool to predict the evolutionarily conserved protein-protein interaction hot-spot residues from the structure of the unbound protein.

    Science.gov (United States)

    Agrawal, Neeraj J; Helk, Bernhard; Trout, Bernhardt L

    2014-01-21

    Identifying hot-spot residues - residues that are critical to protein-protein binding - can help to elucidate a protein's function and assist in designing therapeutic molecules to target those residues. We present a novel computational tool, termed spatial-interaction-map (SIM), to predict the hot-spot residues of an evolutionarily conserved protein-protein interaction from the structure of an unbound protein alone. SIM can predict the protein hot-spot residues with an accuracy of 36-57%. Thus, the SIM tool can be used to predict the yet unknown hot-spot residues for many proteins for which the structure of the protein-protein complexes are not available, thereby providing a clue to their functions and an opportunity to design therapeutic molecules to target these proteins. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. MadR1, a Mycobacterium tuberculosis cell cycle stress response protein that is a member of a widely conserved protein class of prokaryotic, eukaryotic and archeal origin.

    Science.gov (United States)

    Crew, Rebecca; Ramirez, Melissa V; England, Kathleen; Slayden, Richard A

    2015-05-01

    Stress-induced molecular programs designed to stall division progression are nearly ubiquitous in bacteria, with one well-known example being the participation of the SulA septum inhibiting protein in the SOS DNA damage repair response. Mycobacteria similarly demonstrate stress-altered growth kinetics, however no such regulators have been found in these organisms. We therefore set out to identify SulA-like regulatory proteins in Mycobacterium tuberculosis. A bioinformatics modeling-based approach led to the identification of rv2216 as encoding for a protein with weak similarity to SulA, further analysis distinguished this protein as belonging to a group of uncharacterized growth promoting proteins. We have named the mycobacterial protein encoded by rv2216 morphology altering division regulator protein 1, MadR1. Overexpression of madR1 modulated cell length while maintaining growth kinetics similar to wild-type, and increased the proportion of bent or V-form cells in the population. The presence of MadR1-GFP at regions of cellular elongation (poles) and morphological differentiation (V-form) suggests MadR1 involvement in phenotypic heterogeneity and longitudinal cellular growth. Global transcriptional analysis indicated that MadR1 functionality is linked to lipid editing programs required for growth and persistence. This is the first report to differentiate the larger class of these conserved proteins from SulA proteins and characterizes MadR1 effects on the mycobacterial cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Structural insights into the interaction of the conserved mammalian proteins GAPR-1 and Beclin 1, a key autophagy protein

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yue; Zhao, Yuting; Su, Minfei; Glover, Karen; Chakravarthy, Srinivas; Colbert, Christopher L.; Levine, Beth; Sinha, Sangita C.

    2017-08-29

    Mammalian Golgi-associated plant pathogenesis-related protein 1 (GAPR-1) is a negative autophagy regulator that binds Beclin 1, a key component of the autophagosome nucleation complex. Beclin 1 residues 267–284 are required for binding GAPR-1. Here, sequence analyses, structural modeling, mutagenesis combined with pull-down assays, X-ray crystal structure determination and small-angle X-ray scattering were used to investigate the Beclin 1–GAPR-1 interaction. Five conserved residues line an equatorial GAPR-1 surface groove that is large enough to bind a peptide. A model of a peptide comprising Beclin 1 residues 267–284 docked onto GAPR-1, built using theCABS-dockserver, indicates that this peptide binds to this GAPR-1 groove. Mutation of the five conserved residues lining this groove, H54A/E86A/G102K/H103A/N138G, abrogates Beclin 1 binding. The 1.27 Å resolution X-ray crystal structure of this pentad mutant GAPR-1 was determined. Comparison with the wild-type (WT) GAPR-1 structure shows that the equatorial groove of the pentad mutant is shallower and more positively charged, and therefore may not efficiently bind Beclin 1 residues 267–284, which include many hydrophobic residues. Both WT and pentad mutant GAPR-1 crystallize as dimers, and in each case the equatorial groove of one subunit is partially occluded by the other subunit, indicating that dimeric GAPR-1 is unlikely to bind Beclin 1. SAXS analysis of WT and pentad mutant GAPR-1 indicates that in solution the WT forms monomers, while the pentad mutant is primarily dimeric. Thus, changes in the structure of the equatorial groove combined with the improved dimerization of pentad mutant GAPR-1 are likely to abrogate binding to Beclin 1.

  11. Screening and expression of selected taxonomically conserved and unique hypothetical proteins in Burkholderia pseudomallei K96243

    Science.gov (United States)

    Akhir, Nor Azurah Mat; Nadzirin, Nurul; Mohamed, Rahmah; Firdaus-Raih, Mohd

    2015-09-01

    Hypothetical proteins of bacterial pathogens represent a large numbers of novel biological mechanisms which could belong to essential pathways in the bacteria. They lack functional characterizations mainly due to the inability of sequence homology based methods to detect functional relationships in the absence of detectable sequence similarity. The dataset derived from this study showed 550 candidates conserved in genomes that has pathogenicity information and only present in the Burkholderiales order. The dataset has been narrowed down to taxonomic clusters. Ten proteins were selected for ORF amplification, seven of them were successfully amplified, and only four proteins were successfully expressed. These proteins will be great candidates in determining the true function via structural biology.

  12. Competing conservation objectives for predators and prey: estimating killer whale prey requirements for Chinook salmon.

    Directory of Open Access Journals (Sweden)

    Rob Williams

    Full Text Available Ecosystem-based management (EBM of marine resources attempts to conserve interacting species. In contrast to single-species fisheries management, EBM aims to identify and resolve conflicting objectives for different species. Such a conflict may be emerging in the northeastern Pacific for southern resident killer whales (Orcinus orca and their primary prey, Chinook salmon (Oncorhynchus tshawytscha. Both species have at-risk conservation status and transboundary (Canada-US ranges. We modeled individual killer whale prey requirements from feeding and growth records of captive killer whales and morphometric data from historic live-capture fishery and whaling records worldwide. The models, combined with caloric value of salmon, and demographic and diet data for wild killer whales, allow us to predict salmon quantities needed to maintain and recover this killer whale population, which numbered 87 individuals in 2009. Our analyses provide new information on cost of lactation and new parameter estimates for other killer whale populations globally. Prey requirements of southern resident killer whales are difficult to reconcile with fisheries and conservation objectives for Chinook salmon, because the number of fish required is large relative to annual returns and fishery catches. For instance, a U.S. recovery goal (2.3% annual population growth of killer whales over 28 years implies a 75% increase in energetic requirements. Reducing salmon fisheries may serve as a temporary mitigation measure to allow time for management actions to improve salmon productivity to take effect. As ecosystem-based fishery management becomes more prevalent, trade-offs between conservation objectives for predators and prey will become increasingly necessary. Our approach offers scenarios to compare relative influence of various sources of uncertainty on the resulting consumption estimates to prioritise future research efforts, and a general approach for assessing the extent of

  13. Conformational coupling between receptor and kinase binding sites through a conserved salt bridge in a signaling complex scaffold protein.

    Directory of Open Access Journals (Sweden)

    Davi R Ortega

    Full Text Available Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD, NMR spectroscopy, and circular dichroism (CD, we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.

  14. Structure of the conserved hypothetical protein MAL13P1.257 from Plasmodium falciparum

    International Nuclear Information System (INIS)

    Holmes, Margaret A.; Buckner, Frederick S.; Van Voorhis, Wesley C.; Mehlin, Christopher; Boni, Erica; Earnest, Thomas N.; DeTitta, George; Luft, Joseph; Lauricella, Angela; Anderson, Lori; Kalyuzhniy, Oleksandr; Zucker, Frank; Schoenfeld, Lori W.; Hol, Wim G. J.; Merritt, Ethan A.

    2006-01-01

    The crystal structure of a conserved hypothetical protein, MAL13P1.257 from P. falciparum, has been determined at 2.17 Å resolution. The structure represents a new protein fold and is the first structural representative for Pfam sequence family PF05907. The structure of a conserved hypothetical protein, PlasmoDB sequence MAL13P1.257 from Plasmodium falciparum, Pfam sequence family PF05907, has been determined as part of the structural genomics effort of the Structural Genomics of Pathogenic Protozoa consortium. The structure was determined by multiple-wavelength anomalous dispersion at 2.17 Å resolution. The structure is almost entirely β-sheet; it consists of 15 β-strands and one short 3 10 -helix and represents a new protein fold. The packing of the two monomers in the asymmetric unit indicates that the biological unit may be a dimer.

  15. Nitrogen Balance and Protein Requirements for Critically Ill Older Patients

    Directory of Open Access Journals (Sweden)

    Roland N. Dickerson

    2016-04-01

    Full Text Available Critically ill older patients with sarcopenia experience greater morbidity and mortality than younger patients. It is anticipated that unabated protein catabolism would be detrimental for the critically ill older patient. Healthy older subjects experience a diminished response to protein supplementation when compared to their younger counterparts, but this anabolic resistance can be overcome by increasing protein intake. Preliminary evidence suggests that older patients may respond differently to protein intake than younger patients during critical illness as well. If sufficient protein intake is given, older patients can achieve a similar nitrogen accretion response as younger patients even during critical illness. However, there is concern among some clinicians that increasing protein intake in older patients during critical illness may lead to azotemia due to decreased renal functional reserve which may augment the propensity towards worsened renal function and worsened clinical outcomes. Current evidence regarding protein requirements, nitrogen balance, ureagenesis, and clinical outcomes during nutritional therapy for critically ill older patients is reviewed.

  16. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  17. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  18. Protein Requirements and Recommendations for Older People: A Review

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    Caryl Nowson

    2015-08-01

    Full Text Available Declines in skeletal muscle mass and strength are major contributors to increased mortality, morbidity and reduced quality of life in older people. Recommended Dietary Allowances/Intakes have failed to adequately consider the protein requirements of the elderly with respect to function. The aim of this paper was to review definitions of optimal protein status and the evidence base for optimal dietary protein. Current recommended protein intakes for older people do not account for the compensatory loss of muscle mass that occurs on lower protein intakes. Older people have lower rates of protein synthesis and whole-body proteolysis in response to an anabolic stimulus (food or resistance exercise. Recommendations for the level of adequate dietary intake of protein for older people should be informed by evidence derived from functional outcomes. Randomized controlled trials report a clear benefit of increased dietary protein on lean mass gain and leg strength, particularly when combined with resistance exercise. There is good consistent evidence (level III-2 to IV that consumption of 1.0 to 1.3 g/kg/day dietary protein combined with twice-weekly progressive resistance exercise reduces age-related muscle mass loss. Older people appear to require 1.0 to 1.3 g/kg/day dietary protein to optimize physical function, particularly whilst undertaking resistance exercise recommendations.

  19. Biochemical and structural characterization of Cren7, a novel chromatin protein conserved among Crenarchaea.

    Science.gov (United States)

    Guo, Li; Feng, Yingang; Zhang, Zhenfeng; Yao, Hongwei; Luo, Yuanming; Wang, Jinfeng; Huang, Li

    2008-03-01

    Archaea contain a variety of chromatin proteins consistent with the evolution of different genome packaging mechanisms. Among the two main kingdoms in the Archaea, Euryarchaeota synthesize histone homologs, whereas Crenarchaeota have not been shown to possess a chromatin protein conserved at the kingdom level. We report the identification of Cren7, a novel family of chromatin proteins highly conserved in the Crenarchaeota. A small, basic, methylated and abundant protein, Cren7 displays a higher affinity for double-stranded DNA than for single-stranded DNA, constrains negative DNA supercoils and is associated with genomic DNA in vivo. The solution structure and DNA-binding surface of Cren7 from the hyperthermophilic crenarchaeon Sulfolobus solfataricus were determined by NMR. The protein adopts an SH3-like fold. It interacts with duplex DNA through a beta-sheet and a long flexible loop, presumably resulting in DNA distortions through intercalation of conserved hydrophobic residues into the DNA structure. These data suggest that the crenarchaeal kingdom in the Archaea shares a common strategy in chromatin organization.

  20. Extreme sequence divergence but conserved ligand-binding specificity in Streptococcus pyogenes M protein.

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    2006-05-01

    Full Text Available Many pathogenic microorganisms evade host immunity through extensive sequence variability in a protein region targeted by protective antibodies. In spite of the sequence variability, a variable region commonly retains an important ligand-binding function, reflected in the presence of a highly conserved sequence motif. Here, we analyze the limits of sequence divergence in a ligand-binding region by characterizing the hypervariable region (HVR of Streptococcus pyogenes M protein. Our studies were focused on HVRs that bind the human complement regulator C4b-binding protein (C4BP, a ligand that confers phagocytosis resistance. A previous comparison of C4BP-binding HVRs identified residue identities that could be part of a binding motif, but the extended analysis reported here shows that no residue identities remain when additional C4BP-binding HVRs are included. Characterization of the HVR in the M22 protein indicated that two relatively conserved Leu residues are essential for C4BP binding, but these residues are probably core residues in a coiled-coil, implying that they do not directly contribute to binding. In contrast, substitution of either of two relatively conserved Glu residues, predicted to be solvent-exposed, had no effect on C4BP binding, although each of these changes had a major effect on the antigenic properties of the HVR. Together, these findings show that HVRs of M proteins have an extraordinary capacity for sequence divergence and antigenic variability while retaining a specific ligand-binding function.

  1. Some epitopes conservation in non structural 3 protein dengue virus serotype 4

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    Tegar A. P. Siregar

    2016-03-01

    Full Text Available AbstrakLatar belakang: Protein Non Struktural 3 (NS3 virus dengue menginduksi respon antibodi netralisasidan respon sel T CD4+ dan CD8+, serta berperan dalam replikasi virus. Protein NS3 memiliki epitopepitopsel T dan B yang terdapat perbedaan kelestarian pada berbagai strain virus dengue serotipe 4(DENV-4. Penelitian ini bertujuan untuk mengetahui kelestarian epitop sel T dan B pada protein NS3DENV-4 strain-strain dunia dan keempat serotipe virus dengue strain Indonesia.Metode: Penelitian ini dilakukan di Departemen Mikrobiologi Fakultas Kedokteran UI sejak Juni 2013 - April2014. Sekuens asam amino NS3 DENV-4 strain 081 didapatkan setelah produk PCR gen NS3 DENV-4 081disekuensing. Epitop-epitop sel T dan sel B protein NS3 DENV-4 081 dianalisis dan dibandingkan dengansekuens asam amino protein NS3 dari 124 strain DENV-4 di dunia dan keempat serotipe DENV strain Indonesia.Strain-strain dunia merupakan strain yang ada di benua Amerika (Venezuela, Colombia, dll dan Asia (Cina,Singapura, dll. Referensi posisi epitop sel T dan B protein NS3 diperoleh dari laporan penelitian terdahulu.Hasil: Delapan epitop sel T dan 2 epitop sel B dari protein NS3 DENV-4 081 ternyata identik dan lestaripada protein NS3 dari 124 strain DENV-4 dunia. Epitop sel B di posisi asam amino 537-544 pada proteinNS3 DENV-4 081 ternyata identik dan lestari dengan epitop sel B protein NS3 dari keempat serotipeDENV strain Indonesia.Kesimpulan: Kelestarian yang luas dari epitop sel T dan B pada hampir seluruh strain DENV-4 dunia danserotipe-serotipe DENV strain Indonesia. (Health Science Journal of Indonesia 2015;6:126-31Kata kunci: virus dengue, protein NS3, epitop sel T, epitop sel B AbstractBackground: Non Structural 3 (NS3 protein of dengue virus (DENV is known to induce antibody, CD4+and CD8+ T cell responses, and playing role in viral replication. NS3 protein has T and B cell epitopes,which has conservation difference between DENV-4 strains. This study aimed to identify

  2. Managing consequences of climate-driven species redistribution requires integration of ecology, conservation and social science.

    Science.gov (United States)

    Bonebrake, Timothy C; Brown, Christopher J; Bell, Johann D; Blanchard, Julia L; Chauvenet, Alienor; Champion, Curtis; Chen, I-Ching; Clark, Timothy D; Colwell, Robert K; Danielsen, Finn; Dell, Anthony I; Donelson, Jennifer M; Evengård, Birgitta; Ferrier, Simon; Frusher, Stewart; Garcia, Raquel A; Griffis, Roger B; Hobday, Alistair J; Jarzyna, Marta A; Lee, Emma; Lenoir, Jonathan; Linnetved, Hlif; Martin, Victoria Y; McCormack, Phillipa C; McDonald, Jan; McDonald-Madden, Eve; Mitchell, Nicola; Mustonen, Tero; Pandolfi, John M; Pettorelli, Nathalie; Possingham, Hugh; Pulsifer, Peter; Reynolds, Mark; Scheffers, Brett R; Sorte, Cascade J B; Strugnell, Jan M; Tuanmu, Mao-Ning; Twiname, Samantha; Vergés, Adriana; Villanueva, Cecilia; Wapstra, Erik; Wernberg, Thomas; Pecl, Gretta T

    2018-02-01

    Climate change is driving a pervasive global redistribution of the planet's species. Species redistribution poses new questions for the study of ecosystems, conservation science and human societies that require a coordinated and integrated approach. Here we review recent progress, key gaps and strategic directions in this nascent research area, emphasising emerging themes in species redistribution biology, the importance of understanding underlying drivers and the need to anticipate novel outcomes of changes in species ranges. We highlight that species redistribution has manifest implications across multiple temporal and spatial scales and from genes to ecosystems. Understanding range shifts from ecological, physiological, genetic and biogeographical perspectives is essential for informing changing paradigms in conservation science and for designing conservation strategies that incorporate changing population connectivity and advance adaptation to climate change. Species redistributions present challenges for human well-being, environmental management and sustainable development. By synthesising recent approaches, theories and tools, our review establishes an interdisciplinary foundation for the development of future research on species redistribution. Specifically, we demonstrate how ecological, conservation and social research on species redistribution can best be achieved by working across disciplinary boundaries to develop and implement solutions to climate change challenges. Future studies should therefore integrate existing and complementary scientific frameworks while incorporating social science and human-centred approaches. Finally, we emphasise that the best science will not be useful unless more scientists engage with managers, policy makers and the public to develop responsible and socially acceptable options for the global challenges arising from species redistributions. © 2017 Cambridge Philosophical Society.

  3. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhongshan [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom); College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Xiang, Quanju [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Department of Microbiology, College of Resource and Environment Science, Sichuan Agriculture University, Yaan 625000 (China); Zhu, Xiaofeng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Dong, Haohao [Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); He, Chuan [School of Electronics and Information, Wuhan Technical College of Communications, No. 6 Huangjiahu West Road, Hongshan District, Wuhan, Hubei 430065 (China); Wang, Haiyan; Zhang, Yizheng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Wang, Wenjian, E-mail: Wenjian166@gmail.com [Laboratory of Department of Surgery, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080 (China); Dong, Changjiang, E-mail: C.Dong@uea.ac.uk [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom)

    2014-09-26

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

  4. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    International Nuclear Information System (INIS)

    Wang, Zhongshan; Xiang, Quanju; Zhu, Xiaofeng; Dong, Haohao; He, Chuan; Wang, Haiyan; Zhang, Yizheng; Wang, Wenjian; Dong, Changjiang

    2014-01-01

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg 2+ . • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg 2+ , which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics

  5. Conservation and variability of dengue virus proteins: implications for vaccine design.

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    Asif M Khan

    2008-08-01

    Full Text Available Genetic variation and rapid evolution are hallmarks of RNA viruses, the result of high mutation rates in RNA replication and selection of mutants that enhance viral adaptation, including the escape from host immune responses. Variability is uneven across the genome because mutations resulting in a deleterious effect on viral fitness are restricted. RNA viruses are thus marked by protein sites permissive to multiple mutations and sites critical to viral structure-function that are evolutionarily robust and highly conserved. Identification and characterization of the historical dynamics of the conserved sites have relevance to multiple applications, including potential targets for diagnosis, and prophylactic and therapeutic purposes.We describe a large-scale identification and analysis of evolutionarily highly conserved amino acid sequences of the entire dengue virus (DENV proteome, with a focus on sequences of 9 amino acids or more, and thus immune-relevant as potential T-cell determinants. DENV protein sequence data were collected from the NCBI Entrez protein database in 2005 (9,512 sequences and again in 2007 (12,404 sequences. Forty-four (44 sequences (pan-DENV sequences, mainly those of nonstructural proteins and representing approximately 15% of the DENV polyprotein length, were identical in 80% or more of all recorded DENV sequences. Of these 44 sequences, 34 ( approximately 77% were present in >or=95% of sequences of each DENV type, and 27 ( approximately 61% were conserved in other Flaviviruses. The frequencies of variants of the pan-DENV sequences were low (0 to approximately 5%, as compared to variant frequencies of approximately 60 to approximately 85% in the non pan-DENV sequence regions. We further showed that the majority of the conserved sequences were immunologically relevant: 34 contained numerous predicted human leukocyte antigen (HLA supertype-restricted peptide sequences, and 26 contained T-cell determinants identified by

  6. A human protein interaction network shows conservation of aging processes between human and invertebrate species.

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    Russell Bell

    2009-03-01

    Full Text Available We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteome-scale human protein interaction Core Network generated through unbiased high-throughput yeast two-hybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33% extended life span when knocked-down in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins.

  7. A conserved tryptophan within the WRDPLVDID domain of yeast Pah1 phosphatidate phosphatase is required for its in vivo function in lipid metabolism.

    Science.gov (United States)

    Park, Yeonhee; Han, Gil-Soo; Carman, George M

    2017-12-01

    PAH1 -encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase-like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions ( e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Counterproductive Consequences of a Conservative Ideology: Medicaid Expansion and Personal Responsibility Requirements.

    Science.gov (United States)

    Baker, Allison M; Hunt, Linda M

    2016-07-01

    Medicaid expansion, a key part of the Affordable Care Act, has been opposed by conservative politicians despite its fiscal and public health benefits. In response, some Republican-led states have expanded Medicaid with new reforms, including requirements for cost sharing and behavioral incentives, that promote conservative political values tied to an ideology of personal responsibility. We examine this trend using Michigan's Medicaid expansion as a case example. We explore the origins, evidence base, and possible consequences of these reforms. We argue that these reforms prioritize ideology over sound public health knowledge, deflecting attention away from the social, economic, and structural factors that influence the health of the poor, and may ultimately contribute to counterproductive public health and fiscal outcomes.

  9. Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx.

    Science.gov (United States)

    Löschberger, Anna; Niehörster, Thomas; Sauer, Markus

    2014-05-01

    Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites

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    Sibley L David

    2005-12-01

    Full Text Available Abstract Background The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. Results Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8–10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex, and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery. In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. Conclusion The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility.

  11. Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

    Science.gov (United States)

    Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

    2015-01-01

    Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

  12. Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

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    Mihaly Varadi

    Full Text Available Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

  13. Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

    Science.gov (United States)

    Hackenberg, Dieter; McKain, Michael R; Lee, Soon Goo; Roy Choudhury, Swarup; McCann, Tyler; Schreier, Spencer; Harkess, Alex; Pires, J Chris; Wong, Gane Ka-Shu; Jez, Joseph M; Kellogg, Elizabeth A; Pandey, Sona

    2017-10-01

    Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  14. Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

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    Meuwissen Pieter J

    2012-04-01

    Full Text Available Abstract Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2 and non-canonical (B2 and C1422 HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2, the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we

  15. A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  16. Protein requirements for Blue-fronted Amazon (Amazona aestiva) growth.

    Science.gov (United States)

    Carciofi, A C; Sanfilippo, L F; de-Oliveira, L D; do Amaral, P P; Prada, F

    2008-06-01

    The objective of this study was to evaluate the protein requirements for hand-rearing Blue-fronted Amazon parrots (Amazona aestiva). Forty hatchlings were fed semi-purified diets containing one of four (as-fed basis) protein levels: 13%, 18%, 23% and 28%. The experiment was carried out in a randomized block design with the initial weight of the nestling as the blocking factor and 10 parrots per protein level. Regression analysis was used to determine relationships between protein level and biometric measurements. The data indicated that 13% crude protein supported nestling growth with 18% being the minimum tested level required for maximum development. The optimal protein concentration for maximum weight gain was 24.4% (p = 0.08; r(2) = 0.25), tail length 23.7% (p = 0.09; r(2) = 0.19), wing length 23.0% (p = 0.07; r(2) = 0.17), tarsus length 21.3% (p = 0.06; r(2) = 0.10) and tarsus width 21.4% (p = 0.07; r(2) = 0.09). Tarsus measurements were larger in males (p < 0.05), indicating that sex must be considered when studying developing psittacines. These results were obtained using a highly digestible protein and a diet with moderate metabolizable energy levels.

  17. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    Science.gov (United States)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  18. A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize.

    Directory of Open Access Journals (Sweden)

    Matt eGeisler

    2015-06-01

    Full Text Available Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6,004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize.

  19. Gains of ubiquitylation sites in highly conserved proteins in the human lineage

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Post-translational modification of lysine residues of specific proteins by ubiquitin modulates the degradation, localization, and activity of these target proteins. Here, we identified gains of ubiquitylation sites in highly conserved regions of human proteins that occurred during human evolution. Results We analyzed human ubiquitylation site data and multiple alignments of orthologous mammalian proteins including those from humans, primates, other placental mammals, opossum, and platypus. In our analysis, we identified 281 ubiquitylation sites in 252 proteins that first appeared along the human lineage during primate evolution: one protein had four novel sites; four proteins had three sites each; 18 proteins had two sites each; and the remaining 229 proteins had one site each. PML, which is involved in neurodevelopment and neurodegeneration, acquired three sites, two of which have been reported to be involved in the degradation of PML. Thirteen human proteins, including ERCC2 (also known as XPD and NBR1, gained human-specific ubiquitylated lysines after the human-chimpanzee divergence. ERCC2 has a Lys/Gln polymorphism, the derived (major allele of which confers enhanced DNA repair capacity and reduced cancer risk compared with the ancestral (minor allele. NBR1 and eight other proteins that are involved in the human autophagy protein interaction network gained a novel ubiquitylation site. Conclusions The gain of novel ubiquitylation sites could be involved in the evolution of protein degradation and other regulatory networks. Although gains of ubiquitylation sites do not necessarily equate to adaptive evolution, they are useful candidates for molecular functional analyses to identify novel advantageous genetic modifications and innovative phenotypes acquired during human evolution.

  20. Revision of the energy conservation requirements in the manufactured home construction and safety standards

    Energy Technology Data Exchange (ETDEWEB)

    Conner, C C; Lee, A D; Lucas, R G; Taylor, Z T

    1992-02-01

    Thermal requirements were developed for manufactured (mobile) homes in response to legislation requiring the US Department of Housing and Urban Development (HUD) to revise its thermal standards for manufactured homes. A life-cycle cost minimization from the home owner's perspecetive was used to establish an optimum in a large number of cities for several prototype homes. The development of the economic, financial, and energy conservation measure parameters input into the life-cycle cost analysis was documented. The optimization results were aggregated to zones which were expressed as a maximum overall home U-value (thermal transmittance) requirement. The revised standard's costs, benefits, and net value to the consumer were quantified. 50 refs.

  1. Homologous high-throughput expression and purification of highly conserved E coli proteins

    Directory of Open Access Journals (Sweden)

    Duchmann Rainer

    2007-06-01

    Full Text Available Abstract Background Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD. Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies. Results As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP, N-utilizing substance A (NusA, or glutathione S-transferase (GST to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS. Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500

  2. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

    OpenAIRE

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C. G.; Benavente, Ricardo

    2012-01-01

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed...

  3. The conservation pattern of short linear motifs is highly correlated with the function of interacting protein domains

    Directory of Open Access Journals (Sweden)

    Wang Yiguo

    2008-10-01

    Full Text Available Abstract Background Many well-represented domains recognize primary sequences usually less than 10 amino acids in length, called Short Linear Motifs (SLiMs. Accurate prediction of SLiMs has been difficult because they are short (often Results Our combined approach revealed that SLiMs are highly conserved in proteins from functional classes that are known to interact with a specific domain, but that they are not conserved in most other protein groups. We found that SLiMs recognized by SH2 domains were highly conserved in receptor kinases/phosphatases, adaptor molecules, and tyrosine kinases/phosphatases, that SLiMs recognized by SH3 domains were highly conserved in cytoskeletal and cytoskeletal-associated proteins, that SLiMs recognized by PDZ domains were highly conserved in membrane proteins such as channels and receptors, and that SLiMs recognized by S/T kinase domains were highly conserved in adaptor molecules, S/T kinases/phosphatases, and proteins involved in transcription or cell cycle control. We studied Tyr-SLiMs recognized by SH2 domains in more detail, and found that SH2-recognized Tyr-SLiMs on the cytoplasmic side of membrane proteins are more highly conserved than those on the extra-cellular side. Also, we found that SH2-recognized Tyr-SLiMs that are associated with SH3 motifs and a tyrosine kinase phosphorylation motif are more highly conserved. Conclusion The interactome of protein domains is reflected by the evolutionary conservation of SLiMs recognized by these domains. Combining scoring matrixes derived from peptide libraries and conservation analysis, we would be able to find those protein groups that are more likely to interact with specific domains.

  4. Requirements of Slm proteins for proper eisosome organization ...

    Indian Academy of Sciences (India)

    Eisosomes are large immobile assemblies at the cortex of a cell under the membrane ... microscopic analysis of Abp1-RFP revealed that the actin defect in slmts cells was not ... Our data provide evidence for the requirement of Slm proteins in eisosome ..... with a bandwidth of 10 nm, and emission was measured at. 680 nm ...

  5. Macoilin, a conserved nervous system-specific ER membrane protein that regulates neuronal excitability.

    Directory of Open Access Journals (Sweden)

    Fausto Arellano-Carbajal

    2011-03-01

    Full Text Available Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O₂ responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca²(+ transients, at least in some neurons: in maco-1 mutants the O₂-sensing neuron PQR is unable to generate a Ca²(+ response to a rise in O₂. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O₂, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca²(+ channels, also fails to disrupt Ca²(+ responses in the PQR cell body to O₂ stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca²(+ channel α1 subunit, recapitulate the Ca²(+ response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or

  6. Conservation of a proteinase cleavage site between an insect retrovirus (gypsy) Env protein and a baculovirus envelope fusion protein

    International Nuclear Information System (INIS)

    Pearson, Margot N.; Rohrmann, George F.

    2004-01-01

    The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR in the Lymantria dispar nucleopolyhedrovirus fusion protein (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env-green fluorescent protein construct localized to cell membranes, no cell fusion was observed

  7. Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

    Directory of Open Access Journals (Sweden)

    Quevillon-Cheruel Sophie

    2007-01-01

    Full Text Available Abstract The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes.

  8. RNA Binding Proteins in Eye Development and Disease: Implication of Conserved RNA Granule Components

    Science.gov (United States)

    Dash, Soma; Siddam, Archana D.; Barnum, Carrie E.; Janga, Sarath Chandra

    2016-01-01

    The molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1 and Bmp4 are commonly required for their development. In contrast, our understanding of post-transcriptional regulation in eye development and disease, particularly regarding the function of RNA binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila, as well as several vertebrate models such as fish, frog, chicken and mouse. Interestingly, of the 42 RBPs that have been investigated with for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as Processing bodies (P-bodies), Stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate post-transcriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2 and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving post-transcriptional regulatory networks in eye development and disease. PMID:27133484

  9. A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression

    Directory of Open Access Journals (Sweden)

    Amsterdam Adam

    2006-06-01

    identification of approximately 25% of the essential genes required for craniofacial development. The identification of zebrafish models for two human disease syndromes indicates that homologs to the other genes are likely to also be relevant for human craniofacial development. The initial characterization of wdr68 suggests an important role in craniofacial development for the highly conserved Wdr68-Dyrk1 protein complexes.

  10. Crystal Structure of VC0702 at 2.0 Angstrom: Conserved Hypothetical Protein from Vibrio Cholerae

    International Nuclear Information System (INIS)

    Ni, S.; Forouhar, F.; Bussiere, D.; Robinson, H.; Kennedy, M.

    2006-01-01

    VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a three-gene operon containing the MbaA gene that encodes for a GGDEF and EAL domain-containing protein which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0 Angstroms and refined to R work = 22.8% and R free = 26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C2221 space group with dimensions of a = 66.61 Angstroms, b = 88.118 Angstroms, and c = 118.35 Angstroms with a homodimer in the asymmetric unit. VC0702, which forms a mixed α + β three-layered αβα sandwich, belongs to the Pfam DUF84 and COG1986 families of proteins. Sequence conservation within the DUF84 and COG1986 families was used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeschii, which has been identified as a novel NTPase that binds NTP in a deep cleft similarly located to the conserved patch of surface residues that define an analogous cleft in VC0702. Collectively, the data suggest that VC0702 may have a biochemical function that involves NTP binding and phosphatase activity of some kind, and is likely involved in regulation of the signaling pathway that controls biofilm formation and maintenance in Vibrio cholerae

  11. An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

    Directory of Open Access Journals (Sweden)

    Marcin Michalik

    Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

  12. Optimum dietary protein requirement in nondiabetic maintenance hemodialysis patients.

    Science.gov (United States)

    Ohkawa, Sakae; Kaizu, Yukiko; Odamaki, Mari; Ikegaya, Naoki; Hibi, Ikuo; Miyaji, Kunihiko; Kumagai, Hiromichi

    2004-03-01

    There is controversy about whether the dietary protein requirement of 1.2 g/kg/d for hemodialysis (HD) patients, in the nutritional guidelines recommended by the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (NKF-KDOQI), is reasonable. A cross-sectional study was conducted in 129 stable HD patients without diabetes (84 men, 45 women) to investigate the association between the protein equivalent of nitrogen appearance normalized by ideal body weight (nPNAibw), an index of protein intake, and skeletal muscle mass or other metabolic consequences. Patients were divided into 5 groups according to nPNAibw index. Midthigh muscle area (TMA), midthigh subcutaneous fat area (TSFA), abdominal muscle area (AMA), abdominal subcutaneous fat area (ASFA), and visceral fat area (AVFA) were measured using computed tomography, and various nutritional parameters were compared among these groups. TMA and AMA values increased with increasing dietary protein intake from less than 0.7 g/kg/d to 0.9-1.1 g/kg/d and showed a plateau at greater than 0.9 to 1.1 g/kg/d of dietary protein intake. Conversely, fat mass, including TSFA, ASFA, and AVFA, and serum potassium concentration increased with graded protein intake, and no plateau was formed. Patients with nPNAibw greater than 1.3 g/kg/d satisfied the criterion of visceral obesity. Although serum prealbumin levels showed a trend similar to that of muscle mass, there was no significant difference in serum albumin levels among the study groups. Optimal dietary protein requirement for patients undergoing maintenance HD in a stable condition appears to be less than the level recommended by the NKF-KDOQI nutritional guidelines.

  13. Translating conservation genetics into management: Pan-European minimum requirements for dynamic conservation units of forest tree genetic diversity

    OpenAIRE

    Koskela, Jarkko; Lefèvre, François; Schueler, Silvio; Kraigher, Hojka; Olrik, Ditte C.; Hubert, Jason; Longauer, Roman; Bozzano, Michele; Yrjänä, Leena; Alizoti, Paraskevi; Rotach, Peter; Vietto, Lorenzo; Bordács, Sándor; Myking, Tor; Eysteinsson, Thröstur

    2013-01-01

    This paper provides a review of theoretical and practical aspects related to genetic management of forest trees. The implementation of international commitments on forest genetic diversity has been slow and partly neglected. Conservation of forest genetic diversity is still riddled with problems, and complexities of national legal and administrative structures. Europe is an example of a complex region where the dis- tribution ranges of tree species extend across large geographical areas with ...

  14. Optimum dietary protein requirement of Malaysian mahseer (Tor tambroides) fingerling.

    Science.gov (United States)

    Misieng, Josephine Dorin; Kamarudin, Mohd Salleh; Musa, Mazlinda

    2011-02-01

    The optimum dietary protein requirement of the Malaysian mahseer (Tor tambroides) fingerlings was determined in this study. In this completely randomized designed experiment, formulated diets of five levels of dietary protein (30, 35, 40, 45 and 50%) were tested on the T. tambroides fingerlings (initial body weight of 5.85 +/- 0.40 g), reared in aquarium fitted with a biofiltering system. The fingerlings were fed twice daily at 5% of biomass. The fingerling body weight and total length was taken at every two weeks. Mortality was recorded daily. The dietary protein had significant effects on the body weight gain and Specific Growth Rate (SGR) of the fingerlings. The body weight gain and SGR of fingerlings fed with the diet with the dietary protein level of 40% was significantly higher (p<0.05) than that of 30, 35 and 50%. The feed conversion ratio of the 40% dietary protein was the significantly lowest at 2.19 +/- 0.163. The dietary protein level of 40% was the most optimum for T. tambroides fingerlings.

  15. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

    Science.gov (United States)

    Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

    2016-01-01

    Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

  16. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans.

    Science.gov (United States)

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C G; Benavente, Ricardo

    2012-10-09

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans.

  17. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    Science.gov (United States)

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

  18. Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

    Energy Technology Data Exchange (ETDEWEB)

    Liban, Tyler J.; Medina, Edgar M.; Tripathi, Sarvind; Sengupta, Satyaki; Henry, R. William; Buchler, Nicolas E.; Rubin, Seth M. (UCSC); (Duke); (MSU)

    2017-04-24

    The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD–CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein–E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.

  19. A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation.

    Science.gov (United States)

    Zattas, Dimitrios; Berk, Jason M; Kreft, Stefan G; Hochstrasser, Mark

    2016-06-03

    Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Protein import into chloroplasts requires a chloroplast ATPase

    International Nuclear Information System (INIS)

    Pain, D.; Blobel, G.

    1987-01-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the [ 35 S]methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H + , K + , Na + , or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors

  1. Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold.

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    Stefan M Ivanov

    Full Text Available An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners.

  2. Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold.

    Science.gov (United States)

    Ivanov, Stefan M; Cawley, Andrew; Huber, Roland G; Bond, Peter J; Warwicker, Jim

    2017-01-01

    An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge) are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners.

  3. Models of protein and amino acid requirements for cattle

    Directory of Open Access Journals (Sweden)

    Luis Orlindo Tedeschi

    2015-03-01

    Full Text Available Protein supply and requirements by ruminants have been studied for more than a century. These studies led to the accumulation of lots of scientific information about digestion and metabolism of protein by ruminants as well as the characterization of the dietary protein in order to maximize animal performance. During the 1980s and 1990s, when computers became more accessible and powerful, scientists began to conceptualize and develop mathematical nutrition models, and to program them into computers to assist with ration balancing and formulation for domesticated ruminants, specifically dairy and beef cattle. The most commonly known nutrition models developed during this period were the National Research Council (NRC in the United States, Agricultural Research Council (ARC in the United Kingdom, Institut National de la Recherche Agronomique (INRA in France, and the Commonwealth Scientific and Industrial Research Organization (CSIRO in Australia. Others were derivative works from these models with different degrees of modifications in the supply or requirement calculations, and the modeling nature (e.g., static or dynamic, mechanistic, or deterministic. Circa 1990s, most models adopted the metabolizable protein (MP system over the crude protein (CP and digestible CP systems to estimate supply of MP and the factorial system to calculate MP required by the animal. The MP system included two portions of protein (i.e., the rumen-undegraded dietary CP - RUP - and the contributions of microbial CP - MCP as the main sources of MP for the animal. Some models would explicitly account for the impact of dry matter intake (DMI on the MP required for maintenance (MPm; e.g., Cornell Net Carbohydrate and Protein System - CNCPS, the Dutch system - DVE/OEB, while others would simply account for scurf, urinary, metabolic fecal, and endogenous contributions independently of DMI. All models included milk yield and its components in estimating MP required for lactation

  4. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Science.gov (United States)

    Fauteux, François; Strömvik, Martina V

    2009-01-01

    Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs

  5. Conservation and divergence of ADAM family proteins in the Xenopus genome

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    Shah Anoop

    2010-07-01

    Full Text Available Abstract Background Members of the disintegrin metalloproteinase (ADAM family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species. Results Based on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region. Conclusions Our genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some

  6. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Directory of Open Access Journals (Sweden)

    Fauteux François

    2009-10-01

    Full Text Available Abstract Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP gene promoters from three plant families, namely Brassicaceae (mustards, Fabaceae (legumes and Poaceae (grasses using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L. Heynh., soybean (Glycine max (L. Merr. and rice (Oryza sativa L. respectively. We have identified three conserved motifs (two RY-like and one ACGT-like in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination

  7. Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

    Science.gov (United States)

    Kazakov, Alexei S; Sokolov, Andrei S; Vologzhannikova, Alisa A; Permyakova, Maria E; Khorn, Polina A; Ismailov, Ramis G; Denessiouk, Konstantin A; Denesyuk, Alexander I; Rastrygina, Victoria A; Baksheeva, Viktoriia E; Zernii, Evgeni Yu; Zinchenko, Dmitry V; Glazatov, Vladimir V; Uversky, Vladimir N; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

    2017-01-01

    Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

  8. The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

    International Nuclear Information System (INIS)

    Brush, G.S.; Morrow, D.M.; Hieter, P.; Kelly, T.J.

    1996-01-01

    Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T

  9. PML tumor suppressor protein is required for HCV production

    International Nuclear Information System (INIS)

    Kuroki, Misao; Ariumi, Yasuo; Hijikata, Makoto; Ikeda, Masanori; Dansako, Hiromichi; Wakita, Takaji; Shimotohno, Kunitada; Kato, Nobuyuki

    2013-01-01

    Highlights: ► PML tumor suppressor protein is required for HCV production. ► PML is dispensable for HCV RNA replication. ► HCV could not alter formation of PML-NBs. ► INI1 and DDX5, PML-related proteins, are involved in HCV life cycle. -- Abstract: PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.

  10. Functional requirements of the yellow fever virus capsid protein.

    Science.gov (United States)

    Patkar, Chinmay G; Jones, Christopher T; Chang, Yu-hsuan; Warrier, Ranjit; Kuhn, Richard J

    2007-06-01

    Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.

  11. Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

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    Bo Zhang

    2017-08-01

    Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

  12. Roles of the conserved cytoplasmic region and non-conserved carboxy-terminal region of SecE in Escherichia coli protein translocase.

    Science.gov (United States)

    Kontinen, V P; Yamanaka, M; Nishiyama, K; Tokuda, H

    1996-06-01

    SecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 amino acid residues. Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained. The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues. The conservation of positively charged residues corresponding to Arg80 and Lys81 is also substantial. We deleted or replaced these residues to assess their roles in the SecE function. Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function. Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function. These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function. A chimeric SecE possessing the cytoplasmic region of the E. coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG. Although a Leu to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function. The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE. Based on these results, the SecE function in the translocase is discussed.

  13. Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume

    DEFF Research Database (Denmark)

    Heckmann, Anne Birgitte Lau; Lombardo, Fabien; Miwa, Hiroki

    2006-01-01

    and then increased following infection by M. loti. In hairy root transformations, LjNSP1 and MtNSP1 complemented both Mtnsp1-1 and Ljnsp1-1 mutants, demonstrating that these orthologous proteins have a conserved biochemical function. A Nicotiana benthamiana NSP1-like gene (NbNSP1) was shown to restore nodule...

  14. Peptide-chaperone-directed transdermal protein delivery requires energy.

    Science.gov (United States)

    Ruan, Renquan; Jin, Peipei; Zhang, Li; Wang, Changli; Chen, Chuanjun; Ding, Weiping; Wen, Longping

    2014-11-03

    The biologically inspired transdermal enhanced peptide TD1 has been discovered to specifically facilitate transdermal delivery of biological macromolecules. However, the biological behavior of TD1 has not been fully defined. In this study, we find that energy is required for the TD1-mediated transdermal protein delivery through rat and human skins. Our results show that the permeation activity of TD1-hEGF, a fusion protein composed of human epidermal growth factor (hEGF) and the TD1 sequence connected with a glycine-serine linker (GGGGS), can be inhibited by the energy inhibitor, rotenone or oligomycin. In addition, adenosine triphosphate (ATP), the essential energetic molecule in organic systems, can effectively facilitate the TD1 directed permeation of the protein-based drug into the skin in a dose-dependent fashion. Our results here demonstrate a novel energy-dependent permeation process during the TD1-mediated transdermal protein delivery that could be valuable for the future development of promising new transdermal drugs.

  15. Evolutionary conservation of nuclear and nucleolar targeting sequences in yeast ribosomal protein S6A

    International Nuclear Information System (INIS)

    Lipsius, Edgar; Walter, Korden; Leicher, Torsten; Phlippen, Wolfgang; Bisotti, Marc-Angelo; Kruppa, Joachim

    2005-01-01

    Over 1 billion years ago, the animal kingdom diverged from the fungi. Nevertheless, a high sequence homology of 62% exists between human ribosomal protein S6 and S6A of Saccharomyces cerevisiae. To investigate whether this similarity in primary structure is mirrored in corresponding functional protein domains, the nuclear and nucleolar targeting signals were delineated in yeast S6A and compared to the known human S6 signals. The complete sequence of S6A and cDNA fragments was fused to the 5'-end of the LacZ gene, the constructs were transiently expressed in COS cells, and the subcellular localization of the fusion proteins was detected by indirect immunofluorescence. One bipartite and two monopartite nuclear localization signals as well as two nucleolar binding domains were identified in yeast S6A, which are located at homologous regions in human S6 protein. Remarkably, the number, nature, and position of these targeting signals have been conserved, albeit their amino acid sequences have presumably undergone a process of co-evolution with their corresponding rRNAs

  16. Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

    Science.gov (United States)

    2011-01-01

    Background Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules ("salt-in" strategy) or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy). Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs. Results The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and may help the structure preventing aggregation and/or loss of function in hypersaline environments. Conclusions Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules. PMID:22192175

  17. Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

    Directory of Open Access Journals (Sweden)

    Siglioccolo Alessandro

    2011-12-01

    Full Text Available Abstract Background Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules ("salt-in" strategy or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy. Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs. Results The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and may help the structure preventing aggregation and/or loss of function in hypersaline environments. Conclusions Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules.

  18. Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

    DEFF Research Database (Denmark)

    Kragelund, B B; Poulsen, K; Andersen, K V

    1999-01-01

    In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability...

  19. Origin and spread of photosynthesis based upon conserved sequence features in key bacteriochlorophyll biosynthesis proteins.

    Science.gov (United States)

    Gupta, Radhey S

    2012-11-01

    The origin of photosynthesis and how this capability has spread to other bacterial phyla remain important unresolved questions. I describe here a number of conserved signature indels (CSIs) in key proteins involved in bacteriochlorophyll (Bchl) biosynthesis that provide important insights in these regards. The proteins BchL and BchX, which are essential for Bchl biosynthesis, are derived by gene duplication in a common ancestor of all phototrophs. More ancient gene duplication gave rise to the BchX-BchL proteins and the NifH protein of the nitrogenase complex. The sequence alignment of NifH-BchX-BchL proteins contain two CSIs that are uniquely shared by all NifH and BchX homologs, but not by any BchL homologs. These CSIs and phylogenetic analysis of NifH-BchX-BchL protein sequences strongly suggest that the BchX homologs are ancestral to BchL and that the Bchl-based anoxygenic photosynthesis originated prior to the chlorophyll (Chl)-based photosynthesis in cyanobacteria. Another CSI in the BchX-BchL sequence alignment that is uniquely shared by all BchX homologs and the BchL sequences from Heliobacteriaceae, but absent in all other BchL homologs, suggests that the BchL homologs from Heliobacteriaceae are primitive in comparison to all other photosynthetic lineages. Several other identified CSIs in the BchN homologs are commonly shared by all proteobacterial homologs and a clade consisting of the marine unicellular Cyanobacteria (Clade C). These CSIs in conjunction with the results of phylogenetic analyses and pair-wise sequence similarity on the BchL, BchN, and BchB proteins, where the homologs from Clade C Cyanobacteria and Proteobacteria exhibited close relationship, provide strong evidence that these two groups have incurred lateral gene transfers. Additionally, phylogenetic analyses and several CSIs in the BchL-N-B proteins that are uniquely shared by all Chlorobi and Chloroflexi homologs provide evidence that the genes for these proteins have also been

  20. Requirement of Sequences outside the Conserved Kinase Domain of Fission Yeast Rad3p for Checkpoint Control

    Science.gov (United States)

    Chapman, Carolyn Riley; Evans, Sarah Tyler; Carr, Antony M.; Enoch, Tamar

    1999-01-01

    The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein–protein interactions. PMID:10512862

  1. Deorphanization and target validation of cross-tick species conserved novel Amblyomma americanum tick saliva protein.

    Science.gov (United States)

    Mulenga, Albert; Kim, Tae Kwon; Ibelli, Adriana Mércia Guaratini

    2013-05-01

    We previously identified a cross-tick species conserved tick feeding stimuli responsive Amblyomma americanum (Aam) AV422 gene. This study demonstrates that AamAV422 belongs to a novel group of arthropod proteins that is characterized by 14 cysteine amino acid residues: C(23)-X7/9-C(33)-X23/24-C(58)-X8-C(67)-X7-C(75)-X23-C(99)-X15-C(115)-X10-C(126)-X24/25/33-C(150)C(151)-X7-C(159)-X8-C(168)-X23/24-C(192)-X9/10-C(202) predicted to form seven disulfide bonds. We show that AamAV422 protein is a ubiquitously expressed protein that is injected into the host within the first 24h of the tick attaching onto the host as revealed by Western blotting analyses of recombinant (r)AamAV422, tick saliva and dissected tick organ protein extracts using antibodies to 24 and 48 h tick saliva proteins. Native AamAV422 is apparently involved with mediating tick anti-hemostasis and anti-complement functions in that rAamAV422 delayed plasma clotting time in a dose responsive manner by up to ≈ 160 s, prevented platelet aggregation by up to ≈ 16% and caused ≈ 24% reduction in production of terminal complement complexes. Target validation analysis revealed that rAamAV422 is a potential candidate for a cocktail or multivalent tick vaccine preparation in that RNA interference (RNAi)-mediated silencing of AamAV422 mRNA caused a statistically significant (≈ 44%) reduction in tick engorgement weights, which is proxy for amounts of ingested blood. We speculate that AamAV422 is a potential target antigen for development of the highly desired universal tick vaccine in that consistent with high conservation among ticks, antibodies to 24h Ixodes scapularis tick saliva proteins specifically bound rAamAV422. We discuss data in this study in the context of advancing the biology of tick feeding physiology and discovery of potential target antigens for tick vaccine development. Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  2. A conserved histidine in human DNLZ/HEP is required for stimulation of HSPA9 ATPase activity.

    Science.gov (United States)

    Zhai, Peng; Vu, Michael T; Hoff, Kevin G; Silberg, Jonathan J

    2011-05-20

    The DNL-type zinc-finger protein DNLZ regulates the activity and solubility of the human mitochondrial chaperone HSPA9. To identify DNLZ residues that are critical for chaperone regulation, we carried out an alanine mutagenesis scan of charged residues in a W115I mutant of human DNLZ and assessed the effect of each mutation on interactions with HSPA9. All mutants analyzed promote the solubility of HSPA9 upon expression in Escherichia coli. However, binding studies examining the effect of DNLZ mutants on chaperone tryptophan fluorescence identified three mutations (R81A, H107A, and D111A) that decrease DNLZ binding affinity for nucleotide-free chaperone. In addition, ATPase measurements revealed that DNLZ-R81A and DNLZ-D111A both stimulate the catalytic activity HSPA9, whereas DNLZ-H107A does not elicit an increase in activity even when present at a concentration that is 10-fold higher than the level required for half-maximal stimulation by DNLZ. These findings implicate a conserved histidine as critical for DNLZ regulation of mitochondrial HSPA9 catalytic activity. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

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    Qi Zhai

    Full Text Available The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE based on the expressed sequence tag (EST. The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites. The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

  4. The unfolded protein response is required for dendrite morphogenesis

    Science.gov (United States)

    Wei, Xing; Howell, Audrey S; Dong, Xintong; Taylor, Caitlin A; Cooper, Roshni C; Zhang, Jianqi; Zou, Wei; Sherwood, David R; Shen, Kang

    2015-01-01

    Precise patterning of dendritic fields is essential for the formation and function of neuronal circuits. During development, dendrites acquire their morphology by exuberant branching. How neurons cope with the increased load of protein production required for this rapid growth is poorly understood. Here we show that the physiological unfolded protein response (UPR) is induced in the highly branched Caenorhabditis elegans sensory neuron PVD during dendrite morphogenesis. Perturbation of the IRE1 arm of the UPR pathway causes loss of dendritic branches, a phenotype that can be rescued by overexpression of the ER chaperone HSP-4 (a homolog of mammalian BiP/ grp78). Surprisingly, a single transmembrane leucine-rich repeat protein, DMA-1, plays a major role in the induction of the UPR and the dendritic phenotype in the UPR mutants. These findings reveal a significant role for the physiological UPR in the maintenance of ER homeostasis during morphogenesis of large dendritic arbors. DOI: http://dx.doi.org/10.7554/eLife.06963.001 PMID:26052671

  5. PML tumor suppressor protein is required for HCV production

    Energy Technology Data Exchange (ETDEWEB)

    Kuroki, Misao [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Research Fellow of the Japan Society for the Promotion of Science (Japan); Center for AIDS Research, Kumamoto University, Kumamoto 860-0811 (Japan); Ariumi, Yasuo, E-mail: ariumi@kumamoto-u.ac.jp [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Center for AIDS Research, Kumamoto University, Kumamoto 860-0811 (Japan); Hijikata, Makoto [Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan); Ikeda, Masanori; Dansako, Hiromichi [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Wakita, Takaji [Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640 (Japan); Shimotohno, Kunitada [Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516 (Japan); Kato, Nobuyuki [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer PML tumor suppressor protein is required for HCV production. Black-Right-Pointing-Pointer PML is dispensable for HCV RNA replication. Black-Right-Pointing-Pointer HCV could not alter formation of PML-NBs. Black-Right-Pointing-Pointer INI1 and DDX5, PML-related proteins, are involved in HCV life cycle. -- Abstract: PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.

  6. Protein import into chloroplasts requires a chloroplast ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Pain, D.; Blobel, G.

    1987-05-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the (/sup 35/S)methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H/sup +/, K/sup +/, Na/sup +/, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.

  7. Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

    DEFF Research Database (Denmark)

    Pedersen, Søren W; Moran, Griffin E; Sereikaité, Vita

    2016-01-01

    PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting...... drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys...

  8. Conserved binding of GCAC motifs by MEC-8, couch potato, and the RBPMS protein family

    Science.gov (United States)

    Soufari, Heddy

    2017-01-01

    Precise regulation of mRNA processing, translation, localization, and stability relies on specific interactions with RNA-binding proteins whose biological function and target preference are dictated by their preferred RNA motifs. The RBPMS family of RNA-binding proteins is defined by a conserved RNA recognition motif (RRM) domain found in metazoan RBPMS/Hermes and RBPMS2, Drosophila couch potato, and MEC-8 from Caenorhabditis elegans. In order to determine the parameters of RNA sequence recognition by the RBPMS family, we have first used the N-terminal domain from MEC-8 in binding assays and have demonstrated a preference for two GCAC motifs optimally separated by >6 nucleotides (nt). We have also determined the crystal structure of the dimeric N-terminal RRM domain from MEC-8 in the unbound form, and in complex with an oligonucleotide harboring two copies of the optimal GCAC motif. The atomic details reveal the molecular network that provides specificity to all four bases in the motif, including multiple hydrogen bonds to the initial guanine. Further studies with human RBPMS, as well as Drosophila couch potato, confirm a general preference for this double GCAC motif by other members of the protein family and the presence of this motif in known targets. PMID:28003515

  9. Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase‐like (CUS) proteins that are conserved among land plants

    DEFF Research Database (Denmark)

    Yeats, Trevor H.; Huang, Wenlin; Chatterjee, Subhasish

    2014-01-01

    synthases within the large GDSL superfamily. We demonstrate that members of this ancient and conserved family of cutin synthase‐like (CUS) proteins act as polyester synthases with negligible hydrolytic activity. Moreover, solution‐state NMR analysis indicates that CD1 catalyzes the formation of primarily...... of hydroxyacylglycerol precursors, catalyzed by the GDSL‐motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). Here, we present additional biochemical characterization of CD1 and putative orthologs from Arabidopsis thaliana and the moss Physcomitrella patens, which represent a distinct clade of cutin...... linear cutin oligomeric products in vitro. These results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or cross‐linking, may require additional, as yet unknown, factors....

  10. A conserved function in phosphatidylinositol metabolism for mammalian Vps13 family proteins.

    Directory of Open Access Journals (Sweden)

    Jae-Sook Park

    Full Text Available The Vps13 protein family is highly conserved in eukaryotic cells. In humans, mutations in the gene encoding the family member VPS13A lead to the neurodegenerative disorder chorea-acanthocytosis. In the yeast Saccharomyces cerevisiae, there is just a single version of VPS13, thereby simplifying the task of unraveling its molecular function(s. While VPS13 was originally identified in yeast by its role in vacuolar sorting, recent studies have revealed a completely different function for VPS13 in sporulation, where VPS13 regulates phosphatidylinositol-4-phosphate (PtdIns(4P levels in the prospore membrane. This discovery raises the possibility that the disease phenotype associated with vps13A mutants in humans is due to misregulation of PtdIns(4P in membranes. To determine whether VPS13A affects PtdIns(4P in membranes from mammalian neuronal cells, phosphatidylinositol phosphate pools were compared in PC12 tissue culture cells in the absence or presence of VPS13A. Consistent with the yeast results, the localization of PtdIns(4P is specifically altered in VPS13A knockdown cells while other phosphatidylinositol phosphates appear unaffected. In addition, VPS13A is necessary to prevent the premature degeneration of neurites that develop in response to Nerve Growth Factor. The regulation of PtdIns(4P is therefore a conserved function of the Vps13 family and may play a role in the maintenance of neuronal processes in mammals.

  11. Cross-serotype immunity induced by immunization with a conserved rhinovirus capsid protein.

    Directory of Open Access Journals (Sweden)

    Nicholas Glanville

    Full Text Available Human rhinovirus (RV infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2 capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.

  12. An evolutionary model for protein-coding regions with conserved RNA structure

    DEFF Research Database (Denmark)

    Pedersen, Jakob Skou; Forsberg, Roald; Meyer, Irmtraud Margret

    2004-01-01

    in the RNA structure. The overlap of these fundamental dependencies is sufficient to cause "contagious" context dependencies which cascade across many nucleotide sites. Such large-scale dependencies challenge the use of traditional phylogenetic models in evolutionary inference because they explicitly assume...... components of traditional phylogenetic models. We applied this to a data set of full-genome sequences from the hepatitis C virus where five RNA structures are mapped within the coding region. This allowed us to partition the effects of selection on different structural elements and to test various hypotheses......Here we present a model of nucleotide substitution in protein-coding regions that also encode the formation of conserved RNA structures. In such regions, apparent evolutionary context dependencies exist, both between nucleotides occupying the same codon and between nucleotides forming a base pair...

  13. Identification of an evolutionary conserved SURF-6 domain in a family of nucleolar proteins extending from human to yeast

    International Nuclear Information System (INIS)

    Polzikov, Mikhail; Zatsepina, Olga; Magoulas, Charalambos

    2005-01-01

    The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome. We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast. By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus. Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain. We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity. These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells

  14. Fear memory consolidation in sleep requires protein kinase A.

    Science.gov (United States)

    Cho, Jiyeon; Sypniewski, Krzysztof A; Arai, Shoko; Yamada, Kazuo; Ogawa, Sonoko; Pavlides, Constantine

    2018-05-01

    It is well established that protein kinase A (PKA) is involved in hippocampal dependent memory consolidation. Sleep is also known to play an important role in this process. However, whether sleep-dependent memory consolidation involves PKA activation has not been clearly determined. Using behavioral observation, animals were categorized into sleep and awake groups. We show that intrahippocampal injections of the PKA inhibitor Rp-cAMPs in post-contextual fear conditioning sleep produced a suppression of long-term fear memory, while injections of Rp-cAMPs during an awake state, at a similar time point, had no effect. In contrast, injections of the PKA activator Sp-cAMPs in awake state, rescued sleep deprivation-induced memory impairments. These results suggest that following learning, PKA activation specifically in sleep is required for the consolidation of long-term memory. © 2018 Cho et al.; Published by Cold Spring Harbor Laboratory Press.

  15. SecA is required for membrane targeting of the cell division protein DivIVA in vivo

    Directory of Open Access Journals (Sweden)

    Sven eHalbedel

    2014-02-01

    Full Text Available The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localises to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localisation of DivIVA in these mutants. Surprisingly, DivIVA was delocalised, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localisation depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localisation is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.

  16. Overall requirements for an advanced underground coal extraction system. [environment effects, miner health and safety, production cost, and coal conservation

    Science.gov (United States)

    Goldsmith, M.; Lavin, M. L.

    1980-01-01

    Underground mining systems suitable for coal seams expoitable in the year 2000 are examined with particular relevance to the resources of Central Appalachia. Requirements for such systems may be summarized as follows: (1) production cost; (2)miner safety; (3) miner health; (4) environmental impact; and (5) coal conservation. No significant trade offs between production cost and other performance indices were found.

  17. Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

    Directory of Open Access Journals (Sweden)

    Gareth W Morgan

    2010-02-01

    Full Text Available Vaccinia virus (VACV uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC, a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs. Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD motif, which is conserved in the kinesin-1-binding sequence (KBS of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

  18. The Number, Organization, and Size of Polymorphic Membrane Protein Coding Sequences as well as the Most Conserved Pmp Protein Differ within and across Chlamydia Species.

    Science.gov (United States)

    Van Lent, Sarah; Creasy, Heather Huot; Myers, Garry S A; Vanrompay, Daisy

    2016-01-01

    Variation is a central trait of the polymorphic membrane protein (Pmp) family. The number of pmp coding sequences differs between Chlamydia species, but it is unknown whether the number of pmp coding sequences is constant within a Chlamydia species. The level of conservation of the Pmp proteins has previously only been determined for Chlamydia trachomatis. As different Pmp proteins might be indispensible for the pathogenesis of different Chlamydia species, this study investigated the conservation of Pmp proteins both within and across C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci. The pmp coding sequences were annotated in 16 C. trachomatis, 6 C. pneumoniae, 2 C. abortus, and 16 C. psittaci genomes. The number and organization of polymorphic membrane coding sequences differed within and across the analyzed Chlamydia species. The length of coding sequences of pmpA,pmpB, and pmpH was conserved among all analyzed genomes, while the length of pmpE/F and pmpG, and remarkably also of the subtype pmpD, differed among the analyzed genomes. PmpD, PmpA, PmpH, and PmpA were the most conserved Pmp in C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci, respectively. PmpB was the most conserved Pmp across the 4 analyzed Chlamydia species. © 2016 S. Karger AG, Basel.

  19. Conservation of polypyrimidine tract binding proteins and their putative target RNAs in several storage root crops.

    Science.gov (United States)

    Kondhare, Kirtikumar R; Kumar, Amit; Hannapel, David J; Banerjee, Anjan K

    2018-02-07

    Polypyrimidine-tract binding proteins (PTBs) are ubiquitous RNA-binding proteins in plants and animals that play diverse role in RNA metabolic processes. PTB proteins bind to target RNAs through motifs rich in cytosine/uracil residues to fine-tune transcript metabolism. Among tuber and root crops, potato has been widely studied to understand the mobile signals that activate tuber development. Potato PTBs, designated as StPTB1 and StPTB6, function in a long-distance transport system by binding to specific mRNAs (StBEL5 and POTH1) to stabilize them and facilitate their movement from leaf to stolon, the site of tuber induction, where they activate tuber and root growth. Storage tubers and root crops are important sustenance food crops grown throughout the world. Despite the availability of genome sequence for sweet potato, cassava, carrot and sugar beet, the molecular mechanism of root-derived storage organ development remains completely unexplored. Considering the pivotal role of PTBs and their target RNAs in potato storage organ development, we propose that a similar mechanism may be prevalent in storage root crops as well. Through a bioinformatics survey utilizing available genome databases, we identify the orthologues of potato PTB proteins and two phloem-mobile RNAs, StBEL5 and POTH1, in five storage root crops - sweet potato, cassava, carrot, radish and sugar beet. Like potato, PTB1/6 type proteins from these storage root crops contain four conserved RNA Recognition Motifs (characteristic of RNA-binding PTBs) in their protein sequences. Further, 3´ UTR (untranslated region) analysis of BEL5 and POTH1 orthologues revealed the presence of several cytosine/uracil motifs, similar to those present in potato StBEL5 and POTH1 RNAs. Using RT-qPCR assays, we verified the presence of these related transcripts in leaf and root tissues of these five storage root crops. Similar to potato, BEL5-, PTB1/6- and POTH1-like orthologue RNAs from the aforementioned storage root

  20. Comparative biology of the pentraxin protein family: evolutionarily conserved component of innate immune system.

    Science.gov (United States)

    Armstrong, Peter B

    2015-01-01

    The immune system is based on the actions of the collection of specialized immune defense cells and their secreted proteins and peptides that defend the host against infection by parasites. Parasites are organisms that live part or all of their lives in close physical association with the host and extract nutrients from the host and, by releasing toxins and virulence factors, cause disease with the potential for injury and premature death of that host. Parasites of the metazoa can be viruses, eubacteria, fungi, protozoans, and other metazoans. The immune system operates to kill or eliminate parasites and eliminate or detoxify their toxins and virulence factors. Although some of the elements of immune systems are specific to a particular phylum of metazoans, others show extensive evolutionary conservation, being present in several or all major phyla of the metazoa. The pentraxins display this latter character in their roles in immune defense. Pentraxins have been documented in vertebrates, nonvertebrate chordates, arthropods, and mollusks and may be present in other taxa of metazoans. Presumably the pentraxins appeared early in the evolution of metazoa, prior to their evolutionary divergence in the Precambrian epoch into many phyla present today, and have been preserved for the 542 million years since that explosive evolutionary radiation. The fidelity with which these phyla have preserved the pentraxins suggests that the functions of these proteins are important for survival of the members of these diverse taxa of animals. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A conserved small RNA promotes silencing of the outer membrane protein YbfM

    DEFF Research Database (Denmark)

    Rasmussen, Anders Aamann; Johansen, Jesper; Nielsen, Jesper S

    2009-01-01

    important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the outer membrane protein YbfM is silenced by a conserved sRNA......In the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post-transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one......, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on-rate of duplex...

  2. Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development.

    Science.gov (United States)

    Luo, Ting; Xu, Yanhua; Hoffman, Trevor L; Zhang, Tailin; Schilling, Thomas; Sargent, Thomas D

    2007-04-01

    Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin "purse strings", which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.

  3. Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins.

    Science.gov (United States)

    Flavin, William P; Bousset, Luc; Green, Zachary C; Chu, Yaping; Skarpathiotis, Stratos; Chaney, Michael J; Kordower, Jeffrey H; Melki, Ronald; Campbell, Edward M

    2017-10-01

    Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from WT or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis. The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.

  4. GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-05-01

    Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

  5. Assessing the structural conservation of protein pockets to study functional and allosteric sites: implications for drug discovery

    Directory of Open Access Journals (Sweden)

    Daura Xavier

    2010-03-01

    Full Text Available Abstract Background With the classical, active-site oriented drug-development approach reaching its limits, protein ligand-binding sites in general and allosteric sites in particular are increasingly attracting the interest of medicinal chemists in the search for new types of targets and strategies to drug development. Given that allostery represents one of the most common and powerful means to regulate protein function, the traditional drug discovery approach of targeting active sites can be extended by targeting allosteric or regulatory protein pockets that may allow the discovery of not only novel drug-like inhibitors, but activators as well. The wealth of available protein structural data can be exploited to further increase our understanding of allosterism, which in turn may have therapeutic applications. A first step in this direction is to identify and characterize putative effector sites that may be present in already available structural data. Results We performed a large-scale study of protein cavities as potential allosteric and functional sites, by integrating publicly available information on protein sequences, structures and active sites for more than a thousand protein families. By identifying common pockets across different structures of the same protein family we developed a method to measure the pocket's structural conservation. The method was first parameterized using known active sites. We characterized the predicted pockets in terms of sequence and structural conservation, backbone flexibility and electrostatic potential. Although these different measures do not tend to correlate, their combination is useful in selecting functional and regulatory sites, as a detailed analysis of a handful of protein families shows. We finally estimated the numbers of potential allosteric or regulatory pockets that may be present in the data set, finding that pockets with putative functional and effector characteristics are widespread across

  6. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements.

    Directory of Open Access Journals (Sweden)

    Olga V Viktorovskaya

    2016-08-01

    Full Text Available There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses replication.Seventy-nine novel RNA binding proteins for dengue virus (DENV were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated.The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps

  7. Multi-signal sedimentation velocity analysis with mass conservation for determining the stoichiometry of protein complexes.

    Directory of Open Access Journals (Sweden)

    Chad A Brautigam

    Full Text Available Multi-signal sedimentation velocity analytical ultracentrifugation (MSSV is a powerful tool for the determination of the number, stoichiometry, and hydrodynamic shape of reversible protein complexes in two- and three-component systems. In this method, the evolution of sedimentation profiles of macromolecular mixtures is recorded simultaneously using multiple absorbance and refractive index signals and globally transformed into both spectrally and diffusion-deconvoluted component sedimentation coefficient distributions. For reactions with complex lifetimes comparable to the time-scale of sedimentation, MSSV reveals the number and stoichiometry of co-existing complexes. For systems with short complex lifetimes, MSSV reveals the composition of the reaction boundary of the coupled reaction/migration process, which we show here may be used to directly determine an association constant. A prerequisite for MSSV is that the interacting components are spectrally distinguishable, which may be a result, for example, of extrinsic chromophores or of different abundances of aromatic amino acids contributing to the UV absorbance. For interacting components that are spectrally poorly resolved, here we introduce a method for additional regularization of the spectral deconvolution by exploiting approximate knowledge of the total loading concentrations. While this novel mass conservation principle does not discriminate contributions to different species, it can be effectively combined with constraints in the sedimentation coefficient range of uncomplexed species. We show in theory, computer simulations, and experiment, how mass conservation MSSV as implemented in SEDPHAT can enhance or even substitute for the spectral discrimination of components. This should broaden the applicability of MSSV to the analysis of the composition of reversible macromolecular complexes.

  8. An Investigation into Energy Requirements and Conservation Techniques for Sustainable Buildings

    Science.gov (United States)

    Robitaille, Jad

    Traditionally, societies use to design their built environment in a way that was in line with the climate and the geographical location that they evolved in, thereby supporting sustainable lifestyles (i.e. thick walls with small windows in cold climates). With the industrial revolution and the heavy use and reliance on cheap fossil fuels, it can be argued that the built environment has become more focused on aesthetics and cost savings rather than on true sustainability. This, in turn, has led to energy intensive practices associated with the construction of homes, buildings, cities and megalopolises. Environmental concerns with regards to the future have pushed people, entities and industries to search for ways to decrease human's energy dependency and/or to supply the demand in ways that are deemed sustainable. Efforts to address this concern with respect to the built environment were translated into 'green buildings', sustainable building technologies and high performance buildings that can be rated and/or licensed by selected certifying bodies with varying metrics of building construction and performance. The growing number of such systems has brought real concerns: Do certified sustainable buildings really achieve the level of sustainability (i.e. performance) they were intended to? For the purpose of this study, buildings' energy consumption will be analysed, as it is one of the main drivers when taking into consideration greenhouse gas emissions. Heating and cooling in the residential and commercial/institutional sector, combined account for approximately a fifth of the secondary energy use in Canada. For this reason, this research aims at evaluating the main rating systems in Canada based on the efficacy of their rating systems' certification methodology and the weighting and comparison of energy requirements under each scheme. It has been proven through numerous studies that major energy savings can be achieved by focusing primarily on building designs

  9. Protein kinase A and fungal virulence: a sinister side to a conserved nutrient sensing pathway.

    Science.gov (United States)

    Fuller, Kevin K; Rhodes, Judith C

    2012-01-01

    Diverse fungal species are the cause of devastating agricultural and human diseases. As successful pathogenesis is dependent upon the ability of the fungus to adapt to the nutritional and chemical environment of the host, the understanding of signaling pathways required for such adaptation will provide insights into the virulence of these pathogens and the potential identification of novel targets for antifungal intervention. The cAMP-PKA signaling pathway is well conserved across eukaryotes. In the nonpathogenic yeast, S. cerevisiae, PKA is activated in response to extracellular nutrients and subsequently regulates metabolism and growth. Importantly, this pathway is also a regulator of pathogenesis, as defects in PKA signaling lead to an attenuation of virulence in diverse plant and human pathogenic fungi. This review will compare and contrast PKA signaling in S. cerevisiae vs. various pathogenic species and provide a framework for the role of this pathway in regulating fungal virulence.

  10. Biodiversity data requirements for systematic conservation planning in the Mediterranean Sea

    DEFF Research Database (Denmark)

    Levin, Noam; Coll, Marta; Fraschetti, Simonetta

    2014-01-01

    The Mediterranean Sea’s biodiversity and ecosystems face many threats due to anthropogenic factors. Some of these include high human population growth, coastal urbanization, accelerated human activities and climate change. To enhance the formation of a science-based system of marine protected areas......, the Mediterranean Sea is lagging behind other marine regions where conservation planning adopting rigorous criteria has been applied in the past 20 years. Therefore, we call upon scientists, governments and international governmental and non-governmental conservation organizations to harmonize current approaches...

  11. Comparative Genomics Identifies a Novel Conserved Protein, HpaT, in Proteobacterial Type III Secretion Systems that Do Not Possess the Putative Translocon Protein HrpF

    Directory of Open Access Journals (Sweden)

    Céline Pesce

    2017-06-01

    Full Text Available Xanthomonas translucens is the causal agent of bacterial leaf streak, the most common bacterial disease of wheat and barley. To cause disease, most xanthomonads depend on a highly conserved type III secretion system, which translocates type III effectors into host plant cells. Mutagenesis of the conserved type III secretion gene hrcT confirmed that the X. translucens type III secretion system is required to cause disease on the host plant barley and to trigger a non-host hypersensitive response (HR in pepper leaves. Type III effectors are delivered to the host cell by a surface appendage, the Hrp pilus, and a translocon protein complex that inserts into the plant cell plasma membrane. Homologs of the Xanthomonas HrpF protein, including PopF from Ralstonia solanacearum and NolX from rhizobia, are thought to act as a translocon protein. Comparative genomics revealed that X. translucens strains harbor a noncanonical hrp gene cluster, which rather shares features with type III secretion systems from Ralstonia solanacearum, Paraburkholderia andropogonis, Collimonas fungivorans, and Uliginosibacterium gangwonense than other Xanthomonas spp. Surprisingly, none of these bacteria, except R. solanacearum, encode a homolog of the HrpF translocon. Here, we aimed at identifying a candidate translocon from X. translucens. Notably, genomes from strains that lacked hrpF/popF/nolX instead encode another gene, called hpaT, adjacent to and co-regulated with the type III secretion system gene cluster. An insertional mutant in the X. translucens hpaT gene, which is the first gene of a two-gene operon, hpaT-hpaH, was non-pathogenic on barley and did not cause the HR or programmed cell death in non-host pepper similar to the hrcT mutant. The hpaT mutant phenotypes were partially complemented by either hpaT or the downstream gene, hpaH, which has been described as a facilitator of translocation in Xanthomonas oryzae. Interestingly, the hpaT mutant was also complemented

  12. Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

    Science.gov (United States)

    Shen, Shu; Tobery, Cynthia E; Rose, Mark D

    2009-05-01

    Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

  13. Efficient protein production by yeast requires global tuning of metabolism

    DEFF Research Database (Denmark)

    Huang, Mingtao; Bao, Jichen; Hallstrom, Bjorn M.

    2017-01-01

    The biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous...... intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular...... that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion....

  14. Structural and Sequence Similarities of Hydra Xeroderma Pigmentosum A Protein to Human Homolog Suggest Early Evolution and Conservation

    Directory of Open Access Journals (Sweden)

    Apurva Barve

    2013-01-01

    Full Text Available Xeroderma pigmentosum group A (XPA is a protein that binds to damaged DNA, verifies presence of a lesion, and recruits other proteins of the nucleotide excision repair (NER pathway to the site. Though its homologs from yeast, Drosophila, humans, and so forth are well studied, XPA has not so far been reported from protozoa and lower animal phyla. Hydra is a fresh-water cnidarian with a remarkable capacity for regeneration and apparent lack of organismal ageing. Cnidarians are among the first metazoa with a defined body axis, tissue grade organisation, and nervous system. We report here for the first time presence of XPA gene in hydra. Putative protein sequence of hydra XPA contains nuclear localization signal and bears the zinc-finger motif. It contains two conserved Pfam domains and various characterized features of XPA proteins like regions for binding to excision repair cross-complementing protein-1 (ERCC1 and replication protein A 70 kDa subunit (RPA70 proteins. Hydra XPA shows a high degree of similarity with vertebrate homologs and clusters with deuterostomes in phylogenetic analysis. Homology modelling corroborates the very close similarity between hydra and human XPA. The protein thus most likely functions in hydra in the same manner as in other animals, indicating that it arose early in evolution and has been conserved across animal phyla.

  15. JABAWS 2.2 distributed web services for Bioinformatics: protein disorder, conservation and RNA secondary structure.

    Science.gov (United States)

    Troshin, Peter V; Procter, James B; Sherstnev, Alexander; Barton, Daniel L; Madeira, Fábio; Barton, Geoffrey J

    2018-06-01

    JABAWS 2.2 is a computational framework that simplifies the deployment of web services for Bioinformatics. In addition to the five multiple sequence alignment (MSA) algorithms in JABAWS 1.0, JABAWS 2.2 includes three additional MSA programs (Clustal Omega, MSAprobs, GLprobs), four protein disorder prediction methods (DisEMBL, IUPred, Ronn, GlobPlot), 18 measures of protein conservation as implemented in AACon, and RNA secondary structure prediction by the RNAalifold program. JABAWS 2.2 can be deployed on a variety of in-house or hosted systems. JABAWS 2.2 web services may be accessed from the Jalview multiple sequence analysis workbench (Version 2.8 and later), as well as directly via the JABAWS command line interface (CLI) client. JABAWS 2.2 can be deployed on a local virtual server as a Virtual Appliance (VA) or simply as a Web Application Archive (WAR) for private use. Improvements in JABAWS 2.2 also include simplified installation and a range of utility tools for usage statistics collection, and web services querying and monitoring. The JABAWS CLI client has been updated to support all the new services and allow integration of JABAWS 2.2 services into conventional scripts. A public JABAWS 2 server has been in production since December 2011 and served over 800 000 analyses for users worldwide. JABAWS 2.2 is made freely available under the Apache 2 license and can be obtained from: http://www.compbio.dundee.ac.uk/jabaws. g.j.barton@dundee.ac.uk.

  16. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines.

    Science.gov (United States)

    Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

    2014-01-01

    The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

  17. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

    Directory of Open Access Journals (Sweden)

    Tatjana P Kristensen

    2014-03-01

    Full Text Available The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural

  18. 75 FR 652 - Energy Conservation Program: Certification, Compliance, and Enforcement Requirements for Certain...

    Science.gov (United States)

    2010-01-05

    ...; Comment Request; Certification, Compliance, and Enforcement Requirements for Consumer Products and Certain...: Certification, Compliance, and Enforcement Requirements for Certain Consumer Products and Commercial and... certification, compliance, and enforcement requirements for various consumer products and commercial and...

  19. Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli

    Science.gov (United States)

    Johnson, Brant R.; Hymes, Jeffrey; Sanozky-Dawes, Rosemary; Henriksen, Emily DeCrescenzo

    2015-01-01

    The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa. PMID:26475115

  20. Conserved syntenic clusters of protein coding genes are missing in birds.

    Science.gov (United States)

    Lovell, Peter V; Wirthlin, Morgan; Wilhelm, Larry; Minx, Patrick; Lazar, Nathan H; Carbone, Lucia; Warren, Wesley C; Mello, Claudio V

    2014-01-01

    Birds are one of the most highly successful and diverse groups of vertebrates, having evolved a number of distinct characteristics, including feathers and wings, a sturdy lightweight skeleton and unique respiratory and urinary/excretion systems. However, the genetic basis of these traits is poorly understood. Using comparative genomics based on extensive searches of 60 avian genomes, we have found that birds lack approximately 274 protein coding genes that are present in the genomes of most vertebrate lineages and are for the most part organized in conserved syntenic clusters in non-avian sauropsids and in humans. These genes are located in regions associated with chromosomal rearrangements, and are largely present in crocodiles, suggesting that their loss occurred subsequent to the split of dinosaurs/birds from crocodilians. Many of these genes are associated with lethality in rodents, human genetic disorders, or biological functions targeting various tissues. Functional enrichment analysis combined with orthogroup analysis and paralog searches revealed enrichments that were shared by non-avian species, present only in birds, or shared between all species. Together these results provide a clearer definition of the genetic background of extant birds, extend the findings of previous studies on missing avian genes, and provide clues about molecular events that shaped avian evolution. They also have implications for fields that largely benefit from avian studies, including development, immune system, oncogenesis, and brain function and cognition. With regards to the missing genes, birds can be considered ‘natural knockouts’ that may become invaluable model organisms for several human diseases.

  1. A conserved PHD finger protein and endogenous RNAi modulate insulin signaling in Caenorhabditis elegans.

    Science.gov (United States)

    Mansisidor, Andres R; Cecere, Germano; Hoersch, Sebastian; Jensen, Morten B; Kawli, Trupti; Kennedy, Lisa M; Chavez, Violeta; Tan, Man-Wah; Lieb, Jason D; Grishok, Alla

    2011-09-01

    Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16-dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.

  2. Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

    DEFF Research Database (Denmark)

    Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard

    2008-01-01

    CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild...... irradiation. Several proteins (including GAPDH, heterogeneous nuclear ribonucleoprotein (hnRNP) D and A/B) were identified from the MCF-7 cytoplasmic extracts that bound specifically to the CPEs. CONCLUSION: Two conserved CPEs in the p53 3'UTR regulate stability and translation of a reporter mRNA in non...

  3. A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

    DEFF Research Database (Denmark)

    Wong, W T; Schumacher, C; Salcini, A E

    1995-01-01

    In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge......In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several...... heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic...... (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins....

  4. Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion.

    Science.gov (United States)

    Jiang, Yi; Liu, Haican; Wang, Xuezhi; Li, Guilian; Qiu, Yan; Dou, Xiangfeng; Wan, Kanglin

    2015-01-01

    Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion.

  5. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

    NARCIS (Netherlands)

    Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

    1993-01-01

    An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

  6. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Science.gov (United States)

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  7. Energy and protein requirements for growth of the local domestic ...

    African Journals Online (AJOL)

    The diet 3 in the grower phase improved growth with the highest EFU, 0.12, and was better utilized by the birds than in the chick starter phase where it was least utilized. High energy, high protein diet enhanced growth while low energy, high protein diet did not support maximum growth especially in the chick starter phase.

  8. Optimum dietary protein requirement of genetically male tilapia ...

    African Journals Online (AJOL)

    The study was conducted to investigate the optimum dietary protein level needed for growing genetically male tilapia, Oreochromis niloticus. Diets containing crude protein levels 40, 42.5, 45, 47.5 and 50% were formulated and tried in triplicates. Test diets were fed to 20 fish/1m3 floating hapa at 5% of fish body weight daily ...

  9. Computing wheat nitrogen requirements from grain yield and protein maps

    Science.gov (United States)

    Optical protein sensors and mass-flow yield monitors provide the opportunity to continuously measure grain quality and quantity during harvesting. This chapter illustrates how yield monitor and grain protein measurements may provide useful postharvest information for evaluating water or nitrogen (N)...

  10. Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

    Science.gov (United States)

    Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan

    2017-06-07

    Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.

  11. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Leder, Verena [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Lummer, Martina [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Tegeler, Kathrin [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Humpert, Fabian [Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Lewinski, Martin [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany); Schüttpelz, Mark [Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany); Staiger, Dorothee, E-mail: dorothee.staiger@uni-bielefeld.de [Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany)

    2014-10-10

    Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R{sup 49} abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to binding. - Abstract: Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K{sub d} value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R{sup 49} that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.

  12. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

    International Nuclear Information System (INIS)

    Leder, Verena; Lummer, Martina; Tegeler, Kathrin; Humpert, Fabian; Lewinski, Martin; Schüttpelz, Mark; Staiger, Dorothee

    2014-01-01

    Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R 49 abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to binding. - Abstract: Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K d value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R 49 that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding

  13. Maintenance of muscle myosin levels in adult C. elegans requires both the double bromodomain protein BET-1 and sumoylation

    Directory of Open Access Journals (Sweden)

    Kate Fisher

    2013-10-01

    Attenuation of RAS-mediated signalling is a conserved process essential to control cell proliferation, differentiation, and apoptosis. Cooperative interactions between histone modifications such as acetylation, methylation and sumoylation are crucial for proper attenuation in C. elegans, implying that the proteins recognising these histone modifications could also play an important role in attenuation of RAS-mediated signalling. We sought to systematically identify these proteins and found BET-1. BET-1 is a conserved double bromodomain protein that recognises acetyl-lysines on histone tails and maintains the stable fate of various lineages. Unexpectedly, adults lacking both BET-1 and SUMO-1 are depleted of muscle myosin, an essential component of myofibrils. We also show that this muscle myosin depletion does not occur in all animals at a specific time, but rather that the penetrance of the phenotype increases with age. To gain mechanistic insights into this process, we sought to delay the occurrence of the muscle myosin depletion phenotype and found that it requires caspase activity and MEK-dependent signalling. We also performed transcription profiling on these mutants and found an up-regulation of the FGF receptor, egl-15, a tyrosine kinase receptor acting upstream of MEK. Consistent with a MEK requirement, we could delay the muscle phenotype by systemic or hypodermal knock down of egl-15. Thus, this work uncovered a caspase- and MEK-dependent mechanism that acts specifically on ageing adults to maintain the appropriate net level of muscle myosin.

  14. FuncPatch: a web server for the fast Bayesian inference of conserved functional patches in protein 3D structures.

    Science.gov (United States)

    Huang, Yi-Fei; Golding, G Brian

    2015-02-15

    A number of statistical phylogenetic methods have been developed to infer conserved functional sites or regions in proteins. Many methods, e.g. Rate4Site, apply the standard phylogenetic models to infer site-specific substitution rates and totally ignore the spatial correlation of substitution rates in protein tertiary structures, which may reduce their power to identify conserved functional patches in protein tertiary structures when the sequences used in the analysis are highly similar. The 3D sliding window method has been proposed to infer conserved functional patches in protein tertiary structures, but the window size, which reflects the strength of the spatial correlation, must be predefined and is not inferred from data. We recently developed GP4Rate to solve these problems under the Bayesian framework. Unfortunately, GP4Rate is computationally slow. Here, we present an intuitive web server, FuncPatch, to perform a fast approximate Bayesian inference of conserved functional patches in protein tertiary structures. Both simulations and four case studies based on empirical data suggest that FuncPatch is a good approximation to GP4Rate. However, FuncPatch is orders of magnitudes faster than GP4Rate. In addition, simulations suggest that FuncPatch is potentially a useful tool complementary to Rate4Site, but the 3D sliding window method is less powerful than FuncPatch and Rate4Site. The functional patches predicted by FuncPatch in the four case studies are supported by experimental evidence, which corroborates the usefulness of FuncPatch. The software FuncPatch is freely available at the web site, http://info.mcmaster.ca/yifei/FuncPatch golding@mcmaster.ca Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. GEMC1 is a TopBP1 interacting protein required for chromosomal DNA replication

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-01-01

    Many factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication in complex multicellular organisms is poorly understood. Here, we report the identification of GEMC1, a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus egg extract we show that Xenopus GEMC1 (xGEMC1) binds to checkpoint and replication factor TopBP1, which promotes xGEMC1 binding to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 directly interacts with replication factors such as Cdc45 and Cdk2-CyclinE by which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication whereas depletion of xGEMC1 prevents DNA replication onset due to impairment of Cdc45 loading onto chromatin. Likewise, inhibition of GEMC1 expression by morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent recruitment of Cdc45 onto replication origins. PMID:20383140

  16. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome

    Directory of Open Access Journals (Sweden)

    Böhm Siegfried

    2004-07-01

    Full Text Available Background The classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking. Results We found through in silico analysis 176 zinc finger proteins in Arabidopsis thaliana that hence constitute the most abundant family of putative transcriptional regulators in this plant. Only a minority of 33 A. thaliana zinc finger proteins are conserved in other eukaryotes. In contrast, the majority of these proteins (81% are plant specific. They are derived from extensive duplication events and form expanded families. We assigned the proteins to different subgroups and families and focused specifically on the two largest and evolutionarily youngest families (A1 and C1 that are suggested to be primarily involved in transcriptional regulation. The newly defined family A1 (24 members comprises proteins with tandemly arranged zinc finger domains. Family C1 (64 members, earlier described as the EPF-family in Petunia, comprises proteins with one isolated or two to five dispersed fingers and a mostly invariant QALGGH motif in the zinc finger helices. Based on the amino acid pattern in these helices we could describe five different signature sequences prevalent in C1 zinc finger domains. We also found a number of non-finger domains that are conserved in these families. Conclusions Our analysis of the few evolutionarily conserved zinc finger proteins of A. thaliana suggests that most of them could be involved in ancient biological processes like RNA metabolism and chromatin-remodeling. In contrast, the majority of the unique A. thaliana zinc finger proteins are known or

  17. An ancient conserved role for prion protein in learning and memory

    Directory of Open Access Journals (Sweden)

    Patricia L. A. Leighton

    2018-01-01

    Full Text Available The misfolding of cellular prion protein (PrPC to form PrP Scrapie (PrPSc is an exemplar of toxic gain-of-function mechanisms inducing propagated protein misfolding and progressive devastating neurodegeneration. Despite this, PrPC function in the brain is also reduced and subverted during prion disease progression; thus understanding the normal function of PrPC in healthy brains is key. Disrupting PrPC in mice has led to a myriad of controversial functions that sometimes map onto disease symptoms, including a proposed role in memory or learning. Intriguingly, PrPC interaction with amyloid beta (Aβ oligomers at synapses has also linked its function to Alzheimer's disease and dementia in recent years. We set out to test the involvement of PrPC in memory using a disparate animal model, the zebrafish. Here we document an age-dependent memory decline in prp2−/− zebrafish, pointing to a conserved and ancient role of PrPC in memory. Specifically, we found that aged (3-year-old prp2−/− fish performed poorly in an object recognition task relative to age-matched prp2+/+ fish or 1-year-old prp2−/− fish. Further, using a novel object approach (NOA test, we found that aged (3-year-old prp2−/− fish approached the novel object more than either age-matched prp2+/+ fish or 1-year-old prp2−/− fish, but did not have decreased anxiety when we tested them in a novel tank diving test. Taken together, the results of the NOA and novel tank diving tests suggest an altered cognitive appraisal of the novel object in the 3-year-old prp2−/− fish. The learning paradigm established here enables a path forward to study PrPC interactions of relevance to Alzheimer's disease and prion diseases, and to screen for candidate therapeutics for these diseases. The findings underpin a need to consider the relative contributions of loss- versus gain-of-function of PrPC during Alzheimer's and prion diseases, and have implications upon the prospects of several

  18. Radiotherapy for squamous cell carcinoma of maxillary sinus. Requirements for conservative treatment strategy

    International Nuclear Information System (INIS)

    Onishi, Hiroshi; Komiyama, Takafumi; Marino, Kan; Kuriyama, Kengo; Tanaka, Shiho; Araki, Tsutomu; Sano, Naoki

    2004-01-01

    We reviewed the records of 45 patients with squamous cell carcinoma of maxillary sinus who underwent radiotherapy and intra-arterial chemotherapy with or without maxillectomy. Of 45 patients, 10 patients (stage II:III:IV A=1:5:4) were operated with radical radiotherapy without maxillectomy and had no local failure for more than 1 year (conservative and no local failure group), and 9 patients (stage III:IV A=3:6) were performed with partial or total maxillectomy after pre-operative radiotherapy and had no malignant cells in the operated specimen (group B). Complete response on radiological images was shown in 70% of patients in group A, and 67% of patients in group B. In group A, local recurrence was found in 1 of 10 patients in group A and 1 of 9 patients in group B during a further follow-up period. Five-year overall survival rate was 53% and 67% in group A and B, respectively. Five-year cause-specific survival rate was 89% and 67% in group A and B, respectively. Maxillary conservative treatment is appropriate in cases showing good response on radiological images after radiotherapy combined with concurrent intra-arterial infusion chemotherapy. (author)

  19. Optimization of Power Generation Rights Under the Requirements of Energy Conservation and Emission Reduction

    Science.gov (United States)

    Hu-ping, YANY; Chong-wei, ZHONG; Fei-fei, YAN; Cheng-yi, TANG

    2018-03-01

    In recent years, the energy crisis and greenhouse effect problem have caused wide public concern, if these issues cannot be resolved quickly, they will bring troubles to people’s lives.In response, many countries around the world have implemented policies to reduce energy consumption and greenhouse gas emissions. In our country, the electric power industry has made great contribution to the daily life of people and the development of industry, but it is also an industry of high consumption and high emission.In order to realize the sustainable development of society, it is necessary to make energy conservation and emission reduction in the power industry as an important part of the realization of this goal.In this context, power generation trade has become a hot topic in energy conservation and emission reduction.Through the electricity consumption of the units with different power efficiency and coal consumption rate,it can achieve the target of reducing coal consumption, reducing network loss, reducing greenhouse gas emission, and increasing social benefit,and so on. This article put forward a optimal energy model on the basis of guaranteeing safety and environmental protection.In this paper, they used the IEEE30, IEEE39, IEEE57 and IEEE118 node system as an example, and set up the control groups to prove the practicality of the presented model.The solving method of this model was interior-point method.

  20. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    International Nuclear Information System (INIS)

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-01-01

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

  1. Drosophila Vps13 Is Required for Protein Homeostasis in the Brain

    NARCIS (Netherlands)

    Vonk, Jan J.; Yeshaw, Wondwossen M.; Pinto, Francesco; Faber, Anita I. E.; Lahaye, Liza L.; Kanon, Bart; van der Zwaag, Marianne; Velayos-Baeza, Antonio; Freire, Raimundo; van IJzendoorn, Sven C.; Grzeschik, Nicola A.; Sibon, Ody C. M.

    2017-01-01

    Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences of VPS13A

  2. Sequence analysis of the L protein of the Ebola 2014 outbreak: Insight into conserved regions and mutations.

    Science.gov (United States)

    Ayub, Gohar; Waheed, Yasir

    2016-06-01

    The 2014 Ebola outbreak was one of the largest that have occurred; it started in Guinea and spread to Nigeria, Liberia and Sierra Leone. Phylogenetic analysis of the current virus species indicated that this outbreak is the result of a divergent lineage of the Zaire ebolavirus. The L protein of Ebola virus (EBOV) is the catalytic subunit of the RNA‑dependent RNA polymerase complex, which, with VP35, is key for the replication and transcription of viral RNA. Earlier sequence analysis demonstrated that the L protein of all non‑segmented negative‑sense (NNS) RNA viruses consists of six domains containing conserved functional motifs. The aim of the present study was to analyze the presence of these motifs in 2014 EBOV isolates, highlight their function and how they may contribute to the overall pathogenicity of the isolates. For this purpose, 81 2014 EBOV L protein sequences were aligned with 475 other NNS RNA viruses, including Paramyxoviridae and Rhabdoviridae viruses. Phylogenetic analysis of all EBOV outbreak L protein sequences was also performed. Analysis of the amino acid substitutions in the 2014 EBOV outbreak was conducted using sequence analysis. The alignment demonstrated the presence of previously conserved motifs in the 2014 EBOV isolates and novel residues. Notably, all the mutations identified in the 2014 EBOV isolates were tolerant, they were pathogenic with certain examples occurring within previously determined functional conserved motifs, possibly altering viral pathogenicity, replication and virulence. The phylogenetic analysis demonstrated that all sequences with the exception of the 2014 EBOV sequences were clustered together. The 2014 EBOV outbreak has acquired a great number of mutations, which may explain the reasons behind this unprecedented outbreak. Certain residues critical to the function of the polymerase remain conserved and may be targets for the development of antiviral therapeutic agents.

  3. Utilization of conserved Lucerne Hay as a Protein Supplement in the Diet of Calves on Smallholder Farms of Kenya

    International Nuclear Information System (INIS)

    Kiragu, J.W.; Tamminga, S.; Mitaru, B.N.

    1999-01-01

    The nutritive value and the conserved lucerne hay as a protein supplement in the diet of calves reared on the smallholder farms of Kenya was studied in relation to feed intake, growth rate and efficiency of feed utilization. Forty Friesian calves aged one week were allocate in a completely randomised design experiment to the following five dietary treatments: Napier Grass only (N) and supplemented with 33% lucerne (NL33), 50% lucerne (NL50), 67% lucerne (NL67) and 100% lucerne hay (L100) in the diet of calves. Results showed that increasing the level of lucerne supplementation in the ratio of dairy calves from 50 to 100% lucerne hay significantly (P <0.01) increased the crude protein content the mixed ration from 13.8 t o 16.88% CP which would meet the protein, requirement of the growing ruminants. The growth rate of calves increased linearly (P < 0.001) with the levels of lucerne supplements fro 50 to 100 % lucerne hay resulting in 0.02 kg gain more growth per day per kg additional feed supplement given. The cost of production per kg gain decreased with increase in the level of lucerne supplementation and cost of could further reduced by farmers growing lucerne on the smallholder farms for use in compounding home-made ration at the farm level. Result of these study further shows that lucerne as a leguminous forage has ability to fix Nitrogen in the soil which has significantly effect on soil fertility, increased crop yields and reduction in the cost of fertilisers. It is recommended that an acre of lucerne could yield five to seven tons of dry matter per year sufficient to rear a herd of 15 to 20 calves for a period of 1 year. This research has further shown that a pure stand of lucerne crop which is very deep rooted crop has a longevity of five to seven years in the same field and that pest and diseases are not of a serious problem to lucerne, thus making it excellent supplementary legume to the Napier grass in the diet of calves on smallholder farms of Kenya

  4. Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 Regulates Xylem Development and Growth by a Conserved Mechanism That Modulates Hormone Signaling1[W][OPEN

    Science.gov (United States)

    Grienenberger, Etienne; Douglas, Carl J.

    2014-01-01

    Despite a strict conservation of the vascular tissues in vascular plants (tracheophytes), our understanding of the genetic basis underlying the differentiation of secondary cell wall-containing cells in the xylem of tracheophytes is still far from complete. Using coexpression analysis and phylogenetic conservation across sequenced tracheophyte genomes, we identified a number of Arabidopsis (Arabidopsis thaliana) genes of unknown function whose expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 (VUP1) gene encodes a predicted protein of 24 kD with no annotated functional domains but containing domains that are highly conserved in tracheophytes. Here, we show that the VUP1 expression pattern, determined by promoter-β-glucuronidase reporter gene expression, is associated with vascular tissues, while vup1 loss-of-function mutants exhibit collapsed morphology of xylem vessel cells. Constitutive overexpression of VUP1 caused dramatic and pleiotropic developmental defects, including severe dwarfism, dark green leaves, reduced apical dominance, and altered photomorphogenesis, resembling brassinosteroid-deficient mutants. Constitutive overexpression of VUP homologs from multiple tracheophyte species induced similar defects. Whole-genome transcriptome analysis revealed that overexpression of VUP1 represses the expression of many brassinosteroid- and auxin-responsive genes. Additionally, deletion constructs and site-directed mutagenesis were used to identify critical domains and amino acids required for VUP1 function. Altogether, our data suggest a conserved role for VUP1 in regulating secondary wall formation during vascular development by tissue- or cell-specific modulation of hormone signaling pathways. PMID:24567189

  5. Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

    Science.gov (United States)

    Růžička, Kamil; Zhang, Mi; Campilho, Ana; Bodi, Zsuzsanna; Kashif, Muhammad; Saleh, Mária; Eeckhout, Dominique; El-Showk, Sedeer; Li, Hongying; Zhong, Silin; De Jaeger, Geert; Mongan, Nigel P; Hejátko, Jan; Helariutta, Ykä; Fray, Rupert G

    2017-07-01

    N6-adenosine methylation (m 6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m 6 A writer proteins in Arabidopsis thaliana. The components required for m 6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m 6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m 6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m 6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  6. Drosophila Vps13 Is Required for Protein Homeostasis in the Brain.

    Directory of Open Access Journals (Sweden)

    Jan J Vonk

    Full Text Available Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis.

  7. Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation

    International Nuclear Information System (INIS)

    Ambrose, R.L.; Mackenzie, J.M.

    2015-01-01

    The West Nile virus strain Kunjin virus (WNV KUN ) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNV KUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNV KUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein

  8. 77 FR 38266 - Sea Turtle Conservation; Shrimp Trawling Requirements; Public Hearing

    Science.gov (United States)

    2012-06-27

    ... withdraw the alternative tow time restriction and require all skimmer trawls, pusher-head trawls, and wing nets (butterfly trawls) rigged for fishing to use turtle excluder devices (TEDs) in their nets, which...

  9. RNA Processing Factor 5 is required for efficient 5' cleavage at a processing site conserved in RNAs of three different mitochondrial genes in Arabidopsis thaliana.

    Science.gov (United States)

    Hauler, Aron; Jonietz, Christian; Stoll, Birgit; Stoll, Katrin; Braun, Hans-Peter; Binder, Stefan

    2013-05-01

    The 5' ends of many mitochondrial transcripts are generated post-transcriptionally. Recently, we identified three RNA PROCESSING FACTORs required for 5' end maturation of different mitochondrial mRNAs in Arabidopsis thaliana. All of these factors are pentatricopeptide repeat proteins (PPRPs), highly similar to RESTORERs OF FERTILTY (RF), that rescue male fertility in cytoplasmic male-sterile lines from different species. Therefore, we suggested a general role of these RF-like PPRPs in mitochondrial 5' processing. We now identified RNA PROCESSING FACTOR 5, a PPRP not classified as an RF-like protein, required for the efficient 5' maturation of the nad6 and atp9 mRNAs as well as 26S rRNA. The precursor molecules of these RNAs share conserved sequence elements, approximately ranging from positions -50 to +9 relative to mature 5' mRNA termini, suggesting these sequences to be at least part of the cis elements required for processing. The knockout of RPF5 has only a moderate influence on 5' processing of atp9 mRNA, whereas the generation of the mature nad6 mRNA and 26S rRNA is almost completely abolished in the mutant. The latter leads to a 50% decrease of total 26S rRNA species, resulting in an imbalance between the large rRNA and 18S rRNA. Despite these severe changes in RNA levels and in the proportion between the 26S and 18S rRNAs, mitochondrial protein levels appear to be unaltered in the mutant, whereas seed germination capacity is markedly reduced. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  10. Fragile X Mental Retardation Protein Is Required to Maintain Visual Conditioning-Induced Behavioral Plasticity by Limiting Local Protein Synthesis.

    Science.gov (United States)

    Liu, Han-Hsuan; Cline, Hollis T

    2016-07-06

    Fragile X mental retardation protein (FMRP) is thought to regulate neuronal plasticity by limiting dendritic protein synthesis, but direct demonstration of a requirement for FMRP control of local protein synthesis during behavioral plasticity is lacking. Here we tested whether FMRP knockdown in Xenopus optic tectum affects local protein synthesis in vivo and whether FMRP knockdown affects protein synthesis-dependent visual avoidance behavioral plasticity. We tagged newly synthesized proteins by incorporation of the noncanonical amino acid azidohomoalanine and visualized them with fluorescent noncanonical amino acid tagging (FUNCAT). Visual conditioning and FMRP knockdown produce similar increases in FUNCAT in tectal neuropil. Induction of visual conditioning-dependent behavioral plasticity occurs normally in FMRP knockdown animals, but plasticity degrades over 24 h. These results indicate that FMRP affects visual conditioning-induced local protein synthesis and is required to maintain the visual conditioning-induced behavioral plasticity. Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Exaggerated dendritic protein synthesis resulting from loss of fragile X mental retardation protein (FMRP) is thought to underlie cognitive deficits in FXS, but no direct evidence has demonstrated that FMRP-regulated dendritic protein synthesis affects behavioral plasticity in intact animals. Xenopus tadpoles exhibit a visual avoidance behavior that improves with visual conditioning in a protein synthesis-dependent manner. We showed that FMRP knockdown and visual conditioning dramatically increase protein synthesis in neuronal processes. Furthermore, induction of visual conditioning-dependent behavioral plasticity occurs normally after FMRP knockdown, but performance rapidly deteriorated in the absence of FMRP. These studies show that FMRP negatively regulates local protein synthesis and is required to maintain visual conditioning

  11. Temporal requirements of the fragile X mental retardation protein in modulating circadian clock circuit synaptic architecture

    Directory of Open Access Journals (Sweden)

    Cheryl L Gatto

    2009-08-01

    Full Text Available Loss of fragile X mental retardation 1 (FMR1 gene function is the most common cause of inherited mental retardation and autism spectrum disorders, characterized by attention disorder, hyperactivity and disruption of circadian activity cycles. Pursuit of effective intervention strategies requires determining when the FMR1 product (FMRP is required in the regulation of neuronal circuitry controlling these behaviors. In the well-characterized Drosophila disease model, loss of the highly conserved dFMRP causes circadian arrhythmicity and conspicuous abnormalities in the circadian clock circuitry. Here, a novel Sholl Analysis was used to quantify over-elaborated synaptic architecture in dfmr1-null small ventrolateral neurons (sLNvs, a key subset of clock neurons. The transgenic Gene-Switch system was employed to drive conditional neuronal dFMRP expression in the dfmr1-null mutant background in order to dissect temporal requirements within the clock circuit. Introduction of dFMRP during early brain development, including the stages of neurogenesis, neuronal fate specification and early pathfinding, provided no rescue of dfmr1 mutant phenotypes. Similarly, restoring normal dFMRP expression in the adult failed to restore circadian circuit architecture. In sharp contrast, supplying dFMRP during a transient window of very late brain development, wherein synaptogenesis and substantial subsequent synaptic reorganization (e.g. use-dependent pruning occur, provided strong morphological rescue to reestablish normal sLNvs synaptic arbors. We conclude that dFMRP plays a developmentally restricted role in sculpting synaptic architecture in these neurons that cannot be compensated for by later reintroduction of the protein at maturity.

  12. Requirement for a conserved, tertiary interaction in the core of 23S ribosomal RNA

    DEFF Research Database (Denmark)

    Aagaard, C; Douthwaite, S

    1994-01-01

    RNA. Every substitution that disrupts the potential for Watson-Crick base pairing between these positions reduces or abolishes the participation of 23S rRNA in protein synthesis. All mutant 23S rRNAs are assembled into 50S subunits, but the mutant subunits are less able to stably interact with 30S subunits...... is nonfunctional. In contrast to the considerable effect the mutations have on function, they impart only slight structural changes on the naked rRNA, and these are limited to the immediate vicinity of the mutations. The data show that positions 1262 and 2017 pair in a Watson-Crick manner, but the data also...

  13. Contemporary Issues in Protein Requirements and Consumption for Resistance Trained Athletes

    Directory of Open Access Journals (Sweden)

    Wilson Jacob

    2006-06-01

    Full Text Available Abstract In recent years an explosion of research papers concerning protein consumption has been published. The need to consolidate this information has become critical from both practical and future research standpoints. For this reason, the following paper presents an in depth analysis of contemporary issues in protein requirements and consumption for resistance trained athletes. Specifically, the paper covers: 1. protein requirements for resistance trained athletes; 2. the effect of the digestion rate of protein on muscular protein balance; 3. the optimal timing of protein intake relative to exercise; 4. the optimal pattern of protein ingestion, relative to how an individual should consume their protein throughout a 24 hour period, and what sources are utilized during this time frame; 5. protein composition and its interaction with measures of protein balance and strength performance; 6. the combination of protein and carbohydrates on plasma insulin levels and protein balance; 7. the efficacy of protein supplements and whole food protein sources. Our goal is to provide the reader with practical information in optimizing protein intake as well as for provision of sound advice to their clients. Finally, special care was taken to provide future research implications.

  14. Flotillin-1 is an evolutionary-conserved memory-related protein up-regulated in implicit and explicit learning paradigms.

    Science.gov (United States)

    Monje, Francisco J; Divisch, Isabella; Demit, Marvie; Lubec, Gert; Pollak, Daniela D

    2013-06-01

    Studies of synaptic plasticity using the marine mollusk Aplysia californica as model system have been successfully used to identify proteins involved in learning and memory. The importance of molecular elements regulated by the learning- related neurotransmitter serotonin in Aplysia can then be explored in rodent models and finally tested for their relevance for human physiology and pathology. Herein, 2-DE gel-based electrophoresis has been used to investigate protein level changes after treatment with serotonin in Aplysia abdominal ganglia. Twenty-one proteins have been found to be regulated by serotonin, and protein level changes of actin depolymerizing factor (ADF), deleted in azoospermia associated protein (DAZAP-1), and Flotillin-1 have been verified by Western blotting. Flotillin-1, a member of the flotillin/reggie family of scaffolding proteins, has been previously found to be involved in neuritic branching and synapse formation in hippocampal neurons in vitro. However, its importance for hippocampal- dependent learning and memory in the mouse has not been examined. Here, elevated levels of Flotillin-1 in hippocampal tissue of mice trained in the Morris water maze confirmed the relevance of Flotillin-1 for memory-related processes in a mammalian system. Thus, a translational approach-from invertebrates to rodents-led to the identification of Flotillin-1 as evolutionary-conserved memory-related protein.

  15. Functions and requirements of conserved RNA structures in the 3’ untranslated region of Flaviviruses

    NARCIS (Netherlands)

    Agostinho Gonçalves Costa da Silva, Patrícia

    2011-01-01

    The Flavivirus genus contains nearly 80 viruses, including many important human pathogens such as dengue virus, yellow fever virus, West Nile virus and tick-borne encephalitis virus. To reduce and prevent the impact of flavivirus infection on society, vaccines and effective therapies are required.

  16. Assessing common birds' ecological requirements to address nature conservation in permanent crops: Lessons from Italian vineyards.

    Science.gov (United States)

    Assandri, Giacomo; Bogliani, Giuseppe; Pedrini, Paolo; Brambilla, Mattia

    2017-04-15

    Viticulture has contributed to shaping cultural landscapes in several regions across all continents. Recent farming intensification is causing landscape homogenization and biodiversity loss in several of those areas, but knowledge about the impacts on biodiversity in vineyards is still scarce. Simplified agro-ecosystems resulting from intensification host mainly generalist and common species, which still play a key role in the regulation of ecosystems and in the provision of ecosystem services. We assessed the abundance of 11 common bird species at 47 linear transects in a vineyard-dominated landscape in Trentino (NE Italy), in both spring and winter, and analysed abundance variation in relation to three independent groups of predictors: landscape, management, and topographic-climatic variables. In the majority of species (7), abundance was primarily or considerably affected by landscape attributes. However, an additional 5 species were largely affected by management practices, often with conspicuous seasonal differences. Overall, landscape and management heterogeneity positively affected the abundance of 6 species. Vineyard cover (and in particular the new spalliera trellising system) was negatively related with the abundance of 6 species, with the strongest impacts occurring in winter. On the contrary, the cover of marginal habitats had major positive effects over 8 species. Hedgerows, tree rows, and dry stone walls, as well as traditional pergola vineyards and landscape and management heterogeneity should be conserved or restored in viticultural landscapes to promote the abundance of common bird species. This strategy would ensure the maintenance of the ecosystem services they provide, while promoting the general sustainability of the agroecosystem. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Bactericidal activity of M protein conserved region antibodies against group A streptococcal isolates from the Northern Thai population

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    Pruksachatkunakorn Chulabhorn

    2006-08-01

    Full Text Available Abstract Background Most group A streptococcal (GAS vaccine strategies have focused on the surface M protein, a major virulence factor of GAS. The amino-terminus of the M protein elicits antibodies, that are both opsonic and protective, but which are type specific. J14, a chimeric peptide that contains 14 amino acids from the M protein conserved C-region at the carboxy-terminus, offers the possibility of a vaccine which will elicit protective opsonic antibodies against multiple different GAS strains. In this study, we searched for J14 and J14-like sequences and the number of their repeats in the C-region of the M protein from GAS strains isolated from the Northern Thai population. Then, we examined the bactericidal activity of J14, J14.1, J14-R1 and J14-R2 antisera against multiple Thai GAS strains. Results The emm genes of GAS isolates were sequenced and grouped as 14 different J14-types. The most diversity of J14-types was found in the C1-repeat. The J14.1 type was the major sequence in the C2 and C3-repeats. We have shown that antisera raised against the M protein conserved C-repeat region peptides, J14, J14.1, J14-R1 and J14-R2, commonly found in GAS isolates from the Northern Thai population, are able to kill GAS of multiple different emm types derived from an endemic area. The mean percent of bactericidal activities for all J14 and J14-like peptide antisera against GAS isolates were more than 70%. The mean percent of bactericidal activity was highest for J14 antisera followed by J14-R2, J14.1 and J14-R1 antisera. Conclusion Our study demonstrated that antisera raised against the M protein conserved C-repeat region are able to kill multiple different strains of GAS isolated from the Northern Thai population. Therefore, the four conserved "J14" peptides have the potential to be used as GAS vaccine candidates to prevent streptococcal infections in an endemic area.

  18. Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila

    Directory of Open Access Journals (Sweden)

    Aditya Sen

    2015-06-01

    Full Text Available Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD. However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1 and Parkin (Park, either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu, a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.

  19. Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins

    OpenAIRE

    Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

    2015-01-01

    Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their func...

  20. Energie- en eiwitbehoefte van biologisch gehouden pluimvee = Energy and protein requirements of organic housed poultry

    NARCIS (Netherlands)

    Knegsel, van A.T.M.; Krimpen, van M.M.

    2008-01-01

    In this literature review, the physiological basis for possible differences in energy and protein requirements of organic versus conventional poultry is investigated. Energy need for maintenance of organic housed poultry seems to be increased, whereas protein requirements might not differ between

  1. DisoMCS: Accurately Predicting Protein Intrinsically Disordered Regions Using a Multi-Class Conservative Score Approach.

    Directory of Open Access Journals (Sweden)

    Zhiheng Wang

    Full Text Available The precise prediction of protein intrinsically disordered regions, which play a crucial role in biological procedures, is a necessary prerequisite to further the understanding of the principles and mechanisms of protein function. Here, we propose a novel predictor, DisoMCS, which is a more accurate predictor of protein intrinsically disordered regions. The DisoMCS bases on an original multi-class conservative score (MCS obtained by sequence-order/disorder alignment. Initially, near-disorder regions are defined on fragments located at both the terminus of an ordered region connecting a disordered region. Then the multi-class conservative score is generated by sequence alignment against a known structure database and represented as order, near-disorder and disorder conservative scores. The MCS of each amino acid has three elements: order, near-disorder and disorder profiles. Finally, the MCS is exploited as features to identify disordered regions in sequences. DisoMCS utilizes a non-redundant data set as the training set, MCS and predicted secondary structure as features, and a conditional random field as the classification algorithm. In predicted near-disorder regions a residue is determined as an order or a disorder according to the optimized decision threshold. DisoMCS was evaluated by cross-validation, large-scale prediction, independent tests and CASP (Critical Assessment of Techniques for Protein Structure Prediction tests. All results confirmed that DisoMCS was very competitive in terms of accuracy of prediction when compared with well-established publicly available disordered region predictors. It also indicated our approach was more accurate when a query has higher homologous with the knowledge database.The DisoMCS is available at http://cal.tongji.edu.cn/disorder/.

  2. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

    Science.gov (United States)

    Yin, Yanting; de Waal, Parker W.; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H. Eric

    2017-01-01

    The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. PMID:28356352

  3. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

    Science.gov (United States)

    Yin, Yanting; de Waal, Parker W; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H Eric

    2017-06-16

    The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β 2 -adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Detecting remote sequence homology in disordered proteins: discovery of conserved motifs in the N-termini of Mononegavirales phosphoproteins.

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    David Karlin

    Full Text Available Paramyxovirinae are a large group of viruses that includes measles virus and parainfluenza viruses. The viral Phosphoprotein (P plays a central role in viral replication. It is composed of a highly variable, disordered N-terminus and a conserved C-terminus. A second viral protein alternatively expressed, the V protein, also contains the N-terminus of P, fused to a zinc finger. We suspected that, despite their high variability, the N-termini of P/V might all be homologous; however, using standard approaches, we could previously identify sequence conservation only in some Paramyxovirinae. We now compared the N-termini using sensitive sequence similarity search programs, able to detect residual similarities unnoticeable by conventional approaches. We discovered that all Paramyxovirinae share a short sequence motif in their first 40 amino acids, which we called soyuz1. Despite its short length (11-16aa, several arguments allow us to conclude that soyuz1 probably evolved by homologous descent, unlike linear motifs. Conservation across such evolutionary distances suggests that soyuz1 plays a crucial role and experimental data suggest that it binds the viral nucleoprotein to prevent its illegitimate self-assembly. In some Paramyxovirinae, the N-terminus of P/V contains a second motif, soyuz2, which might play a role in blocking interferon signaling. Finally, we discovered that the P of related Mononegavirales contain similarly overlooked motifs in their N-termini, and that their C-termini share a previously unnoticed structural similarity suggesting a common origin. Our results suggest several testable hypotheses regarding the replication of Mononegavirales and suggest that disordered regions with little overall sequence similarity, common in viral and eukaryotic proteins, might contain currently overlooked motifs (intermediate in length between linear motifs and disordered domains that could be detected simply by comparing orthologous proteins.

  5. Conservative, unconditionally stable discretization methods for Hamiltonian equations, applied to wave motion in lattice equations modeling protein molecules

    Science.gov (United States)

    LeMesurier, Brenton

    2012-01-01

    A new approach is described for generating exactly energy-momentum conserving time discretizations for a wide class of Hamiltonian systems of DEs with quadratic momenta, including mechanical systems with central forces; it is well-suited in particular to the large systems that arise in both spatial discretizations of nonlinear wave equations and lattice equations such as the Davydov System modeling energetic pulse propagation in protein molecules. The method is unconditionally stable, making it well-suited to equations of broadly “Discrete NLS form”, including many arising in nonlinear optics. Key features of the resulting discretizations are exact conservation of both the Hamiltonian and quadratic conserved quantities related to continuous linear symmetries, preservation of time reversal symmetry, unconditional stability, and respecting the linearity of certain terms. The last feature allows a simple, efficient iterative solution of the resulting nonlinear algebraic systems that retain unconditional stability, avoiding the need for full Newton-type solvers. One distinction from earlier work on conservative discretizations is a new and more straightforward nearly canonical procedure for constructing the discretizations, based on a “discrete gradient calculus with product rule” that mimics the essential properties of partial derivatives. This numerical method is then used to study the Davydov system, revealing that previously conjectured continuum limit approximations by NLS do not hold, but that sech-like pulses related to NLS solitons can nevertheless sometimes arise.

  6. Habitat requirements of endangered species in a former coppice of high conservation value

    Czech Academy of Sciences Publication Activity Database

    Roleček, Jan; Vild, Ondřej; Sladký, J.; Řepka, R.

    2017-01-01

    Roč. 52, č. 1 (2017), s. 59-69 ISSN 1211-9520 R&D Projects: GA MŠk(CZ) EE2.3.20.0267 EU Projects: European Commission(XE) 278065 - LONGWOOD Institutional support: RVO:67985939 Keywords : abandoned coppice * environmental requirements * red-list species Subject RIV: EH - Ecology, Behaviour OBOR OECD: Plant sciences, botany Impact factor: 1.017, year: 2016

  7. Information requirements of young women with breast cancer treated with mastectomy or breast conserving surgery: A systematic review.

    Science.gov (United States)

    Recio-Saucedo, Alejandra; Gerty, Sue; Foster, Claire; Eccles, Diana; Cutress, Ramsey I

    2016-02-01

    Young women with breast cancer have poorer prognosis, greater lifetime risk of local recurrence, contralateral recurrence, and distant disease, regardless of surgery received. Here we systematically review published evidence relating to the information requirements and preferences of young women diagnosed with early-stage breast cancer offered a choice between mastectomy and Breast Conservation Surgery (BCS). Findings will inform the development of a surgical decision aid for young women. Eight databases were searched to identify research examining information requirements of young women facing breast oncological surgery treatment decisions (MESH headings). Twelve studies met the inclusion criteria. Data were extracted and summarised in a narrative synthesis. Findings indicate that young women prefer greater and more detailed information regarding treatment side effects, sexuality, and body image. Younger age of diagnosis leads to an increased risk perception of developing a second breast cancer. Young women's choices are influenced by factors associated with family and career. Information is required in a continuum throughout the treatment experience and not only at diagnosis when treatment decisions are made. Young women show differing levels of participation preferences. Young women find decision-making challenging when the characteristics of diagnosis provide a surgical choice between mastectomy and breast conservation surgery. Efforts should be made to provide information regarding sexuality, body image, reconstruction, fertility and likelihood of familial predisposition. Further research is needed to identify the specific level and information requirements of this young-onset group. The low number of studies indicate a need to design studies targeting specifically this age group of breast cancer patients. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Recording Information on Architectural Heritage Should Meet the Requirements for Conservation Digital Recording Practices at the Summer Palace

    Science.gov (United States)

    Zhang, L.; Cong, Y.; Wu, C.; Bai, C.; Wu, C.

    2017-08-01

    The recording of Architectural heritage information is the foundation of research, conservation, management, and the display of architectural heritage. In other words, the recording of architectural heritage information supports heritage research, conservation, management and architectural heritage display. What information do we record and collect and what technology do we use for information recording? How do we determine the level of accuracy required when recording architectural information? What method do we use for information recording? These questions should be addressed in relation to the nature of the particular heritage site and the specific conditions for the conservation work. In recent years, with the rapid development of information acquisition technology such as Close Range Photogrammetry, 3D Laser Scanning as well as high speed and high precision Aerial Photogrammetry, many Chinese universities, research institutes and heritage management bureaux have purchased considerable equipment for information recording. However, the lack of understanding of both the nature of architectural heritage and the purpose for which the information is being collected has led to several problems. For example: some institutions when recording architectural heritage information aim solely at high accuracy. Some consider that advanced measuring methods must automatically replace traditional measuring methods. Information collection becomes the purpose, rather than the means, of architectural heritage conservation. Addressing these issues, this paper briefly reviews the history of architectural heritage information recording at the Summer Palace (Yihe Yuan, first built in 1750), Beijing. Using the recording practices at the Summer Palace during the past ten years as examples, we illustrate our achievements and lessons in recording architectural heritage information with regard to the following aspects: (buildings') ideal status desired, (buildings') current status

  9. A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies.

    Directory of Open Access Journals (Sweden)

    Cécile Fourrage

    Full Text Available Poc1 (Protein of Centriole 1 proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.

  10. Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

    Science.gov (United States)

    Jacobs, Susan C; Taylor, Adam; Herrero, Lara J; Mahalingam, Suresh; Fazakerley, John K

    2017-10-20

    Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

  11. Preparation, crystallization and preliminary X-ray characterization of a conserved hypothetical protein XC1692 from Xanthomonas campestris

    International Nuclear Information System (INIS)

    Chin, Ko-Hsin; Huang, Zhao-Wei; Wei, Kun-Chou; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Gao, Fei Philip; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-01-01

    A conserved hypothetical protein XC1692 from X. campestris pv. campestris has been overexpressed in E. coli. The purified recombinant protein crystallized in a variety of forms and diffracted to a resolution of at least 1.45 Å. Xanthomonas campestris pv. campestris strain 17 is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, one third of which have no known structure and/or function yet are highly conserved among several different bacterial genuses. One of these gene products is XC1692 protein, containing 141 amino acids. It was overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. The crystals diffract to at least 1.45 Å resolution. They are hexagonal and belong to space group P6 3 , with unit-cell parameters a = b = 56.9, c = 71.0 Å. They contain one molecule per asymmetric unit

  12. Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Ji Qiongmei; Huang, C.-H.; Peng Jianbin; Hashmi, Sarwar; Ye Tianzhang; Chen Ying

    2007-01-01

    We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans

  13. Phylogeny and molecular signatures (conserved proteins and indels that are specific for the Bacteroidetes and Chlorobi species

    Directory of Open Access Journals (Sweden)

    Lorenzini Emily

    2007-05-01

    Full Text Available Abstract Background The Bacteroidetes and Chlorobi species constitute two main groups of the Bacteria that are closely related in phylogenetic trees. The Bacteroidetes species are widely distributed and include many important periodontal pathogens. In contrast, all Chlorobi are anoxygenic obligate photoautotrophs. Very few (or no biochemical or molecular characteristics are known that are distinctive characteristics of these bacteria, or are commonly shared by them. Results Systematic blast searches were performed on each open reading frame in the genomes of Porphyromonas gingivalis W83, Bacteroides fragilis YCH46, B. thetaiotaomicron VPI-5482, Gramella forsetii KT0803, Chlorobium luteolum (formerly Pelodictyon luteolum DSM 273 and Chlorobaculum tepidum (formerly Chlorobium tepidum TLS to search for proteins that are uniquely present in either all or certain subgroups of Bacteroidetes and Chlorobi. These studies have identified > 600 proteins for which homologues are not found in other organisms. This includes 27 and 51 proteins that are specific for most of the sequenced Bacteroidetes and Chlorobi genomes, respectively; 52 and 38 proteins that are limited to species from the Bacteroidales and Flavobacteriales orders, respectively, and 5 proteins that are common to species from these two orders; 185 proteins that are specific for the Bacteroides genus. Additionally, 6 proteins that are uniquely shared by species from the Bacteroidetes and Chlorobi phyla (one of them also present in the Fibrobacteres have also been identified. This work also describes two large conserved inserts in DNA polymerase III (DnaE and alanyl-tRNA synthetase that are distinctive characteristics of the Chlorobi species and a 3 aa deletion in ClpB chaperone that is mainly found in various Bacteroidales, Flavobacteriales and Flexebacteraceae, but generally not found in the homologs from other organisms. Phylogenetic analyses of the Bacteroidetes and Chlorobi species is also

  14. Relief memory consolidation requires protein synthesis within the nucleus accumbens.

    Science.gov (United States)

    Bruning, Johann E A; Breitfeld, Tino; Kahl, Evelyn; Bergado-Acosta, Jorge R; Fendt, Markus

    2016-06-01

    Relief learning refers to the association of a stimulus with the relief from an aversive event. The thus-learned relief stimulus then can induce, e.g., an attenuation of the startle response or approach behavior, indicating positive valence. Previous studies revealed that the nucleus accumbens is essential for the acquisition and retrieval of relief memory. Here, we ask whether the nucleus accumbens is also the brain site for consolidation of relief memory into a long-term form. In rats, we blocked local protein synthesis within the nucleus accumbens by local infusions of anisomycin at different time points during a relief conditioning experiment. Accumbal anisomycin injections immediately after the relief conditioning session, but not 4 h later, prevented the consolidation into long-term relief memory. The retention of already consolidated relief memory was not affected by anisomycin injections. This identifies a time window and site for relief memory consolidation. These findings should complement our understanding of the full range of effects of adverse experiences, including cases of their distortion in humans such as post-traumatic stress disorder and/or phobias. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

    Science.gov (United States)

    Kemmer, Danielle; Podowski, Raf M; Arenillas, David; Lim, Jonathan; Hodges, Emily; Roth, Peggy; Sonnhammer, Erik LL; Höög, Christer; Wasserman, Wyeth W

    2006-01-01

    Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families. PMID:16533400

  16. NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

    Directory of Open Access Journals (Sweden)

    Sonnhammer Erik LL

    2006-03-01

    Full Text Available Abstract Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families.

  17. On stability and monotonicity requirements of finite difference approximations of stochastic conservation laws with random viscosity

    KAUST Repository

    Pettersson, Per

    2013-05-01

    The stochastic Galerkin and collocation methods are used to solve an advection-diffusion equation with uncertain and spatially varying viscosity. We investigate well-posedness, monotonicity and stability for the extended system resulting from the Galerkin projection of the advection-diffusion equation onto the stochastic basis functions. High-order summation-by-parts operators and weak imposition of boundary conditions are used to prove stability of the semi-discrete system.It is essential that the eigenvalues of the resulting viscosity matrix of the stochastic Galerkin system are positive and we investigate conditions for this to hold. When the viscosity matrix is diagonalizable, stochastic Galerkin and stochastic collocation are similar in terms of computational cost, and for some cases the accuracy is higher for stochastic Galerkin provided that monotonicity requirements are met. We also investigate the total spatial operator of the semi-discretized system and its impact on the convergence to steady-state. © 2013 Elsevier B.V.

  18. On stability and monotonicity requirements of finite difference approximations of stochastic conservation laws with random viscosity

    KAUST Repository

    Pettersson, Per; Doostan, Alireza; Nordströ m, Jan

    2013-01-01

    The stochastic Galerkin and collocation methods are used to solve an advection-diffusion equation with uncertain and spatially varying viscosity. We investigate well-posedness, monotonicity and stability for the extended system resulting from the Galerkin projection of the advection-diffusion equation onto the stochastic basis functions. High-order summation-by-parts operators and weak imposition of boundary conditions are used to prove stability of the semi-discrete system.It is essential that the eigenvalues of the resulting viscosity matrix of the stochastic Galerkin system are positive and we investigate conditions for this to hold. When the viscosity matrix is diagonalizable, stochastic Galerkin and stochastic collocation are similar in terms of computational cost, and for some cases the accuracy is higher for stochastic Galerkin provided that monotonicity requirements are met. We also investigate the total spatial operator of the semi-discretized system and its impact on the convergence to steady-state. © 2013 Elsevier B.V.

  19. Oligomerisation status and evolutionary conservation of interfaces of protein structural domain superfamilies.

    Science.gov (United States)

    Sukhwal, Anshul; Sowdhamini, Ramanathan

    2013-07-01

    Protein-protein interactions are important in carrying out many biological processes and functions. These interactions may be either permanent or of temporary nature. Several studies have employed tools like solvent accessibility and graph theory to identify these interactions, but still more studies need to be performed to quantify and validate them. Although we now have many databases available with predicted and experimental results on protein-protein interactions, we still do not have many databases which focus on providing structural details of the interacting complexes, their oligomerisation state and homologues. In this work, protein-protein interactions have been thoroughly investigated within the structural regime and quantified for their strength using calculated pseudoenergies. The PPCheck server, an in-house webserver, has been used for calculating the pseudoenergies like van der Waals, hydrogen bonds and electrostatic energy based on distances between atoms of amino acids from two interacting proteins. PPCheck can be visited at . Based on statistical data, as obtained by studying established protein-protein interacting complexes from earlier studies, we came to a conclusion that an average protein-protein interface consisted of about 51 to 150 amino acid residues and the generalized energy per residue ranged from -2 kJ mol(-1) to -6 kJ mol(-1). We found that some of the proteins have an exceptionally higher number of amino acids at the interface and it was purely because of their elaborate interface or extended topology i.e. some of their secondary structure regions or loops were either inter-mixing or running parallel to one another or they were taking part in domain swapping. Residue networks were prepared for all the amino acids of the interacting proteins involved in different types of interactions (like van der Waals, hydrogen-bonding, electrostatic or intramolecular interactions) and were analysed between the query domain-interacting partner pair

  20. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    International Nuclear Information System (INIS)

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F.

    2009-01-01

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  1. Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

    Directory of Open Access Journals (Sweden)

    Guan-Feng Wang

    2015-02-01

    Full Text Available Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR. Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC, a nucleotide binding (NB-ARC and a leucine rich repeat (LRR domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

  2. Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

    Science.gov (United States)

    Wang, Guan-Feng; Ji, Jiabing; El-Kasmi, Farid; Dangl, Jeffery L; Johal, Guri; Balint-Kurti, Peter J

    2015-02-01

    Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

  3. Studies on the protein and sulfur amino acid requirements of young bobwhite quail

    Science.gov (United States)

    Serafin, J.A.

    1977-01-01

    Four experiments were conducted with purified diets to examine the influence of protein level and to estimate the sulfur amino acid (S.A.A.) requirement of young Bobwhite quail (Colinus virginianus). These studies demonstrated (I) that 26% protein was sufficient for rapid growth when the diet was supplemented with methionine; (2) that diets containing higher levels of protein (29.3% and 31.3%) failed to support satisfactory growth unless they contained supplemental methionine; and (3) that young Bobwhite quail require no more than 1.0% sulfur-containing amino acids for optimal growth and efficiency of feed utilization. A fifth experiment was conducted to examine the protein and S.A.A. requirements of young Bobwhite quail using practical rations and to compare results with those obtained with purified diets. Diets containing 24%, 26% and 28% protein were supplied with and without supplemental methionine in a five week study. Results showed significant growth responses to protein and supplemental methionine. Responses showed that Bobwhite quail require no more than 26% protein for maximum growth and efficiency of feed utilization when the S.A.A. level of the diet was approximately 1.0%. The results were in close agreement with those obtained with purified diets. These findings define more precisely than had been known the quantitative requirements of young Bobwhite quail for protein and for the S.A.A. necessary for optimal growth.

  4. A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Justin D. Fellows

    2017-06-01

    Full Text Available Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium. Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL, an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.

  5. LRPPRC is a mitochondrial matrix protein that is conserved in metazoans

    International Nuclear Information System (INIS)

    Sterky, Fredrik H.; Ruzzenente, Benedetta; Gustafsson, Claes M.; Samuelsson, Tore; Larsson, Nils-Goeran

    2010-01-01

    Research highlights: → LRPPRC orthologs are restricted to metazoans. → LRPPRC is imported to the mitochondrial matrix. → No evidence of nuclear isoform. -- Abstract: LRPPRC (also called LRP130) is an RNA-binding pentatricopeptide repeat protein. LRPPRC has been recognized as a mitochondrial protein, but has also been shown to regulate nuclear gene transcription and to bind specific RNA molecules in both the nucleus and the cytoplasm. We here present a bioinformatic analysis of the LRPPRC primary sequence, which reveals that orthologs to the LRPPRC gene are restricted to metazoan cells and that all of the corresponding proteins contain mitochondrial targeting signals. To address the subcellular localization further, we have carefully analyzed LRPPRC in mammalian cells and identified a single isoform that is exclusively localized to mitochondria. The LRPPRC protein is imported to the mitochondrial matrix and its mitochondrial targeting sequence is cleaved upon entry.

  6. Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

    KAUST Repository

    Skinner, John J.

    2017-12-05

    Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or “theft” mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

  7. Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

    KAUST Repository

    Skinner, John J.; Wang, Sheng; Lee, Jiyoung; Ong, Colin; Sommese, Ruth; Sivaramakrishnan, Sivaraj; Koelmel, Wolfgang; Hirschbeck, Maria; Schindelin, Hermann; Kisker, Caroline; Lorenz, Kristina; Sosnick, Tobin R.; Rosner, Marsha Rich

    2017-01-01

    Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or “theft” mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

  8. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

    Science.gov (United States)

    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

    1996-07-19

    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  9. Protein level affects the relative lysine requirement of growing rainbow trout (Oncorhynchus mykiss) fry.

    Science.gov (United States)

    Bodin, Noelie; Govaerts, Bernadette; Abboudi, Tarik; Detavernier, Christel; De Saeger, Sarah; Larondelle, Yvan; Rollin, Xavier

    2009-07-01

    The effect of two digestible protein levels (310 and 469 g/kg DM) on the relative lysine (Lys; g Lys/kg DM or g Lys/100 g protein) and the absolute Lys (g Lys intake/kg 0.75 per d) requirements was studied in rainbow trout fry using a dose-response trial. At each protein level, sixteen isoenergetic (22-23 MJ digestible energy/kg DM) diets were tested, involving a full range (2-70 g/kg DM) of sixteen Lys levels. Each diet was given to one group of sixty rainbow trout fry (mean initial body weight 0.78 g) reared at 15 degrees C for 31 feeding d. The Lys requirements were estimated based on the relationships between weight, protein, and Lys gains (g/kg 0.75 per d) and Lys concentration (g/kg DM or g/100 g protein) or Lys intake (g/kg 0.75 per d), using the broken-line model (BLM) and the non-linear four-parameter saturation kinetics model (SKM-4). Both the model and the response criterion chosen markedly impacted the relative Lys requirement. The relative Lys requirement for Lys gain of rainbow trout estimated with the BLM (and SKM-4 at 90 % of the maximum response) increased from 16.8 (19.6) g/kg DM at a low protein level to 23.4 (24.5) g/kg DM at a high protein level. However, the dietary protein content affected neither the absolute Lys requirement nor the relative Lys requirement expressed as g Lys/100 g protein nor the Lys requirement for maintenance (21 mg Lys/kg 0.75 per d).

  10. Context Memory Formation Requires Activity-Dependent Protein Degradation in the Hippocampus

    Science.gov (United States)

    Cullen, Patrick K.; Ferrara, Nicole C.; Pullins, Shane E.; Helmstetter, Fred J.

    2017-01-01

    Numerous studies have indicated that the consolidation of contextual fear memories supported by an aversive outcome like footshock requires de novo protein synthesis as well as protein degradation mediated by the ubiquitin-proteasome system (UPS). Context memory formed in the absence of an aversive stimulus by simple exposure to a novel…

  11. The formation of a native-like structure containing eight conserved hydrophobic residues is rate limiting in two-state protein folding of ACBP

    DEFF Research Database (Denmark)

    Kragelund, Birthe Brandt; Osmark, Peter; Neergaard, Thomas B.

    1999-01-01

    The acyl-coenzyme A-binding proteins (ACBPs) contain 26 highly conserved sequence positions. The majority of these have been mutated in the bovine protein, and their influence on the rate of two-state folding and unfolding has been measured. The results identify eight sequence positions, out of 24...... probed, that are critical for fast productive folding. The residues are all hydrophobic and located in the interface between the N- and C-terminal helices. The results suggest that one specific site dominated by conserved hydrophobic residues forms the structure of the productive rate-determining folding...... step and that a sequential framework model can describe the protein folding reaction....

  12. Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

    Science.gov (United States)

    Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G

    2015-06-01

    The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. © 2015 Wiley Periodicals, Inc.

  13. Structure-based functional annotation of putative conserved proteins having lyase activity from Haemophilus influenzae.

    Science.gov (United States)

    Shahbaaz, Mohd; Ahmad, Faizan; Imtaiyaz Hassan, Md

    2015-06-01

    Haemophilus influenzae is a small pleomorphic Gram-negative bacteria which causes several chronic diseases, including bacteremia, meningitis, cellulitis, epiglottitis, septic arthritis, pneumonia, and empyema. Here we extensively analyzed the sequenced genome of H. influenzae strain Rd KW20 using protein family databases, protein structure prediction, pathways and genome context methods to assign a precise function to proteins whose functions are unknown. These proteins are termed as hypothetical proteins (HPs), for which no experimental information is available. Function prediction of these proteins would surely be supportive to precisely understand the biochemical pathways and mechanism of pathogenesis of Haemophilus influenzae. During the extensive analysis of H. influenzae genome, we found the presence of eight HPs showing lyase activity. Subsequently, we modeled and analyzed three-dimensional structure of all these HPs to determine their functions more precisely. We found these HPs possess cystathionine-β-synthase, cyclase, carboxymuconolactone decarboxylase, pseudouridine synthase A and C, D-tagatose-1,6-bisphosphate aldolase and aminodeoxychorismate lyase-like features, indicating their corresponding functions in the H. influenzae. Lyases are actively involved in the regulation of biosynthesis of various hormones, metabolic pathways, signal transduction, and DNA repair. Lyases are also considered as a key player for various biological processes. These enzymes are critically essential for the survival and pathogenesis of H. influenzae and, therefore, these enzymes may be considered as a potential target for structure-based rational drug design. Our structure-function relationship analysis will be useful to search and design potential lead molecules based on the structure of these lyases, for drug design and discovery.

  14. Acyl-CoA binding proteins; structural and functional conservation over 2000 MYA

    DEFF Research Database (Denmark)

    Faergeman, Nils J; Wadum, Majken; Feddersen, Søren

    2007-01-01

    -CoA binding protein, ACBP, has been proposed to play a pivotal role in the intracellular trafficking and utilization of long-chain fatty acyl-CoA esters. Depletion of acyl-CoA binding protein in yeast results in aberrant organelle morphology incl. fragmented vacuoles, multi-layered plasma membranes...... and accumulation of vesicles of variable sizes. In contrast to synthesis and turn-over of glycerolipids, the levels of very-long-chain fatty acids, long-chain bases and ceramide are severely affected by Acb1p depletion, suggesting that Acb1p, rather than playing a general role, serves specific roles in cellular...

  15. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundance of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.

  16. FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

    Directory of Open Access Journals (Sweden)

    Cobos Everardo

    2005-01-01

    Full Text Available Abstract Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20 with three members (FAM20A, FAM20B and FAM20C in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating

  17. A Conserved EAR Motif Is Required for Avirulence and Stability of the Ralstonia solanacearum Effector PopP2 In Planta

    Directory of Open Access Journals (Sweden)

    Cécile Segonzac

    2017-08-01

    Full Text Available Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease in many high value Solanaceae crops. R. solanacearum secretes around 70 effectors into host cells in order to promote infection. Plants have, however, evolved specialized immune receptors that recognize corresponding effectors and confer qualitative disease resistance. In the model species Arabidopsis thaliana, the paired immune receptors RRS1 (resistance to Ralstonia solanacearum 1 and RPS4 (resistance to Pseudomonas syringae 4 cooperatively recognize the R. solanacearum effector PopP2 in the nuclei of infected cells. PopP2 is an acetyltransferase that binds to and acetylates the RRS1 WRKY DNA-binding domain resulting in reduced RRS1-DNA association thereby activating plant immunity. Here, we surveyed the naturally occurring variation in PopP2 sequence among the R. solanacearum strains isolated from diseased tomato and pepper fields across the Republic of Korea. Our analysis revealed high conservation of popP2 sequence with only three polymorphic alleles present amongst 17 strains. Only one variation (a premature stop codon caused the loss of RPS4/RRS1-dependent recognition in Arabidopsis. We also found that PopP2 harbors a putative eukaryotic transcriptional repressor motif (ethylene-responsive element binding factor-associated amphiphilic repression or EAR, which is known to be involved in the recruitment of transcriptional co-repressors. Remarkably, mutation of the EAR motif disabled PopP2 avirulence function as measured by the development of hypersensitive response, electrolyte leakage, defense marker gene expression and bacterial growth in Arabidopsis. This lack of recognition was partially but significantly reverted by the C-terminal addition of a synthetic EAR motif. We show that the EAR motif-dependent gain of avirulence correlated with the stability of the PopP2 protein. Furthermore, we demonstrated the requirement of the PopP2 EAR motif for PTI

  18. The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Assia Hijazi

    Full Text Available BACKGROUND: Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. METHODOLOGY/PRINCIPAL FINDINGS: In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. CONCLUSION/SIGNIFICANCE: We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.

  19. The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila.

    Science.gov (United States)

    Hijazi, Assia; Haenlin, Marc; Waltzer, Lucas; Roch, Fernando

    2011-03-15

    Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.

  20. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera

    International Nuclear Information System (INIS)

    Aris, J.P.; Blobel, G.

    1991-01-01

    The authors have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is ∼1,300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain ∼75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis

  1. The MCM-binding protein ETG1 aids sister chromatid cohesion required for postreplicative homologous recombination repair.

    Directory of Open Access Journals (Sweden)

    Naoki Takahashi

    2010-01-01

    Full Text Available The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

  2. The conjugation-specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena.

    Science.gov (United States)

    Matsuda, Atsushi; Shieh, Annie Wan-Yi; Chalker, Douglas L; Forney, James D

    2010-07-01

    Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena DeltaDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway.

  3. The Conjugation-Specific Die5 Protein Is Required for Development of the Somatic Nucleus in both Paramecium and Tetrahymena▿

    Science.gov (United States)

    Matsuda, Atsushi; Shieh, Annie Wan-Yi; Chalker, Douglas L.; Forney, James D.

    2010-01-01

    Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena ΔDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway. PMID:20495055

  4. NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

    Science.gov (United States)

    de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

    2013-01-01

    NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1–33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species. PMID:23516598

  5. Presynaptic protein synthesis required for NT-3-induced long-term synaptic modulation

    Directory of Open Access Journals (Sweden)

    Je H

    2011-01-01

    Full Text Available Abstract Background Neurotrophins elicit both acute and long-term modulation of synaptic transmission and plasticity. Previously, we demonstrated that the long-term synaptic modulation requires the endocytosis of neurotrophin-receptor complex, the activation of PI3K and Akt, and mTOR mediated protein synthesis. However, it is unclear whether the long-term synaptic modulation by neurotrophins depends on protein synthesis in pre- or post-synaptic cells. Results Here we have developed an inducible protein translation blocker, in which the kinase domain of protein kinase R (PKR is fused with bacterial gyrase B domain (GyrB-PKR, which could be dimerized upon treatment with a cell permeable drug, coumermycin. By genetically targeting GyrB-PKR to specific cell types, we show that NT-3 induced long-term synaptic modulation requires presynaptic, but not postsynaptic protein synthesis. Conclusions Our results provide mechanistic insights into the cell-specific requirement for protein synthesis in the long-term synaptic modulation by neurotrophins. The GyrB-PKR system may be useful tool to study protein synthesis in a cell-specific manner.

  6. Outer Membrane Protein A Conservation among Orientia tsutsugamushi Isolates Suggests Its Potential as a Protective Antigen and Diagnostic Target

    Directory of Open Access Journals (Sweden)

    Sean M. Evans

    2018-06-01

    Full Text Available Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium Orientia tsutsugamushi. A vaccine that affords heterologous protection and a commercially-available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA are highly conserved among 51 O. tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of the phylogenetically-related pathogen, Anaplasma phagocytophilum, including the location of a helix that contains residues functionally essential for A. phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. tsutsugamushi strains, but not from negative control bacteria. Using these primers in quantitative PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from organs and blood of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly-protective scrub typhus vaccine.

  7. Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope.

    Science.gov (United States)

    Zhang, Limeng; Zhang, Hua; Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Zhu, Zhanbo; Cui, Yudong

    2015-02-01

    Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

    Directory of Open Access Journals (Sweden)

    José I. Labella

    2017-07-01

    Full Text Available Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essential PLP-holoenzymes or synthetic lethality with cysK. In addition, the related phenotypes of pipY and pipX mutants are consistent with genetic interactions in the contexts of survival to PLP-targeting antibiotics and transcriptional regulation. We also showed that PipY overexpression increased the length of S. elongatus cells. Taken together, our results support a universal regulatory role for COG0325 proteins, paving the way to a better understanding of these proteins and of their connections with other biological processes.

  9. Spike protein assembly into the coronavirion: exploring the limits of its sequence requirements

    International Nuclear Information System (INIS)

    Bosch, Berend Jan; Haan, Cornelis A.M. de; Smits, Saskia L.; Rottier, Peter J.M.

    2005-01-01

    The coronavirus spike (S) protein, required for receptor binding and membrane fusion, is incorporated into the assembling virion by interactions with the viral membrane (M) protein. Earlier we showed that the ectodomain of the S protein is not involved in this process. Here we further defined the requirements of the S protein for virion incorporation. We show that the cytoplasmic domain, not the transmembrane domain, determines the association with the M protein and suffices to effect the incorporation into viral particles of chimeric spikes as well as of foreign viral glycoproteins. The essential sequence was mapped to the membrane-proximal region of the cytoplasmic domain, which is also known to be of critical importance for the fusion function of the S protein. Consistently, only short C-terminal truncations of the S protein were tolerated when introduced into the virus by targeted recombination. The important role of the about 38-residues cytoplasmic domain in the assembly of and membrane fusion by this approximately 1300 amino acids long protein is discussed

  10. Strategic study on energy-protein requirements for local sheep: 5. Ewes during lactation phase

    Directory of Open Access Journals (Sweden)

    I-W Mathius

    2004-03-01

    Full Text Available Thirty-six Javanese thin-tail ewes in the end of late pregnancy phase were set out to study the energy and crude protein requirements during the first eight-week of lactation phase. The ewes were penned individually in doors and randomly assigned to a 3 x 3 factorial arrangement, consisting of three levels of energy (low, medium and high and three levels of crude protein (low, medium and high diets with four ewes per treatment. The diets were pelleted and offered four times daily in approximately equal amount. Feed intake, nutrient digestibility, body weight and milk production were recorded. Results showed that, total lamb birth weights was not affected, but protein content on the ration treatments significantly altered (P0.05, while crude protein content on the ration highly significantly affected (P<0.01. Based on data recorded, the energy and protein requirements for ewes during lactation phase are highly significantly depended on ewes’ live weight, milk production and the ratio of energy metabolism and crude protein of the ration. It was concluded that in order to fulfil the crude protein and energy needs of the ewes during lactation phase, the ration given should contain crude protein and energy as much as 16% (based on dry matter and 13.4 MJ/kg dry matter respectively.

  11. 75 FR 35338 - Implementation of Regulations Required Under Title XI of the Food, Conservation and Energy Act of...

    Science.gov (United States)

    2010-06-22

    ... Under Title XI of the Food, Conservation and Energy Act of 2008; Conduct in Violation of the Act AGENCY... fairer market place. DATES: We will consider comments we receive by August 23, 2010. ADDRESSES: We invite... Title XI of the Food, Conservation and Energy Act of 2008 (Farm Bill) (Pub. L. 110-246), Congress...

  12. On the interconnection of stable protein complexes: inter-complex hubs and their conservation in Saccharomyces cerevisiae and Homo sapiens networks.

    Science.gov (United States)

    Guerra, Concettina

    2015-01-01

    Protein complexes are key molecular entities that perform a variety of essential cellular functions. The connectivity of proteins within a complex has been widely investigated with both experimental and computational techniques. We developed a computational approach to identify and characterise proteins that play a role in interconnecting complexes. We computed a measure of inter-complex centrality, the crossroad index, based on disjoint paths connecting proteins in distinct complexes and identified inter-complex hubs as proteins with a high value of the crossroad index. We applied the approach to a set of stable complexes in Saccharomyces cerevisiae and in Homo sapiens. Just as done for hubs, we evaluated the topological and biological properties of inter-complex hubs addressing the following questions. Do inter-complex hubs tend to be evolutionary conserved? What is the relation between crossroad index and essentiality? We found a good correlation between inter-complex hubs and both evolutionary conservation and essentiality.

  13. Prone Accelerated Partial Breast Irradiation After Breast-Conserving Surgery: Compliance to the Dosimetry Requirements of RTOG-0413

    Energy Technology Data Exchange (ETDEWEB)

    Wen Bixiu [Department of Radiation Oncology, New York University Medical Center, New York, New York (United States); Department of Radiation Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Hsu, Howard; Formenti-Ujlaki, George F.; Lymberis, Stella; Magnolfi, Chiara; Zhao Xuan; Chang Jenghwa; DeWyngaert, J. Keith; Jozsef, Gabor [Department of Radiation Oncology, New York University Medical Center, New York, New York (United States); Formenti, Silvia C., E-mail: silvia.formenti@nyumc.org [Department of Radiation Oncology, New York University Medical Center, New York, New York (United States)

    2012-11-15

    Purpose: The dosimetric results from our institution's trials of prone accelerated partial breast irradiation are compared with the dosimetric requirements of RTOG-0413. Methods and Materials: Trial 1 and Trial 2 are 2 consecutive trials of prone-accelerated partial breast irradiation. Eligible for both trials were stage I breast cancer patients with negative margins after breast-conserving surgery. The planning target tumor volume (PTV) was created by extending the surgical cavity 2.0 cm for Trial 1 and 1.5 cm for Trial 2, respectively. Contralateral breast, heart, lungs, and thyroid were contoured. Thirty Gray was delivered in five daily fractions of 6 Gy by a three-dimensional conformal radiation therapy technique in Trial 1 and were by image-guided radiation therapy/intensity-modulated radiation therapy in Trial 2. Dosimetric results from the trials are reported and compared with RTOG 0413 requirements. Results: One hundred forty-six consecutive plans were analyzed: 67 left and 79 right breast cancers. The plans from the trials complied with the required >90% of prescribed dose covering 90% of PTV{sub E}VAL (=generated from the PTV by cropping 0.5 cm from the skin edge and excluding the chest wall): V90% was 98.1 {+-} 3.0% (with V100% and V95%, 89.4 {+-} 12.8%, 96.4 {+-} 5.1%, respectively). No significant difference between laterality was found (Student's t test). The dose constraints criteria of the RTOG-0413 protocol for ipsilateral and contralateral lung (V30 <15% and Dmax <3%), heart (V5 <40%), and thyroid (Dmax <3%) were satisfied because the plans showed an average V5% of 0.6% (range, 0-13.4) for heart, an average V30% of 0.6% (range, 0-9.1%) for ipsilateral lung, and <2% maximum dose to the thyroid. However, our partial breast irradiation plans demonstrated a higher dose to contralateral breast than that defined by RTOG constraints, with a median value of maximum doses of 4.1% (1.2 Gy), possibly as a result of contouring differences

  14. Expression and genomic analysis of midasin, a novel and highly conserved AAA protein distantly related to dynein

    Directory of Open Access Journals (Sweden)

    Gibbons I R

    2002-07-01

    Full Text Available Abstract Background The largest open reading frame in the Saccharomyces genome encodes midasin (MDN1p, YLR106p, an AAA ATPase of 560 kDa that is essential for cell viability. Orthologs of midasin have been identified in the genome projects for Drosophila, Arabidopsis, and Schizosaccharomyces pombe. Results Midasin is present as a single-copy gene encoding a well-conserved protein of ~600 kDa in all eukaryotes for which data are available. In humans, the gene maps to 6q15 and encodes a predicted protein of 5596 residues (632 kDa. Sequence alignments of midasin from humans, yeast, Giardia and Encephalitozoon indicate that its domain structure comprises an N-terminal domain (35 kDa, followed by an AAA domain containing six tandem AAA protomers (~30 kDa each, a linker domain (260 kDa, an acidic domain (~70 kDa containing 35–40% aspartate and glutamate, and a carboxy-terminal M-domain (30 kDa that possesses MIDAS sequence motifs and is homologous to the I-domain of integrins. Expression of hemagglutamin-tagged midasin in yeast demonstrates a polypeptide of the anticipated size that is localized principally in the nucleus. Conclusions The highly conserved structure of midasin in eukaryotes, taken in conjunction with its nuclear localization in yeast, suggests that midasin may function as a nuclear chaperone and be involved in the assembly/disassembly of macromolecular complexes in the nucleus. The AAA domain of midasin is evolutionarily related to that of dynein, but it appears to lack a microtubule-binding site.

  15. Feature-Based Classification of Amino Acid Substitutions outside Conserved Functional Protein Domains

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    Branislava Gemovic

    2013-01-01

    Full Text Available There are more than 500 amino acid substitutions in each human genome, and bioinformatics tools irreplaceably contribute to determination of their functional effects. We have developed feature-based algorithm for the detection of mutations outside conserved functional domains (CFDs and compared its classification efficacy with the most commonly used phylogeny-based tools, PolyPhen-2 and SIFT. The new algorithm is based on the informational spectrum method (ISM, a feature-based technique, and statistical analysis. Our dataset contained neutral polymorphisms and mutations associated with myeloid malignancies from epigenetic regulators ASXL1, DNMT3A, EZH2, and TET2. PolyPhen-2 and SIFT had significantly lower accuracies in predicting the effects of amino acid substitutions outside CFDs than expected, with especially low sensitivity. On the other hand, only ISM algorithm showed statistically significant classification of these sequences. It outperformed PolyPhen-2 and SIFT by 15% and 13%, respectively. These results suggest that feature-based methods, like ISM, are more suitable for the classification of amino acid substitutions outside CFDs than phylogeny-based tools.

  16. Sponge non-metastatic Group I Nme gene/protein - structure and function is conserved from sponges to humans

    Science.gov (United States)

    2011-01-01

    Background Nucleoside diphosphate kinases NDPK are evolutionarily conserved enzymes present in Bacteria, Archaea and Eukarya, with human Nme1 the most studied representative of the family and the first identified metastasis suppressor. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestor's genomic features. Recent studies show that sponges have a wide repertoire of genes many of which are involved in diseases in more complex metazoans. The original function of those genes and the way it has evolved in the animal lineage is largely unknown. Here we report new results on the metastasis suppressor gene/protein homolog from the marine sponge Suberites domuncula, NmeGp1Sd. The purpose of this study was to investigate the properties of the sponge Group I Nme gene and protein, and compare it to its human homolog in order to elucidate the evolution of the structure and function of Nme. Results We found that sponge genes coding for Group I Nme protein are intron-rich. Furthermore, we discovered that the sponge NmeGp1Sd protein has a similar level of kinase activity as its human homolog Nme1, does not cleave negatively supercoiled DNA and shows nonspecific DNA-binding activity. The sponge NmeGp1Sd forms a hexamer, like human Nme1, and all other eukaryotic Nme proteins. NmeGp1Sd interacts with human Nme1 in human cells and exhibits the same subcellular localization. Stable clones expressing sponge NmeGp1Sd inhibited the migratory potential of CAL 27 cells, as already reported for human Nme1, which suggests that Nme's function in migratory processes was engaged long before the composition of true tissues. Conclusions This study suggests that the ancestor of all animals possessed a NmeGp1 protein with properties and functions similar to evolutionarily recent versions of the protein, even before the appearance of true tissues

  17. Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

    Directory of Open Access Journals (Sweden)

    Veronika N Bade

    Full Text Available The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls, ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation. ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no

  18. Conserved nucleation sites reinforce the significance of Phi value analysis in protein-folding studies.

    Science.gov (United States)

    Gianni, Stefano; Jemth, Per

    2014-07-01

    The only experimental strategy to address the structure of folding transition states, the so-called Φ value analysis, relies on the synergy between site directed mutagenesis and the measurement of reaction kinetics. Despite its importance, the Φ value analysis has been often criticized and its power to pinpoint structural information has been questioned. In this hypothesis, we demonstrate that comparing the Φ values between proteins not only allows highlighting the robustness of folding pathways but also provides per se a strong validation of the method. © 2014 International Union of Biochemistry and Molecular Biology.

  19. Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

    Science.gov (United States)

    Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

    1990-02-25

    The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

  20. Poly (ADP-ribose polymerase 1 is required for protein localization to Cajal body.

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    Elena Kotova

    2009-02-01

    Full Text Available Recently, the nuclear protein known as Poly (ADP-ribose Polymerase1 (PARP1 was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose, PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

  1. Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

    Science.gov (United States)

    Yang, Jae-Seong; Kwon, Oh Sung; Kim, Sanguk; Jang, Sung Key

    2013-01-01

    Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets. PMID:23593195

  2. Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

    OpenAIRE

    Roy, C R; Isberg, R R

    1997-01-01

    The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 ...

  3. Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections.

    Directory of Open Access Journals (Sweden)

    Alexandra Rockstroh

    2015-11-01

    Full Text Available Dengue virus (DENV is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV, Yellow Fever virus (YFV or Tick-borne encephalitis virus (TBEV. Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL domain in the viral envelope (E protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.

  4. Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells

    International Nuclear Information System (INIS)

    Kwon, Y.K.; Hecht, N.B.

    1991-01-01

    The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA

  5. UK114, a YjgF/Yer057p/UK114 family protein highly conserved from bacteria to mammals, is localized in rat liver peroxisomes

    International Nuclear Information System (INIS)

    Antonenkov, Vasily D.; Ohlmeier, Steffen; Sormunen, Raija T.; Hiltunen, J. Kalervo

    2007-01-01

    Mammalian UK114 belongs to a highly conserved family of proteins with unknown functions. Although it is believed that UK114 is a cytosolic or mitochondrial protein there is no detailed study of its intracellular localization. Using analytical subcellular fractionation, electron microscopic colloidal gold technique, and two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with mass spectrometric analysis we show here that a large portion of UK114 is present in rat liver peroxisomes. The peroxisomal UK114 is a soluble matrix protein and it is not inducible by the peroxisomal proliferator clofibrate. The data predict involvement of UK114 in peroxisomal metabolism

  6. The fission yeast heterochromatin protein Rik1 is required for telomere clustering during meiosis

    DEFF Research Database (Denmark)

    Tuzon, Creighton T; Borgstrøm, Britta; Weilguny, Dietmar

    2004-01-01

    Telomeres share the ability to silence nearby transcription with heterochromatin, but the requirement of heterochromatin proteins for most telomere functions is unknown. The fission yeast Rik1 protein is required for heterochromatin formation at centromeres and the mating-type locus, as it recrui...... meiosis. However, Rik1 is dispensable for the protective roles of telomeres in preventing chromosome end-fusion. Thus, a Swi6-independent heterochromatin function distinct from that at centromeres and the mating-type locus operates at telomeres during sexual differentiation....

  7. Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains.

    Science.gov (United States)

    Armas, Pablo; Agüero, Tristán H; Borgognone, Mariana; Aybar, Manuel J; Calcaterra, Nora B

    2008-10-17

    Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the

  8. Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain.

    Science.gov (United States)

    Richards, Mark W; O'Regan, Laura; Roth, Daniel; Montgomery, Jessica M; Straube, Anne; Fry, Andrew M; Bayliss, Richard

    2015-05-01

    Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

  9. Net protein and metabolizable protein requirements for maintenance and growth of early-weaned Dorper crossbred male lambs

    Institute of Scientific and Technical Information of China (English)

    Tao Ma; Kaidong Deng; Yan Tu; Naifeng Zhang; Bingwen Si; Guishan Xu; Qiyu Diao

    2017-01-01

    Background:Dorper is an important breed for meat purpose and widely used in the livestock industry of the world.However,the protein requirement of Dorper crossbred has not been investigated.The current paper reports the net protein (NP) and metabolizable protein (MP) requirements of Dorper crossbred ram lambs from 20 to 35 kg BW.Methods:Thirty-five Dorper × thin-tailed Han crossbred lambs weaned at approximately 50 d of age (20.3 ± 2.15 kg of BW) were used.Seven lambs of 25 kg BW were slaughtered as the baseline animals at the start of the trial.An intermediate group of seven randomly selected lambs fed ad libitum was slaughtered at 28.6 kg BW.The remaining 21 lambs were randomly divided into three levels of dry matter intake:ad libitum or 70% or 40% of ad libitum intake.Those lambs were slaughtered when the lambs fed ad libitum reached 35 kg BW.Total body N and N retention were measured.Results:The daily NP and MP requirements for maintenance were 1.89 and 4.52 g/kg metabolic shrunk BW (SBW0.75).The partial efficiency of MP utilization for maintenance was 0.42.The NP requirement for growth ranged from 12.1 to 43.5 g/d,for the lambs gaining 100 to 350 g/d,and the partial efficiency of MP utilization for growth was 0.86.Conclusions:The NP and MP requirements for the maintenance and growth of Dorper crossbred male lambs were lower than the recommendations of American and British nutritional systems.

  10. Policy objectives for conserving freshwater ecosystems: How many rivers, which ones, and what level of protection are required

    CSIR Research Space (South Africa)

    Roux, D

    2006-05-01

    Full Text Available A current initiative aims to develop a set of operational policy objectives for facilitating national-level coordination in the conservation of freshwater ecosystems and their associated biodiversity. This initiative draws from the relatively new...

  11. The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants

    Directory of Open Access Journals (Sweden)

    Colasanti Joseph

    2006-06-01

    Full Text Available Abstract Background The maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD, which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation. Results Comparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only. Conclusion Comparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein

  12. hPOC5 is a centrin-binding protein required for assembly of full-length centrioles.

    Science.gov (United States)

    Azimzadeh, Juliette; Hergert, Polla; Delouvée, Annie; Euteneuer, Ursula; Formstecher, Etienne; Khodjakov, Alexey; Bornens, Michel

    2009-04-06

    Centrin has been shown to be involved in centrosome biogenesis in a variety of eukaryotes. In this study, we characterize hPOC5, a conserved centrin-binding protein that contains Sfi1p-like repeats. hPOC5 is localized, like centrin, in the distal portion of human centrioles. hPOC5 recruitment to procentrioles occurs during G2/M, a process that continues up to the full maturation of the centriole during the next cell cycle and is correlated with hyperphosphorylation of the protein. In the absence of hPOC5, RPE1 cells arrest in G1 phase, whereas HeLa cells show an extended S phase followed by cell death. We show that hPOC5 is not required for the initiation of procentriole assembly but is essential for building the distal half of centrioles. Interestingly, the hPOC5 family reveals an evolutionary divergence between vertebrates and organisms like Drosophila melanogaster or Caenorhabditis elegans, in which the loss of hPOC5 may correlate with the conspicuous differences in centriolar structure.

  13. Protein synthetic requirements for caffeine amelioration of radiation-induced G/sub 2/-arrest

    International Nuclear Information System (INIS)

    Rowley, R.; Colkitt, D.

    1984-01-01

    Irradiated cells are arrested in G/sub 2/ (transition point [TP] = 32 min before cell selection in mitosis). Irradiated cells do not recover from G/sub 2/ arrest in the presence of cycloheximide (CHM) indicating dependence of recovery on protein synthesis. Irradiated cells in the presence of caffeine progress to mitosis without arrest. The authors investigate whether irradiated cells in the presence of caffeine require protein synthesis to progress to mitosis. Mitotic cell selection was used to monitor the progression of irradiated CHO cells (150 rad) during exposure to 5 mM caffeine and/or 50 μg/ml CHM. Protein synthesis inhibition was confirmed using /sup 3/H-leucine incorporation. Cells exposed to CHM alone are arrested in G/sub 2/ (TP=49 min), thus cells beyond this point have synthesized all proteins necessary for entry into mitosis. In the presence of caffeine, unirradiated cells exposed to CHM are not arrested at all in G/sub 2/, instead arrest occurs near the S/G/sub 2/ boundary (TP=95 min) indicating that caffeine alleviates the dependence of G/sub 2/ cell progression on protein synthesis. However, irradiated cells exposed to both caffeine and CHM are only able to progress to mitosis if beyond the CHM-TP. Irradiated cells in the presence of caffeine thus behave as untreated cells and require protein synthesis for progression to mitosis when prior to the CHM-TP

  14. GAS2L1 Is a Centriole-Associated Protein Required for Centrosome Dynamics and Disjunction.

    NARCIS (Netherlands)

    Au, F.K.; Jia, Y.; Jiang, K.; Grigoriev, I.S.; Hau, B.K.; Shen, Y.; Du, S.; Akhmanova, A.S.; Qi, R.Z.

    2017-01-01

    Mitotic spindle formation and chromosome segregation require timely separation of the two duplicated centrosomes, and this process is initiated in late G2 by centrosome disjunction. Here we report that GAS2L1, a microtubule- and actin-binding protein, associates with the proximal end of mature

  15. Determination of energy and protein requirements for crossbred Holstein × Gyr preweaned dairy calves

    NARCIS (Netherlands)

    Silva, A.L.; Marcondes, M.I.; Detmann, E.; Campos, M.M.; Machado, F.S.; Filho, S.C.V.; Castro, M.M.D.; Dijkstra, J.

    2017-01-01

    The objective was to quantify the energy and protein nutritional requirements of Holstein × Gyr crossbred preweaned dairy calves until 64 d of age. Thirty-nine Holstein × Gyr crossbred male calves with an average initial live weight (mean ± SEM; for all next values) of 36 ± 1.0 kg were used. Five

  16. 40 CFR 180.1204 - Harpin protein; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Harpin protein; exemption from the requirement of a tolerance. 180.1204 Section 180.1204 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1204 Harpi...

  17. Primary structure and localization of a conserved immunogenic Plasmodium falciparum glutamate rich protein (GLURP) expressed in both the preerythrocytic and erythrocytic stages of the vertebrate life cycle

    DEFF Research Database (Denmark)

    Borre, M B; Dziegiel, M; Høgh, B

    1991-01-01

    A gene coding for a 220-kDa glutamate rich protein (GLURP), an exoantigen of Plasmodium falciparum, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence contains 2 repeat regions. The sequence of one of these was shown to be conserved among geographically...

  18. A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

    Science.gov (United States)

    Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

    2014-01-01

    Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

  19. Duplication of the IGFBP-2 gene in teleost fish: protein structure and functionality conservation and gene expression divergence.

    Directory of Open Access Journals (Sweden)

    Jianfeng Zhou

    Full Text Available BACKGROUND: Insulin-like growth factor binding protein-2 (IGFBP-2 is a secreted protein that binds and regulates IGF actions in controlling growth, development, reproduction, and aging. Elevated expression of IGFBP-2 is often associated with progression of many types of cancers. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification and characterization of two IGFBP-2 genes in zebrafish and four other teleost fish. Comparative genomics and structural analyses suggest that they are co-orthologs of the human IGFBP-2 gene. Biochemical assays show that both zebrafish igfbp-2a and -2b encode secreted proteins that bind IGFs. These two genes exhibit distinct spatiotemporal expression patterns. During embryogenesis, IGFBP-2a mRNA is initially detected in the lens, then in the brain boundary vasculature, and subsequently becomes highly expressed in the liver. In the adult stage, liver has the highest levels of IGFBP-2a mRNA, followed by the brain. Low levels of IGFBP-2a mRNA were detected in muscle and in the gonad in male adults only. IGFBP-2b mRNA is detected initially in all tissues at low levels, but later becomes abundant in the liver. In adult males, IGFBP-2b mRNA is only detected in the liver. In adult females, it is also found in the gut, kidney, ovary, and muscle. To gain insights into how the IGFBP-2 genes may have evolved through partitioning of ancestral functions, functional and mechanistic studies were carried out. Expression of zebrafish IGFBP-2a and -2b caused significant decreases in the growth and developmental rates and their effects are comparable to that of human IGFBP-2. IGFBP-2 mutants with altered IGF binding-, RGD-, and heparin-binding sites were generated and their actions examined. While mutating the RGD and heparin binding sites had little effect, altering the IGF binding site abolished its biological activity. CONCLUSIONS/SIGNIFICANCE: These results suggest that IGFBP-2 is a conserved regulatory protein and it inhibits

  20. The Heat Shock Protein 26 Gene is Required for Ethanol Tolerance in Drosophila

    Directory of Open Access Journals (Sweden)

    Awoyemi A. Awofala

    2011-01-01

    Full Text Available Stress plays an important role in drug- and addiction-related behaviours. However, the mechanisms underlying these behavioural responses are still poorly understood. In the light of recent reports that show consistent regulation of many genes encoding stress proteins including heat shock proteins following ethanol exposure in Drosophila , it was hypothesised that transition to alcohol dependence may involve the dysregulation of the circuits that mediate behavioural responses to stressors. Thus, behavioural genetic methodologies were used to investigate the role of the Drosophila hsp26 gene, a small heat shock protein coding gene which is induced in response to various stresses, in the development of rapid tolerance to ethanol sedation. Rapid tolerance was quantified as the percentage difference in the mean sedation times between the second and first ethanol exposure. Two independently isolated P-element mutations near the hsp26 gene eliminated the capacity for tolerance. In addition, RNAi-mediated functional knockdown of hsp26 expression in the glial cells and the whole nervous system also caused a defect in tolerance development. The rapid tolerance phenotype of the hsp26 mutants was rescued by the expression of the wild-type hsp26 gene in the nervous system. None of these manipulations of the hsp26 gene caused changes in the rate of ethanol absorption. Hsp26 genes are evolutionary conserved, thus the role of hsp26 in ethanol tolerance may present a new direction for research into alcohol dependency.

  1. The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile-X protein ortholog in Drosophila neurons

    Science.gov (United States)

    Bienkowski, Rick S.; Banerjee, Ayan; Rounds, J. Christopher; Rha, Jennifer; Omotade, Omotola F.; Gross, Christina; Morris, Kevin J.; Leung, Sara W.; Pak, ChangHui; Jones, Stephanie K.; Santoro, Michael R.; Warren, Stephen T.; Zheng, James Q.; Bassell, Gary J.; Corbett, Anita H.; Moberg, Kenneth H.

    2017-01-01

    Summary The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA-binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with and the Fragile-X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII but not futsch mRNA, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A)-tail length similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. In sum these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14. PMID:28793261

  2. The Conserved, Disease-Associated RNA Binding Protein dNab2 Interacts with the Fragile X Protein Ortholog in Drosophila Neurons

    Directory of Open Access Journals (Sweden)

    Rick S. Bienkowski

    2017-08-01

    Full Text Available The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.

  3. Increased Protein Requirements in Female Athletes after Variable-Intensity Exercise.

    Science.gov (United States)

    Wooding, Denise J; Packer, Jeff E; Kato, Hiroyuki; West, Daniel W D; Courtney-Martin, Glenda; Pencharz, Paul B; Moore, Daniel R

    2017-11-01

    Protein requirements are primarily studied in the context of resistance or endurance exercise with little research devoted to variable-intensity intermittent exercise characteristic of many team sports. Further, female populations are underrepresented in dietary sports science studies. We aimed to determine a dietary protein requirement in active females performing variable-intensity intermittent exercise using the indicator amino acid oxidation (IAAO) method. We hypothesized that these requirements would be greater than current IAAO-derived estimates in nonactive adult males. Six females (21.2 ± 0.8 yr, 68.8 ± 4.1 kg, 47.1 ± 1.2 mL O2·kg·min; mean ± SE) completed five to seven metabolic trials during the luteal phase of the menstrual cycle. Participants performed a modified Loughborough Intermittent Shuttle Test before consuming eight hourly mixed meals providing the test protein intake (0.2-2.66 g·kg·d), 6 g·kg·d CHO and sufficient energy for resting and exercise-induced energy expenditure. Protein was provided as crystalline amino acid modeling egg protein with [C]phenylalanine as the indicator amino acid. Phenylalanine turnover (Q) was determined from urinary [C]phenylalanine enrichment. Breath CO2 excretion (FCO2) was analyzed using mixed effects biphase linear regression with the breakpoint and upper 95% confidence interval approximating the estimated average requirement and recommended dietary allowance, respectively. Protein intake had no effect on Q (68.7 ± 7.3 μmol·kg·h; mean ± SE). FCO2 displayed a robust biphase response (R = 0.66) with an estimated average requirement of 1.41 g·kg·d and recommended dietary allowance of 1.71 g·kg·d. The protein requirement estimate of 1.41 and 1.71 g·kg·d for females performing variable-intensity intermittent exercise is greater than the IAAO-derived estimates of adult males (0.93 and 1.2 g·kg·d) and at the upper range of the American College of Sports Medicine athlete recommendations (1.2-2.0 g·kg·d).

  4. RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

    Science.gov (United States)

    Grill, Brock; Chen, Lizhen; Tulgren, Erik D; Baker, Scott T; Bienvenut, Willy; Anderson, Matthew; Quadroni, Manfredo; Jin, Yishi; Garner, Craig C

    2012-02-22

    Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

  5. Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD

    Directory of Open Access Journals (Sweden)

    Ackermann Martin

    2011-05-01

    Full Text Available Abstract Background The essential Escherichia coli gene ygjD belongs to a universally conserved group of genes whose function has been the focus of a number of recent studies. Here, we put ygjD under control of an inducible promoter, and used time-lapse microscopy and single cell analysis to investigate the phenotypic consequences of the depletion of YgjD protein from growing cells. Results We show that loss of YgjD leads to a marked decrease in cell size and termination of cell division. The transition towards smaller size occurs in a controlled manner: cell elongation and cell division remain coupled, but cell size at division decreases. We also find evidence that depletion of YgjD leads to the synthesis of the intracellular signaling molecule (pppGpp, inducing a cellular reaction resembling the stringent response. Concomitant deletion of the relA and spoT genes - leading to a strain that is uncapable of synthesizing (pppGpp - abrogates the decrease in cell size, but does not prevent termination of cell division upon YgjD depletion. Conclusions Depletion of YgjD protein from growing cells leads to a decrease in cell size that is contingent on (pppGpp, and to a termination of cell division. The combination of single-cell timelapse microscopy and statistical analysis can give detailed insights into the phenotypic consequences of the loss of essential genes, and can thus serve as a new tool to study the function of essential genes.

  6. Insights into the molecular evolution of the PDZ/LIM family and identification of a novel conserved protein motif.

    Directory of Open Access Journals (Sweden)

    Aartjan J W Te Velthuis

    Full Text Available The PDZ and LIM domain-containing protein family is encoded by a diverse group of genes whose phylogeny has currently not been analyzed. In mammals, ten genes are found that encode both a PDZ- and one or several LIM-domains. These genes are: ALP, RIL, Elfin (CLP36, Mystique, Enigma (LMP-1, Enigma homologue (ENH, ZASP (Cypher, Oracle, LMO7 and the two LIM domain kinases (LIMK1 and LIMK2. As conventional alignment and phylogenetic procedures of full-length sequences fell short of elucidating the evolutionary history of these genes, we started to analyze the PDZ and LIM domain sequences themselves. Using information from most sequenced eukaryotic lineages, our phylogenetic analysis is based on full-length cDNA-, EST-derived- and genomic- PDZ and LIM domain sequences of over 25 species, ranging from yeast to humans. Plant and protozoan homologs were not found. Our phylogenetic analysis identifies a number of domain duplication and rearrangement events, and shows a single convergent event during evolution of the PDZ/LIM family. Further, we describe the separation of the ALP and Enigma subfamilies in lower vertebrates and identify a novel consensus motif, which we call 'ALP-like motif' (AM. This motif is highly-conserved between ALP subfamily proteins of diverse organisms. We used here a combinatorial approach to define the relation of the PDZ and LIM domain encoding genes and to reconstruct their phylogeny. This analysis allowed us to classify the PDZ/LIM family and to suggest a meaningful model for the molecular evolution of the diverse gene architectures found in this multi-domain family.

  7. Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry.

    Directory of Open Access Journals (Sweden)

    Yaw Shin Ooi

    Full Text Available The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ, a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.

  8. A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

    Science.gov (United States)

    Prattes, Michael; Loibl, Mathias; Zisser, Gertrude; Luschnig, Daniel; Kappel, Lisa; Rössler, Ingrid; Grassegger, Manuela; Hromic, Altijana; Krieger, Elmar; Gruber, Karl; Pertschy, Brigitte; Bergler, Helmut

    2017-03-17

    AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

  9. Conserved region at the COOH terminus of human immunodeficiency virus gp120 envelope protein contains an immunodominant epitope

    International Nuclear Information System (INIS)

    Palker, T.J.; Matthews, T.J.; Clark, M.E.

    1987-01-01

    A highly immunogenic epitope from a conserved COOH-terminal region of the human immunodeficiency virus (HIV) gp120 envelope protein has been identified with antisera from HIV-seropositive subjects and a synthetic peptide (SP-22) containing 15 amino acids from this region (Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg). Peptide SP-22 absorbed up to 100% of anti-gp120 antibody reactivity from select HIV + patient sera in immunoblot assays and up to 79% of serum anti-gp120 antibody reactivity in competition RIA. In RIA, 45% of HIV-seropositive subjects had antibodies that bound to peptide SP-22. Human anti-SP-22 antibodies that bound to and were eluted from an SP-22 affinity column reacted with gp120 in RIA and immunoblot assays but did not neutralize HIV or inhibit HIV-induced syncytium formation in vitro, even though these antibodies comprised 70% of all anti-gp120 antibodies in the test serum. In contrast, the remaining 30% of SP-22 nonreactive anti-gp120 antibodies did not react with gp120 in immunoblot assays but did react in RIA and neutralized HIV in vitro. Thus, ≅ 50% of HIV-seropositive patients make high titers of nonneutralizing antibodies to an immunodominant antigen on gp120 defined by SP-22. Moreover, the COOH terminus of gp120 contains the major antigen or antigens identified by human anti-gp120 antibodies in immunoblot assays

  10. 75 FR 44163 - Implementation of Regulations Required Under Title XI of the Food, Conservation and Energy Act of...

    Science.gov (United States)

    2010-07-28

    ... Under Title XI of the Food, Conservation and Energy Act of 2008; Conduct in Violation of the Act AGENCY... Act and provide for a fairer market place. DATES: We will consider comments we receive by November 22... clarify conditions for industry compliance with the P&S Act and provide for a fairer market place. We have...

  11. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

    OpenAIRE

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related long-term memo...

  12. Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25.

    Directory of Open Access Journals (Sweden)

    Victor L Jensen

    2010-11-01

    Full Text Available In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis, but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia, implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT (required to build cilia is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.

  13. Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

    Science.gov (United States)

    Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

    2011-01-01

    The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

  14. The conserved 12-amino acid stretch in the inter-bromodomain region of BET family proteins functions as a nuclear localization signal.

    Science.gov (United States)

    Fukazawa, Hidesuke; Masumi, Atsuko

    2012-01-01

    The bromodomain and extraterminal (BET) family is a group of chromatin-binding proteins characterized by two bromodomains, an extraterminal (ET) domain, and several other conserved regions of unknown function. In humans, the BET family consists of four members, BRD2, BRD3, BRD4 and BRDT, that all normally localize to the nucleus. We identified a 12-amino acid stretch in the inter-bromodomain region that is perfectly conserved among the BET family members. We deleted these residues and expressed the mutant proteins in HEK293T cells to investigate the function of this motif. We found that the deletion of this motif alters the localization of BET proteins. Mutated BRD3 and BRD4 were excluded from the nucleus, and BRDT was found to be diffused throughout the nucleus and cytoplasm. Although the mutant BRD2 remained predominantly in the nucleus, a punctate distribution was also observed in the cytosol. It has been reported that a conserved motif between the second bromodomain and the ET domain serves as a nuclear localization signal for BRD2. Nevertheless, BET mutants lacking the reported nuclear localization signal motif but retaining the 12-amino acid stretch resided in the nucleus. Furthermore, these mutants were diffused throughout the cytoplasm when the 12 residues were removed. These results indicate that the conserved amino acid stretch in the inter-bromodomain region of the BET family functions as a nuclear localization signal.

  15. Late protein synthesis-dependent phases in CTA long-term memory: BDNF requirement

    Directory of Open Access Journals (Sweden)

    Araceli eMartínez-Moreno

    2011-09-01

    Full Text Available It has been proposed that long-term memory persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related long-term memory when protein synthesis was inhibited. Our previous studies on the insular cortex (IC, a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA, have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis dependent in different time-windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 hours after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes.

  16. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement.

    Science.gov (United States)

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F; Escobar, Martha L

    2011-01-01

    It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes.

  17. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

    Science.gov (United States)

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes. PMID:21960964

  18. 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

  19. Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity.

    Science.gov (United States)

    Tron, Cecile M; McNae, Iain W; Nutley, Margaret; Clarke, David J; Cooper, Alan; Walkinshaw, Malcolm D; Baxter, Robert L; Campopiano, Dominic J

    2009-03-20

    Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5'-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 A resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed beta- and gamma-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the alpha- and beta-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg(2+), and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPDelta67 fragment and BSA, and is subject to self-biotinylation.

  20. Human T-cell recognition of synthetic peptides representing conserved and variant sequences from the merozoite surface protein 2 of Plasmodium falciparum

    DEFF Research Database (Denmark)

    Theander, T G; Hviid, L; Dodoo, D

    1997-01-01

    Merozoite surface protein 2 (MSP2) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the peripheral blood mononuclear cell (PBMC) response to synthetic peptides corresponding to conserved and variant regions of the FCQ-27 allelic form of MSP2 in Ghanaian individuals....... The findings are encouraging for the development of a vaccine based on these T-epitope containing regions of MSP2, as the peptides were broadly recognized suggesting that they can bind to diverse HLA alleles and also because they include conserved MSP2 sequences. Immunisation with a vaccine construct...

  1. Domain architecture conservation in orthologs

    Science.gov (United States)

    2011-01-01

    Background As orthologous proteins are expected to retain function more often than other homologs, they are often used for functional annotation transfer between species. However, ortholog identification methods do not take into account changes in domain architecture, which are likely to modify a protein's function. By domain architecture we refer to the sequential arrangement of domains along a protein sequence. To assess the level of domain architecture conservation among orthologs, we carried out a large-scale study of such events between human and 40 other species spanning the entire evolutionary range. We designed a score to measure domain architecture similarity and used it to analyze differences in domain architecture conservation between orthologs and paralogs relative to the conservation of primary sequence. We also statistically characterized the extents of different types of domain swapping events across pairs of orthologs and paralogs. Results The analysis shows that orthologs exhibit greater domain architecture conservation than paralogous homologs, even when differences in average sequence divergence are compensated for, for homologs that have diverged beyond a certain threshold. We interpret this as an indication of a stronger selective pressure on orthologs than paralogs to retain the domain architecture required for the proteins to perform a specific function. In general, orthologs as well as the closest paralogous homologs have very similar domain architectures, even at large evolutionary separation. The most common domain architecture changes observed in both ortholog and paralog pairs involved insertion/deletion of new domains, while domain shuffling and segment duplication/deletion were very infrequent. Conclusions On the whole, our results support the hypothesis that function conservation between orthologs demands higher domain architecture conservation than other types of homologs, relative to primary sequence conservation. This supports the

  2. Conservation endocrinology

    Science.gov (United States)

    McCormick, Stephen; Romero, L. Michael

    2017-01-01

    Endocrinologists can make significant contributions to conservation biology by helping to understand the mechanisms by which organisms cope with changing environments. Field endocrine techniques have advanced rapidly in recent years and can provide substantial information on the growth, stress, and reproductive status of individual animals, thereby providing insight into current and future responses of populations to changes in the environment. Environmental stressors and reproductive status can be detected nonlethally by measuring a number of endocrine-related endpoints, including steroids in plasma, living and nonliving tissue, urine, and feces. Information on the environmental or endocrine requirements of individual species for normal growth, development, and reproduction will provide critical information for species and ecosystem conservation. For many taxa, basic information on endocrinology is lacking, and advances in conservation endocrinology will require approaches that are both “basic” and “applied” and include integration of laboratory and field approaches.

  3. Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions

    International Nuclear Information System (INIS)

    Jhun, E.; Jhun, B.H.; Jones, L.R.; Jung, C.Y.

    1991-01-01

    The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100 degree C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein

  4. Sas-4 proteins are required during basal body duplication in Paramecium

    Science.gov (United States)

    Gogendeau, Delphine; Hurbain, Ilse; Raposo, Graca; Cohen, Jean; Koll, France; Basto, Renata

    2011-01-01

    Centrioles and basal bodies are structurally related organelles composed of nine microtubule (MT) triplets. Studies performed in Caenorhabditis elegans embryos have shown that centriole duplication takes place in sequential way, in which different proteins are recruited in a specific order to assemble a procentriole. ZYG-1 initiates centriole duplication by triggering the recruitment of a complex of SAS-5 and SAS-6, which then recruits the final player, SAS-4, to allow the incorporation of MT singlets. It is thought that a similar mechanism (that also involves additional proteins) is present in other animal cells, but it remains to be investigated whether the same players and their ascribed functions are conserved during basal body duplication in cells that exclusively contain basal bodies. To investigate this question, we have used the multiciliated protist Paramecium tetraurelia. Here we show that in the absence of PtSas4, two types of defects in basal body duplication can be identified. In the majority of cases, the germinative disk and cartwheel, the first structures assembled during duplication, are not detected. In addition, if daughter basal bodies were formed, they invariably had defects in MT recruitment. Our results suggest that PtSas4 has a broader function than its animal orthologues. PMID:21289083

  5. Current issues in determining dietary protein and amino-acid requirements

    DEFF Research Database (Denmark)

    Pencharz, P; Jahoor, F; Kurpad, A

    2014-01-01

    Pregnancy and the first two years of life are periods of rapid growth and yet the knowledge of requirements for protein and dietary indispensable amino acids is very limited. The development of carbon oxidation methods opens the way to studies that should fill these important gaps in knowledge.Eu.......European Journal of Clinical Nutrition advance online publication, 15 January 2014; doi:10.1038/ejcn.2013.297....

  6. Energy requirements, protein-energy metabolism and balance, and carbohydrates in preterm infants.

    Science.gov (United States)

    Hay, William W; Brown, Laura D; Denne, Scott C

    2014-01-01

    Energy is necessary for all vital functions of the body at molecular, cellular, organ, and systemic levels. Preterm infants have minimum energy requirements for basal metabolism and growth, but also have requirements for unique physiology and metabolism that influence energy expenditure. These include body size, postnatal age, physical activity, dietary intake, environmental temperatures, energy losses in the stool and urine, and clinical conditions and diseases, as well as changes in body composition. Both energy and protein are necessary to produce normal rates of growth. Carbohydrates (primarily glucose) are principle sources of energy for the brain and heart until lipid oxidation develops over several days to weeks after birth. A higher protein/energy ratio is necessary in most preterm infants to approximate normal intrauterine growth rates. Lean tissue is predominantly produced during early gestation, which continues through to term. During later gestation, fat accretion in adipose tissue adds increasingly large caloric requirements to the lean tissue growth. Once protein intake is sufficient to promote net lean body accretion, additional energy primarily produces more body fat, which increases almost linearly at energy intakes >80-90 kcal/kg/day in normal, healthy preterm infants. Rapid gains in adiposity have the potential to produce later life obesity, an increasingly recognized risk of excessive energy intake. In addition to fundamental requirements for glucose, protein, and fat, a variety of non-glucose carbohydrates found in human milk may have important roles in promoting growth and development, as well as production of a gut microbiome that could protect against necrotizing enterocolitis. © 2014 S. Karger AG, Basel.

  7. Variable-Intensity Simulated Team-Sport Exercise Increases Daily Protein Requirements in Active Males.

    Science.gov (United States)

    Packer, Jeffrey E; Wooding, Denise J; Kato, Hiroyuki; Courtney-Martin, Glenda; Pencharz, Paul B; Moore, Daniel R

    2017-01-01

    Protein requirements are generally increased in strength and endurance trained athletes relative to their sedentary peers. However, less is known about the daily requirement for this important macronutrient in individuals performing variable intensity, stop-and-go type exercise that is typical for team sport athletes. The objective of the present study was to determine protein requirements in active, trained adult males performing a simulated soccer match using the minimally invasive indicator amino acid oxidation (IAAO) method. After 2 days of controlled diet (1.2 g⋅kg -1 ⋅day -1 protein), seven trained males (23 ± 1 years; 177.5 ± 6.7 cm; 82.3 ± 6.1 kg; 13.5% ± 4.7% body fat; 52.3 ± 5.9 ml O 2 ⋅kg -1 ⋅min -1 ; mean ± SD) performed an acute bout of variable intensity exercise in the form of a modified Loughborough Intermittent Shuttle Test (4 × 15 min of exercise over 75 min). Immediately after exercise, hourly meals were consumed providing a variable amount of protein (0.2-2.6 g⋅kg -1 ⋅day -1 ) and sufficient energy and carbohydrate (6 g⋅kg -1 ⋅day -1 ). Protein was provided as a crystalline amino acids modeled after egg protein with the exception of phenylalanine and tyrosine, which were provided in excess to ensure the metabolic partitioning of the indicator amino acid (i.e., [1- 13 C]phenylalanine included within the phenylalanine intake) was directed toward oxidation when protein intake was limiting. Whole body phenylalanine flux and 13 CO 2 excretion (F 13 CO 2 ) were determined at metabolic and isotopic steady state from urine and breath samples, respectively. Biphasic linear regression analysis was performed on F 13 CO 2 to determine the estimated average requirement (EAR) for protein with a safe intake defined as the upper 95% confidence interval. Phenylalanine flux was not impacted by protein intake ( P  = 0.45). Bi-phase linear regression ( R 2  = 0.64) of F 13 CO 2 resulted

  8. Variable-Intensity Simulated Team-Sport Exercise Increases Daily Protein Requirements in Active Males

    Directory of Open Access Journals (Sweden)

    Jeffrey E. Packer

    2017-12-01

    Full Text Available Protein requirements are generally increased in strength and endurance trained athletes relative to their sedentary peers. However, less is known about the daily requirement for this important macronutrient in individuals performing variable intensity, stop-and-go type exercise that is typical for team sport athletes. The objective of the present study was to determine protein requirements in active, trained adult males performing a simulated soccer match using the minimally invasive indicator amino acid oxidation (IAAO method. After 2 days of controlled diet (1.2 g⋅kg−1⋅day−1 protein, seven trained males (23 ± 1 years; 177.5 ± 6.7 cm; 82.3 ± 6.1 kg; 13.5% ± 4.7% body fat; 52.3 ± 5.9 ml O2⋅kg−1⋅min-1; mean ± SD performed an acute bout of variable intensity exercise in the form of a modified Loughborough Intermittent Shuttle Test (4 × 15 min of exercise over 75 min. Immediately after exercise, hourly meals were consumed providing a variable amount of protein (0.2–2.6 g⋅kg−1⋅day−1 and sufficient energy and carbohydrate (6 g⋅kg−1⋅day−1. Protein was provided as a crystalline amino acids modeled after egg protein with the exception of phenylalanine and tyrosine, which were provided in excess to ensure the metabolic partitioning of the indicator amino acid (i.e., [1-13C]phenylalanine included within the phenylalanine intake was directed toward oxidation when protein intake was limiting. Whole body phenylalanine flux and 13CO2 excretion (F13CO2 were determined at metabolic and isotopic steady state from urine and breath samples, respectively. Biphasic linear regression analysis was performed on F13CO2 to determine the estimated average requirement (EAR for protein with a safe intake defined as the upper 95% confidence interval. Phenylalanine flux was not impacted by protein intake (P = 0.45. Bi-phase linear regression (R2 = 0.64 of F13CO2 resulted in an EAR

  9. Long-term aversive taste memory requires insular and amygdala protein degradation.

    Science.gov (United States)

    Rodriguez-Ortiz, Carlos J; Balderas, Israela; Saucedo-Alquicira, Fernando; Cruz-Castañeda, Paulina; Bermudez-Rattoni, Federico

    2011-03-01

    Some reports have shown that the ubiquitin-proteasome system (UPS) is necessary to degrade repressor factors to produce new proteins essential to memory consolidation. Furthermore, recent evidence suggests that memory updating also relies on protein degradation through the UPS. To evaluate whether degradation of proteins is part of the cellular events needed for long-term storage of taste aversion, we injected lactacystin--an UPS inhibitor--into the amygdala and/or insular cortex 30 min before the first or second training trials. The results revealed that degradation of proteins in either the amygdala or insular cortex suffices for long-term stabilization of first-time encounter taste aversion. On the other hand, lactacystin applied in the insula, but not in the amygdala, before the second training prevented long-term storage of updated information. Our results support that degradation of proteins by means of the UPS is required every time taste aversion is to be stored in long-term memory. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. Acquisition, consolidation, reconsolidation, and extinction of eyelid conditioning responses require de novo protein synthesis.

    Science.gov (United States)

    Inda, Mari Carmen; Delgado-García, José María; Carrión, Angel Manuel

    2005-02-23

    Memory, as measured by changes in an animal's behavior some time after learning, is a reflection of many processes. Here, using a trace paradigm, in mice we show that de novo protein synthesis is required for acquisition, consolidation, reconsolidation, and extinction of classically conditioned eyelid responses. Two critical periods of protein synthesis have been found: the first, during training, the blocking of which impaired acquisition; and the second, lasting the first 4 h after training, the blocking of which impaired consolidation. The process of reconsolidation was sensitive to protein synthesis inhibition if anisomycin was injected before or just after the reactivation session. Furthermore, extinction was also dependent on protein synthesis, following the same temporal course as that followed during acquisition and consolidation. This last fact reinforces the idea that extinction is an active learning process rather than a passive event of forgetting. Together, these findings demonstrate that all of the different stages of memory formation involved in the classical conditioning of eyelid responses are dependent on protein synthesis.

  11. Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs

    International Nuclear Information System (INIS)

    Horst, M.N.

    1990-01-01

    Chitin synthesis in crustaceans involves the deposition of a protein-polysaccharide complex at the apical surface of epithelial cells which secrete the cuticle or exoskeleton. The present study involves an examination of in vivo incorporation of radiolabeled amino acids and amino sugars into the cuticle of postmolt blue crabs, Callinectes sapidus. Rates of incorporation of both 3H leucine and 3H threonine were linear with respect to time of incubation. Incorporation of 3H threonine into the endocuticle was inhibited greater than 90% in the presence of the protein synthesis inhibitor, puromycin. Linear incorporation of 14C glucosamine into the cuticle was also demonstrated; a significant improvement of radiolabeling was achieved by using 14C-N-acetylglucosamine as the labeled precursor. Incorporation of 3H-N-acetylglucosamine into the cuticle of postmolt blue crabs was inhibited 89% by puromycin, indicating that concurrent protein synthesis is required for the deposition of chitin in the blue crab. Autoradiographic analysis of control vs. puromycin-treated crabs indicates that puromycin totally blocks labeling of the new endocuticle with 3H glucosamine. These results are consistent with the notion that crustacean chitin is synthesized as a protein-polysaccharide complex. Analysis of the postmolt and intermolt blue crab cuticle indicates that the exoskeleton contains about 60% protein and 40% chitin. The predominant amino acids are arginine, glutamic acid, alanine, aspartic acid, and threonine

  12. Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

    Science.gov (United States)

    Aguilera, Cristina; Fernández-Majada, Vanessa; Inglés-Esteve, Julia; Rodilla, Verónica; Bigas, Anna; Espinosa, Lluís

    2006-09-01

    IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

  13. Efficient overproduction of membrane proteins in Lactococcus lactis requires the cell envelope stress sensor/regulator couple CesSR

    NARCIS (Netherlands)

    Pinto, Joao P C; Kuipers, Oscar P; Marreddy, Ravi K R; Poolman, Bert; Kok, Jan

    2011-01-01

    BACKGROUND: Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert

  14. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

    Science.gov (United States)

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-12-01

    Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  15. The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

    Science.gov (United States)

    Huang, Danzhi; Caflisch, Amedeo

    2015-01-01

    Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP(Sc)) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP(C) proteins have provided evidence of a conformational equilibrium between a 3(10)-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165-175). In vitro cell-free experiments of the seeded conversion of PrP(C) indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3(10)-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3(10)-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.

  16. 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.

  17. Evidence for in vitro and in vivo expression of the conserved VAR3 (type 3) plasmodium falciparum erythrocyte membrane protein 1

    DEFF Research Database (Denmark)

    Wang, Christian W; Lavstsen, Thomas; Bengtsson, Dominique C

    2012-01-01

    ABSTRACT: BACKGROUND: Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion antigen family are major contributors to the pathogenesis of P. falciparum malaria infections. The PfEMP1-encoding var genes are among the most diverse sequences in nature, but three genes......, var1, var2csa and var3 are found conserved in most parasite genomes. The most severe forms of malaria disease are caused by parasites expressing a subset of antigenically conserved PfEMP1 variants. Thus the ubiquitous and conserved VAR3 PfEMP1 is of particular interest to the research field. Evidence...... of VAR3 expression on the infected erythrocyte surface has never been presented, and var3 genes have been proposed to be transcribed and expressed differently from the rest of the var gene family members. METHODS: In this study, parasites expressing VAR3 PfEMP1 were generated using anti-VAR3 antibodies...

  18. Residues of the UL25 Protein of Herpes Simplex Virus That Are Required for Its Stable Interaction with Capsids ▿

    Science.gov (United States)

    Cockrell, Shelley K.; Huffman, Jamie B.; Toropova, Katerina; Conway, James F.; Homa, Fred L.

    2011-01-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes. PMID:21411517

  19. A prefoldin-associated WD-repeat protein (WDR92) is required for the correct architectural assembly of motile cilia

    Science.gov (United States)

    Patel-King, Ramila S.; King, Stephen M.

    2016-01-01

    WDR92 is a highly conserved WD-repeat protein that has been proposed to be involved in apoptosis and also to be part of a prefoldin-like cochaperone complex. We found that WDR92 has a phylogenetic signature that is generally compatible with it playing a role in the assembly or function of specifically motile cilia. To test this hypothesis, we performed an RNAi-based knockdown of WDR92 gene expression in the planarian Schmidtea mediterranea and were able to achieve a robust reduction in mRNA expression to levels undetectable under our standard RT-PCR conditions. We found that this treatment resulted in a dramatic reduction in the rate of organismal movement that was caused by a switch in the mode of locomotion from smooth, cilia-driven gliding to muscle-based, peristaltic contractions. Although the knockdown animals still assembled cilia of normal length and in similar numbers to controls, these structures had reduced beat frequency and did not maintain hydrodynamic coupling. By transmission electron microscopy we observed that many cilia had pleiomorphic defects in their architecture, including partial loss of dynein arms, incomplete closure of the B-tubule, and occlusion or replacement of the central pair complex by accumulated electron-dense material. These observations suggest that WDR92 is part of a previously unrecognized cytoplasmic chaperone system that is specifically required to fold key components necessary to build motile ciliary axonemes. PMID:26912790

  20. 40 CFR 174.531 - Coat protein of plum pox virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of plum pox virus...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.531 Coat protein of plum pox virus; exemption from the requirement of a tolerance. Residues of the coat protein of plum pox virus in or on the...

  1. 40 CFR 174.512 - Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Potato Virus Y...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.512 Coat Protein of Potato Virus Y; exemption from the requirement of a tolerance. Residues of Coat Protein of Potato Virus Y are exempt from...

  2. Low-protein diet for conservative management of chronic kidney disease: a systematic review and meta-analysis of controlled trials.

    Science.gov (United States)

    Rhee, Connie M; Ahmadi, Seyed-Foad; Kovesdy, Csaba P; Kalantar-Zadeh, Kamyar

    2018-04-01

    Recent data pose the question whether conservative management of chronic kidney disease (CKD) by means of a low-protein diet can be a safe and effective means to avoid or defer transition to dialysis therapy without causing protein-energy wasting or cachexia. We aimed to systematically review and meta-analyse the controlled clinical trials with adequate participants in each trial, providing rigorous contemporary evidence of the impact of a low-protein diet in the management of uraemia and its complications in patients with CKD. We searched MEDLINE (PubMed) and other sources for controlled trials on CKD to compare clinical management of CKD patients under various levels of dietary protein intake or to compare restricted protein intake with other interventions. Studies with similar patients, interventions, and outcomes were included in the meta-analyses. We identified 16 controlled trials of low-protein diet in CKD that met the stringent qualification criteria including having 30 or more participants. Compared with diets with protein intake of >0.8 g/kg/day, diets with restricted protein intake (disease, and a trend towards lower rates of all-cause death. In addition, very-low-protein diets (protein intake kidney function and reduction in the rate of progression to end-stage renal disease. Safety and adherence to a low-protein diet was not inferior to a normal protein diet, and there was no difference in the rate of malnutrition or protein-energy wasting. In this pooled analysis of moderate-size controlled trials, a low-protein diet appears to enhance the conservative management of non-dialysis-dependent CKD and may be considered as a potential option for CKD patients who wish to avoid or defer dialysis initiation and to slow down the progression of CKD, while the risk of protein-energy wasting and cachexia remains minimal. © 2017 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia

  3. A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

    DEFF Research Database (Denmark)

    Kostenis, Evi; Martini, Lene; Ellis, James

    2004-01-01

    Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor...... recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one...

  4. Protein clustering and RNA phylogenetic reconstruction of the influenza A [corrected] virus NS1 protein allow an update in classification and identification of motif conservation.

    Science.gov (United States)

    Sevilla-Reyes, Edgar E; Chavaro-Pérez, David A; Piten-Isidro, Elvira; Gutiérrez-González, Luis H; Santos-Mendoza, Teresa

    2013-01-01

    The non-structural protein 1 (NS1) of influenza A virus (IAV), coded by its third most diverse gene, interacts with multiple molecules within infected cells. NS1 is involved in host immune response regulation and is a potential contributor to the virus host range. Early phylogenetic analyses using 50 sequences led to the classification of NS1 gene variants into groups (alleles) A and B. We reanalyzed NS1 diversity using 14,716 complete NS IAV sequences, downloaded from public databases, without host bias. Removal of sequence redundancy and further structured clustering at 96.8% amino acid similarity produced 415 clusters that enhanced our capability to detect distinct subgroups and lineages, which were assigned a numerical nomenclature. Maximum likelihood phylogenetic reconstruction using RNA sequences indicated the previously identified deep branching separating group A from group B, with five distinct subgroups within A as well as two and five lineages within the A4 and A5 subgroups, respectively. Our classification model proposes that sequence patterns in thirteen amino acid positions are sufficient to fit >99.9% of all currently available NS1 sequences into the A subgroups/lineages or the B group. This classification reduces host and virus bias through the prioritization of NS1 RNA phylogenetics over host or virus phenetics. We found significant sequence conservation within the subgroups and lineages with characteristic patterns of functional motifs, such as the differential binding of CPSF30 and crk/crkL or the availability of a C-terminal PDZ-binding motif. To understand selection pressures and evolution acting on NS1, it is necessary to organize the available data. This updated classification may help to clarify and organize the study of NS1 interactions and pathogenic differences and allow the drawing of further functional inferences on sequences in each group, subgroup and lineage rather than on a strain-by-strain basis.

  5. A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

    Science.gov (United States)

    Mir, Riyaz A; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A; Ammons, Shalis A; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B; Qiu, Fang; Band, Hamid; Band, Vimla

    2015-12-28

    Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities

    OpenAIRE

    Maréchal Eric; Ortet Philippe; Roy Sylvaine; Bastien Olivier

    2005-01-01

    Abstract Background Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic recon...

  7. Attachment and entry of Chlamydia have distinct requirements for host protein disulfide isomerase.

    Directory of Open Access Journals (Sweden)

    Stephanie Abromaitis

    2009-04-01

    Full Text Available Chlamydia is an obligate intracellular pathogen that causes a wide range of diseases in humans. Attachment and entry are key processes in infectivity and subsequent pathogenesis of Chlamydia, yet the mechanisms governing these interactions are unknown. It was recently shown that a cell line, CHO6, that is resistant to attachment, and thus infectivity, of multiple Chlamydia species has a defect in protein disulfide isomerase (PDI N-terminal signal sequence processing. Ectopic expression of PDI in CHO6 cells led to restoration of Chlamydia attachment and infectivity; however, the mechanism leading to this recovery was not ascertained. To advance our understanding of the role of PDI in Chlamydia infection, we used RNA interference to establish that cellular PDI is essential for bacterial attachment to cells, making PDI the only host protein identified as necessary for attachment of multiple species of Chlamydia. Genetic complementation and PDI-specific inhibitors were used to determine that cell surface PDI enzymatic activity is required for bacterial entry into cells, but enzymatic function was not required for bacterial attachment. We further determined that it is a PDI-mediated reduction at the cell surface that triggers bacterial uptake. While PDI is necessary for Chlamydia attachment to cells, the bacteria do not appear to utilize plasma membrane-associated PDI as a receptor, suggesting that Chlamydia binds a cell surface protein that requires structural association with PDI. Our findings demonstrate that PDI has two essential and independent roles in the process of chlamydial infectivity: it is structurally required for chlamydial attachment, and the thiol-mediated oxido-reductive function of PDI is necessary for entry.

  8. Levels of antibody to conserved parts of Plasmodium falciparum merozoite surface protein 1 in Ghanaian children are not associated with protection from clinical malaria

    DEFF Research Database (Denmark)

    Dodoo, D; Theander, T G; Kurtzhals, J A

    1999-01-01

    malaria season in April and after the season in November. Using enzyme-linked immunosorbent assay, we measured antibody responses to recombinant gluthathione S-transferase-PfMSP119 fusion proteins corresponding to the Wellcome and MAD20 allelic variants in these samples. Prevalence of antibodies......The 19-kDa conserved C-terminal part of the Plasmodium falciparum merozoite surface protein 1 (PfMSP119) is a malaria vaccine candidate antigen, and human antibody responses to PfMSP119 have been associated with protection against clinical malaria. In this longitudinal study carried out in an area...

  9. Host ESCRT proteins are required for bromovirus RNA replication compartment assembly and function.

    Directory of Open Access Journals (Sweden)

    Arturo Diaz

    2015-03-01

    Full Text Available Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV RNA replication occurs on perinuclear endoplasmic reticulum (ER membranes in ~70 nm vesicular invaginations (spherules. BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.

  10. Synaptojanin 1 is required for endolysosomal trafficking of synaptic proteins in cone photoreceptor inner segments.

    Directory of Open Access Journals (Sweden)

    Ashley A George

    Full Text Available Highly polarized cells such as photoreceptors require precise and efficient strategies for establishing and maintaining the proper subcellular distribution of proteins. The signals and molecular machinery that regulate trafficking and sorting of synaptic proteins within cone inner segments is mostly unknown. In this study, we show that the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1 is critical for this process. We used transgenic markers for trafficking pathways, electron microscopy, and immunocytochemistry to characterize trafficking defects in cones of the zebrafish mutant, nrc(a14 , which is deficient in phosphoinositide phosphatase, SynJ1. The outer segments and connecting cilia of nrc(a14 cone photoreceptors are normal, but RibeyeB and VAMP2/synaptobrevin, which normally localize to the synapse, accumulate in the nrc(a14 inner segment. The structure of the Endoplasmic Reticulum in nrc(a14 mutant cones is normal. Golgi develop normally, but later become disordered. Large vesicular structures accumulate within nrc(a14 cone photoreceptor inner segments, particularly after prolonged incubation in darkness. Cone inner segments of nrc (a14 mutants also have enlarged acidic vesicles, abnormal late endosomes, and a disruption in autophagy. This last pathway also appears exacerbated by darkness. Taken altogether, these findings show that SynJ1 is required in cones for normal endolysosomal trafficking of synaptic proteins.

  11. TYLCV-Is movement in planta does not require V2 protein

    Energy Technology Data Exchange (ETDEWEB)

    Hak, Hagit [Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel); Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem (Israel); Levy, Yael; Chandran, Sam A.; Belausov, Eduard [Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel); Loyter, Abraham [Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem (Israel); Lapidot, Moshe [Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel); Gafni, Yedidya, E-mail: ygafni@volcani.agri.gov.il [Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, Bet Dagan (Israel)

    2015-03-15

    Tomato yellow leaf curl virus (TYLCV), a major tomato pathogen causing extensive crop losses, is a whitefly-transmitted geminivirus. V2 mutants of TYLCV-Is and related viruses tend to induce symptomless infection with attenuated viral DNA levels, while accumulating close to wild-type DNA levels in protoplasts, suggesting V2 as a movement protein. The discovery of plant-silencing mechanisms and viral silencing suppressors, V2 included, led us to reconsider V2's involvement in viral movement. We studied two mutant versions of the virus, one impaired in V2 silencing-suppression activity, and another carrying a non-translatable V2. While both mutant viruses spread in the infected plant to newly emerged leaves at the same rate as the wild-type virus, their DNA-accumulation levels were tenfold lower than in the wild-type virus. Thus, we suggest that the setback in virus proliferation, previously ascribed to a movement impediment, is due to lack of silencing-suppression activity. - Highlights: • TYLCV-Is V2 protein is localized in distinct microbodies throughout the cell cytoplasm, around the nucleus and in association with cytoplasmic strands but is not associated with the plasmodesmata. • Disruption of RNA-silencing suppression activity of TYLCV-Is V2 protein causes low titer of the virus in the infected plants. • The movement of TYLCV-Is in planta does not require a functional V2 protein.

  12. The YPLGVG sequence of the Nipah virus matrix protein is required for budding

    Directory of Open Access Journals (Sweden)

    Yan Lianying

    2008-11-01

    Full Text Available Abstract Background Nipah virus (NiV is a recently emerged paramyxovirus capable of causing fatal disease in a broad range of mammalian hosts, including humans. Together with Hendra virus (HeV, they comprise the genus Henipavirus in the family Paramyxoviridae. Recombinant expression systems have played a crucial role in studying the cell biology of these Biosafety Level-4 restricted viruses. Henipavirus assembly and budding occurs at the plasma membrane, although the details of this process remain poorly understood. Multivesicular body (MVB proteins have been found to play a role in the budding of several enveloped viruses, including some paramyxoviruses, and the recruitment of MVB proteins by viral proteins possessing late budding domains (L-domains has become an important concept in the viral budding process. Previously we developed a system for producing NiV virus-like particles (VLPs and demonstrated that the matrix (M protein possessed an intrinsic budding ability and played a major role in assembly. Here, we have used this system to further explore the budding process by analyzing elements within the M protein that are critical for particle release. Results Using rationally targeted site-directed mutagenesis we show that a NiV M sequence YPLGVG is required for M budding and that mutation or deletion of the sequence abrogates budding ability. Replacement of the native and overlapping Ebola VP40 L-domains with the NiV sequence failed to rescue VP40 budding; however, it did induce the cellular morphology of extensive filamentous projection consistent with wild-type VP40-expressing cells. Cells expressing wild-type NiV M also displayed this morphology, which was dependent on the YPLGVG sequence, and deletion of the sequence also resulted in nuclear localization of M. Dominant-negative VPS4 proteins had no effect on NiV M budding, suggesting that unlike other viruses such as Ebola, NiV M accomplishes budding independent of MVB cellular proteins

  13. Provision of protein and energy in relation to measured requirements in intensive care patients

    DEFF Research Database (Denmark)

    Allingstrup, Matilde Jo; Esmailzadeh, Negar; Knudsen, Anne Wilkens

    2012-01-01

    , also when adjusted for baseline prognostic variables (APACHE II, SOFA scores and age). Provision of energy, measured resting energy expenditure or energy and nitrogen balance was not related to mortality. The possible cause-effect relationship is discussed after a more detailed analysis of the initial......BACKGROUND & AIMS: Adequacy of nutritional support in intensive care patients is still a matter of investigation. This study aimed to relate mortality to provision, measured requirements and balances for energy and protein in ICU patients. DESIGN: Prospective observational cohort study of 113 ICU...... part of the admission. CONCLUSION: In these severely ill ICU patients, a higher provision of protein and amino acids was associated with a lower mortality. This was not the case for provision of energy or measured resting energy expenditure or energy or nitrogen balances. The hypothesis that higher...

  14. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

    Science.gov (United States)

    Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

    2017-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.

  15. Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

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    Koji Mizuhashi

    Full Text Available Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119, encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice.First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS and osteoid maturation time (Omt, and significantly decreased mineral apposition rate (MAR and bone formation rate per bone surface (BFR/BS. In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant.Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

  16. Obif, a Transmembrane Protein, Is Required for Bone Mineralization and Spermatogenesis in Mice.

    Science.gov (United States)

    Mizuhashi, Koji; Chaya, Taro; Kanamoto, Takashi; Omori, Yoshihiro; Furukawa, Takahisa

    2015-01-01

    Various kinds of transmembrane and secreted proteins play pivotal roles in development through cell-cell communication. We previously reported that Obif (Osteoblast induction factor, Tmem119), encoding a single transmembrane protein, is expressed in differentiating osteoblasts, and that Obif-/- mice exhibit significantly reduced bone volume in the femur. In the current study, we characterized the Obif protein and further investigated the biological phenotypes of a variety of tissues in Obif-/- mice. First, we found that O-glycosylation of the Obif protein occurs at serine residue 36 in the Obif extracellular domain. Next, we observed that Obif-/- mice exhibit bone dysplasia in association with significantly increased osteoid volume per osteoid surface (OV/OS) and osteoid maturation time (Omt), and significantly decreased mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS). In addition, we observed that Obif-/- mice show a significant decrease in testis weight as well as in sperm number. By histological analysis, we found that Obif is expressed in spermatocytes and spermatids in the developing testis and that spermatogenesis is halted at the round spermatid stage in the Obif-/- testis that lacks sperm. However, the number of litters fathered by male mice was slightly reduced in Obif-/- mice compared with wild-type mice, although this was not statistically significant. Our results, taken together with previous observations, indicate that Obif is a type Ia transmembrane protein whose N-terminal region is O-glycosylated. In addition, we found that Obif is required for normal bone mineralization and late testicular differentiation in vivo. These findings suggest that Obif plays essential roles in the development of multiple tissues.

  17. Fat storage-inducing transmembrane protein 2 is required for normal fat storage in adipose tissue.

    Science.gov (United States)

    Miranda, Diego A; Kim, Ji-Hyun; Nguyen, Long N; Cheng, Wang; Tan, Bryan C; Goh, Vera J; Tan, Jolene S Y; Yaligar, Jadegoud; Kn, Bhanu Prakash; Velan, S Sendhil; Wang, Hongyan; Silver, David L

    2014-04-04

    Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.

  18. The yeast cell fusion protein Prm1p requires covalent dimerization to promote membrane fusion.

    Directory of Open Access Journals (Sweden)

    Alex Engel

    2010-05-01

    Full Text Available Prm1p is a multipass membrane protein that promotes plasma membrane fusion during yeast mating. The mechanism by which Prm1p and other putative regulators of developmentally controlled cell-cell fusion events facilitate membrane fusion has remained largely elusive. Here, we report that Prm1p forms covalently linked homodimers. Covalent Prm1p dimer formation occurs via intermolecular disulfide bonds of two cysteines, Cys-120 and Cys-545. PRM1 mutants in which these cysteines have been substituted are fusion defective. These PRM1 mutants are normally expressed, retain homotypic interaction and can traffic to the fusion zone. Because prm1-C120S and prm1-C545S mutants can form covalent dimers when coexpressed with wild-type PRM1, an intermolecular C120-C545 disulfide linkage is inferred. Cys-120 is adjacent to a highly conserved hydrophobic domain. Mutation of a charged residue within this hydrophobic domain abrogates formation of covalent dimers, trafficking to the fusion zone, and fusion-promoting activity. The importance of intermolecular disulfide bonding informs models regarding the mechanism of Prm1-mediated cell-cell fusion.

  19. The Unfolded Protein Response Is Induced by the Cell Wall Integrity Mitogen-activated Protein Kinase Signaling Cascade and Is Required for Cell Wall Integrity in Saccharomyces cerevisiae

    OpenAIRE

    Scrimale, Thomas; Didone, Louis; de Mesy Bentley, Karen L.; Krysan, Damian J.

    2009-01-01

    The yeast cell wall is an extracellular structure that is dependent on secretory and membrane proteins for its construction. We investigated the role of protein quality control mechanisms in cell wall integrity and found that the unfolded protein response (UPR) and, to a lesser extent, endoplasmic reticulum (ER)-associated degradation (ERAD) pathways are required for proper cell wall construction. Null mutation of IRE1, double mutation of ERAD components (hrd1Δ and ubc7Δ) and ire1Δ, or expres...

  20. Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein.

    Science.gov (United States)

    Garrity, P A; Rao, Y; Salecker, I; McGlade, J; Pawson, T; Zipursky, S L

    1996-05-31

    Mutations in the Drosophila gene dreadlocks (dock) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. Dock protein contains one SH2 and three SH3 domains, implicating it in tyrosine kinase signaling, and is highly related to the human proto-oncogene Nck. Dock expression is detected in R cell growth cones in the target region. We propose Dock transmits signals in the growth cone in response to guidance and targeting cues. These findings provide an important step for dissection of signaling pathways regulating growth cone motility.

  1. The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

    Science.gov (United States)

    Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

    2017-07-15

    Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

  2. Adrenocorticotropic Hormone (ACTH) Responses Require Actions of the Melanocortin-2 Receptor Accessory Protein on the Extracellular Surface of the Plasma Membrane.

    Science.gov (United States)

    Malik, Sundeep; Dolan, Terrance M; Maben, Zachary J; Hinkle, Patricia M

    2015-11-13

    The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with the MC2 receptor accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual antiparallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two antiparallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino terminus of the MC2 receptor. The data firmly establish that only the extracellular amino terminus (Nout) copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the Nout orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor was rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP-MRAP-receptor signaling unit forms higher order complexes and that these multimers signal. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    Science.gov (United States)

    Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K

    2014-07-01

    Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have

  4. Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins.

    Science.gov (United States)

    Schneiter, Roger; Cole, Charles N

    2010-01-01

    The nuclear envelope harbors numerous large proteinaceous channels, the nuclear pore complexes (NPCs), through which macromolecular exchange between the cytosol and the nucleoplasm occurs. This double-membrane nuclear envelope is continuous with the endoplasmic reticulum and thus functionally connected to such diverse processes as vesicular transport, protein maturation and lipid synthesis. Recent results obtained from studies in Saccharomyces cerevisiae indicate that assembly of the nuclear pore complex is functionally dependent upon maintenance of lipid homeostasis of the ER membrane. Previous work from one of our laboratories has revealed that an integral membrane protein Apq12 is important for the assembly of functional nuclear pores. Cells lacking APQ12 are viable but cannot grow at low temperatures, have aberrant NPCs and a defect in mRNA export. Remarkably, these defects in NPC assembly can be overcome by supplementing cells with a membrane fluidizing agent, benzyl alcohol, suggesting that Apq12 impacts the flexibility of the nuclear membrane, possibly by adjusting its lipid composition when cells are shifted to a reduced temperature. Our new study now expands these findings and reveals that an essential membrane protein, Brr6, shares at least partially overlapping functions with Apq12 and is also required for assembly of functional NPCs. A third nuclear envelope membrane protein, Brl1, is related to Brr6, and is also required for NPC assembly. Because maintenance of membrane homeostasis is essential for cellular survival, the fact that these three proteins are conserved in fungi that undergo closed mitoses, but are not found in metazoans or plants, may indicate that their functions are performed by proteins unrelated at the primary sequence level to Brr6, Brl1 and Apq12 in cells that disassemble their nuclear envelopes during mitosis.

  5. Dietary protein and carbohydrate requirement of juvenile Hawaiian limpet (Cellana sandwicensis Pease, 1861 fed practical diet

    Directory of Open Access Journals (Sweden)

    Nhan Thai Hua

    2016-10-01

    Full Text Available Abstract This study was conducted to evaluate dietary protein and carbohydrate requirement of juvenile Hawaiian limpets Cellana sandwincensis. A total of 64 juvenile limpets (3.12 ± 0.86 g were fed five different dietary protein levels ranging from 270 to 470 g kg−1 for 90 days. Carbohydrate and lipid levels were held constant at 180 and 49.7 g kg−1, respectively. Weight gain and growth rates of the animals did not differ significantly (P > 0.05 among the protein levels ranging from 270 g kg−1 (0.30 % day−1 to 470 g kg−1 (0.23 % day−1. Next, opihi were fed four diets with protein levels from 210 to 500 g kg−1 with a constant carbohydrate level at 120 g kg−1. Weight gain and specific growth rates of opihi increased with increasing dietary protein from 210 to 350 g kg−1, and significantly (P < 0.05 decreased at the 500 g kg−1 diet. Highest weight gain, growth rates, and protein efficiency ratio were achieved at 350 g kg−1. Elevated carbohydrate levels (180–370 g kg−1 produced a significant difference (P < 0.05 in growth. The fastest growth rates of animals were obtained with 270 g kg−1 (0.27 % day−1 and 320 g kg−1 (0.26 % day−1. The weight gain of animals fed 180 and 370 g kg−1 carbohydrate diets were significantly (P < 0.05 lower than those of animals fed 270 and 320 g kg−1. We conclude that about 350 g kg−1 protein and 320 g kg−1 carbohydrate levels could be used for opihi.

  6. Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

    DEFF Research Database (Denmark)

    Dherin, Claudine; Gueneau, Emeric; Francin, Mathilde

    2009-01-01

    Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2......, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting...... protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K(d) values ranging from 8.1 to 17.4 microM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2...

  7. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

    Directory of Open Access Journals (Sweden)

    Cody Caba

    2018-02-01

    Full Text Available Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI, the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin. A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

  8. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation.

    Science.gov (United States)

    Caba, Cody; Ali Khan, Hyder; Auld, Janeen; Ushioda, Ryo; Araki, Kazutaka; Nagata, Kazuhiro; Mutus, Bulent

    2018-01-01

    Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys 57 and Lys 401 of human PDI in vitro . Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys 57 and Lys 401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys 57 and acLys 401 . The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

  9. Energy and protein requirements of weaned male and female Saanen goats.

    Science.gov (United States)

    Figueiredo, F O M; Leite, R F; St-Pierre, N R; Resende, K T; Almeida, A K; Souza, A P; Teixeira, I A M A

    2017-10-01

    The objective of this research was to estimate the energy and protein requirements for maintenance and growth in male (castrated and intact) and female Saanen goat kids between 15 and 30 kg BW. To determine the net energy requirements for maintenance (NE m ) and the net protein requirements for maintenance (NP m ), 75 goats (25 castrated and 26 intact males and 24 females) were used. Twenty-one goats (seven castrated and eight intact males and six females) were randomly assigned for slaughter to estimate the initial empty body composition. The 54 remaining animals (18 castrated and 18 intact males and 18 females) were randomly assigned in a split-plot design using a 3 × 3 factorial arrangement with three sexes and three levels of intake (ad libitum and restricted feed to 75% or 50% of the ad libitum intake). Within each sex, six blocks (three goats per block) were formed and one goat was randomly assigned to each level of intake. The 75% and the 50% of ad libitum rationing were determined daily, based on the DMI of the animal fed ad libitum on the previous day. All animals within block were slaughtered when the animal fed ad libitum reached 30 kg BW. The net energy requirements for gain (NE g ) and the net protein requirements for gain (NP g ) were obtained using 58 animals (20 castrated and 20 intact males and 18 females). The animals were fed ad libitum and slaughtered at targeted BW (15, 23 or 30 kg). Sex did not affect NE g and NP m (277.8 kJ/kg 0.75  BW day and 2.98 g CP/kg 0.75  BW day respectively), as well as NP g (180.9 ± 6.48 g/kg EBW gain) in Saanen goat kids. However, castrated males and females had similar NE g (varied from 12.6 ± 0.424 to 17.9 ± 1.38 MJ/kg EBW gain), greater than intact males (varied from 9.74 ± 0.420 to 10.7 ± 0.984 MJ/kg EBW gain), as the BW increased from 15 to 30 kg. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

  10. Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production.

    Science.gov (United States)

    Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion.

    Directory of Open Access Journals (Sweden)

    Loïc Etienne

    Full Text Available Hepatitis C virus (HCV assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis.

  12. G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis.

    Directory of Open Access Journals (Sweden)

    Run Lei

    Full Text Available The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development.

  13. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.

    2010-05-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  14. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.; Ausí n, Israel; Johnson, Lianna M.; Vashisht, Ajay  A Amar; Zhu, Jian-Kang; Wohlschlegel, James  A A.; Jacobsen, Steven E.

    2010-01-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  15. Mitochondrial energy metabolism is required for lifespan extension by the spastic paraplegia-associated protein spartin

    Directory of Open Access Journals (Sweden)

    Julia Ring

    2017-11-01

    Full Text Available Hereditary spastic paraplegias, a group of neurodegenerative disorders, can be caused by loss-of-function mutations in the protein spartin. However, the physiological role of spartin remains largely elusive. Here we show that heterologous expression of human or Drosophila spartin extends chronological lifespan of yeast, reducing age-associated ROS production, apoptosis, and necrosis. We demonstrate that spartin localizes to the proximity of mitochondria and physically interacts with proteins related to mitochondrial and respiratory metabolism. Interestingly, Nde1, the mitochondrial external NADH dehydrogenase, and Pda1, the core enzyme of the pyruvate dehydrogenase complex, are required for spartin-mediated cytoprotection. Furthermore, spartin interacts with the glycolysis enhancer phospo-fructo-kinase-2,6 (Pfk26 and is sufficient to complement for PFK26-deficiency at least in early aging. We conclude that mitochondria-related energy metabolism is crucial for spartin’s vital function during aging and uncover a network of specific interactors required for this function.

  16. Structural requirements for cub domain containing protein 1 (CDCP1 and Src dependent cell transformation.

    Directory of Open Access Journals (Sweden)

    Gwendlyn Kollmorgen

    Full Text Available Cub domain containing protein 1 (CDCP1 is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762 abolished transformation, and mutation of a palmitoylation motif (C689,690G strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.

  17. Glycosylphosphatidylinositol-anchored proteins are required for cell wall synthesis and morphogenesis in Arabidopsis.

    Science.gov (United States)

    Gillmor, C Stewart; Lukowitz, Wolfgang; Brininstool, Ginger; Sedbrook, John C; Hamann, Thorsten; Poindexter, Patricia; Somerville, Chris

    2005-04-01

    Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of pectins, xyloglucans, and callose. Furthermore, pnt1 pollen is less viable than the wild type, and pnt1 embryos were delayed in morphogenesis and showed defects in shoot and root meristems. The PNT1 gene encodes the Arabidopsis thaliana homolog of mammalian PIG-M, an endoplasmic reticulum-localized mannosyltransferase that is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. All five pnt mutants showed strongly reduced accumulation of GPI-anchored proteins, suggesting that they all have defects in GPI anchor synthesis. Although the mutants are seedling lethal, pnt1 cells are able to proliferate for a limited time as undifferentiated callus and do not show the massive deposition of ectopic cell wall material seen in pnt1 embryos. The different phenotype of pnt1 cells in embryos and callus suggest a differential requirement for GPI-anchored proteins in cell wall synthesis in these two tissues and points to the importance of GPI anchoring in coordinated multicellular growth.

  18. Mosaic amino acid conservation in 3D-structures of surface protein and polymerase of hepatitis B virus

    NARCIS (Netherlands)

    van Hemert, Formijn J.; Zaaijer, Hans L.; Berkhout, Ben; Lukashov, Vladimir V.

    2008-01-01

    Surface protein and polymerase of hepatitis B virus provide a striking example of gene overlap. Inclusion of more coding constraints in the phylogenetic analysis forces the tree toward accepted topology. Three-dimensional protein modeling demonstrates that participation in local protein function

  19. 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

  20. Niche-Specific Requirement for Hyphal Wall protein 1 in Virulence of Candida albicans

    Science.gov (United States)

    Staab, Janet F.; Datta, Kausik; Rhee, Peter

    2013-01-01

    Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa. PMID:24260489

  1. The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

    International Nuclear Information System (INIS)

    Shen, Peter S.; Enderlein, Dirk; Nelson, Christian D.S.; Carter, Weston S.; Kawano, Masaaki; Xing Li; Swenson, Robert D.; Olson, Norman H.; Baker, Timothy S.; Cheng, R. Holland; Atwood, Walter J.; Johne, Reimar; Belnap, David M.

    2011-01-01

    Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting β-hairpin observed in other polyomaviruses. We postulate that the terminal β-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

  2. A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities.

    Science.gov (United States)

    Bastien, Olivier; Ortet, Philippe; Roy, Sylvaine; Maréchal, Eric

    2005-03-10

    Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic reconstruction. We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space) and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP) allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny. The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations.

  3. A configuration space of homologous proteins conserving mutual information and allowing a phylogeny inference based on pair-wise Z-score probabilities

    Directory of Open Access Journals (Sweden)

    Maréchal Eric

    2005-03-01

    Full Text Available Abstract Background Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons and be the basis for a novel method of consistent and stable phylogenetic reconstruction. Results We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny. Conclusion The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations.

  4. The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses

    NARCIS (Netherlands)

    K. Overweg (Karin); A. Kerr; M. Sluijter (Marcel); M.H. Jackson; T.J. Mitchell; A.P. de Jong; R. de Groot (Ronald); P.W.M. Hermans (Peter)

    2000-01-01

    textabstractSurface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune

  5. Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

    Science.gov (United States)

    Clemens, Michael J; Elia, Androulla; Morley, Simon J

    2013-01-01

    The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

  6. Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics.

    Directory of Open Access Journals (Sweden)

    Ram H Nagaraj

    Full Text Available Methylglyoxal (MGO is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12 is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 µM, R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.

  7. Phylogenetic analysis and protein structure modelling identifies distinct Ca(2+)/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species.

    Science.gov (United States)

    Pittman, Jon K; Hirschi, Kendal D

    2016-12-01

    The Ca(2+)/Cation Antiporter (CaCA) superfamily is an ancient and widespread family of ion-coupled cation transporters found in nearly all kingdoms of life. In animals, K(+)-dependent and K(+)-indendent Na(+)/Ca(2+) exchangers (NCKX and NCX) are important CaCA members. Recently it was proposed that all rice and Arabidopsis CaCA proteins should be classified as NCX proteins. Here we performed phylogenetic analysis of CaCA genes and protein structure homology modelling to further characterise members of this transporter superfamily. Phylogenetic analysis of rice and Arabidopsis CaCAs in comparison with selected CaCA members from non-plant species demonstrated that these genes form clearly distinct families, with the H(+)/Cation exchanger (CAX) and cation/Ca(2+) exchanger (CCX) families dominant in higher plants but the NCKX and NCX families absent. NCX-related Mg(2+)/H(+) exchanger (MHX) and CAX-related Na(+)/Ca(2+) exchanger-like (NCL) proteins are instead present. Analysis of genomes of ten closely-related rice species and four Arabidopsis-related species found that CaCA gene family structures are highly conserved within related plants, apart from minor variation. Protein structures were modelled for OsCAX1a and OsMHX1. Despite exhibiting broad structural conservation, there are clear structural differences observed between the different CaCA types. Members of the CaCA superfamily form clearly distinct families with different phylogenetic, structural and functional characteristics, and therefore should not be simply classified as NCX proteins, which should remain as a separate gene family.

  8. Two waves of proteasome-dependent protein degradation in the hippocampus are required for recognition memory consolidation.

    Science.gov (United States)

    Figueiredo, Luciana S; Dornelles, Arethuza S; Petry, Fernanda S; Falavigna, Lucio; Dargél, Vinicius A; Köbe, Luiza M; Aguzzoli, Cristiano; Roesler, Rafael; Schröder, Nadja

    2015-04-01

    Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection.

    Science.gov (United States)

    Bakre, Abhijeet A; Harcourt, Jennifer L; Haynes, Lia M; Anderson, Larry J; Tripp, Ralph A

    2017-07-03

    Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182-186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting.

  10. Dsl1p, Tip20p, and the novel Dsl3(Sec39) protein are required for the stability of the Q/t-SNARE complex at the endoplasmic reticulum in yeast

    DEFF Research Database (Denmark)

    Kraynack, Bryan A; Chan, Angela; Rosenthal, Eva Helga

    2005-01-01

    The "Dsl1p complex" in Saccharomyces cerevisiae, consisting of Dsl1p and Tip20p, is involved in Golgi-ER retrograde transport and it is functionally conserved from yeast to mammalian cells. To further characterize this complex, we analyzed the function of Dsl3p, a protein that interacts with Dsl1p...... in yeast two hybrids screens. DSL3, recently identified in a genome wide analysis of essential genes as SEC39, encodes a cytosolic protein of 82 kDa that is peripherally associated with membranes derived from the ER. There is strong genetic interaction between DSL3 and other factors required for Golgi...

  11. Impaired mitotic progression and preimplantation lethality in mice lacking OMCG1, a new evolutionarily conserved nuclear protein

    DEFF Research Database (Denmark)

    Artus, Jérôme; Vandormael-Pournin, Sandrine; Frödin, Morten

    2005-01-01

    While highly conserved through evolution, the cell cycle has been extensively modified to adapt to new developmental programs. Recently, analyses of mouse mutants revealed that several important cell cycle regulators are either dispensable for development or have a tissue- or cell-type-specific f...

  12. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

    Directory of Open Access Journals (Sweden)

    Moffatt Barbara A

    2010-08-01

    Full Text Available Abstract Background Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB for coplanar aromatic motifs similar to those found in known glycan-binding proteins. Results The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192 in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Conclusions Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  13. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d.

    Science.gov (United States)

    Doxey, Andrew C; Cheng, Zhenyu; Moffatt, Barbara A; McConkey, Brendan J

    2010-08-03

    Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  14. Folding behavior of four silks of giant honey bee reflects the evolutionary conservation of aculeate silk proteins.

    Science.gov (United States)

    Maitip, Jakkrawut; Trueman, Holly E; Kaehler, Benjamin D; Huttley, Gavin A; Chantawannakul, Panuwan; Sutherland, Tara D

    2015-04-01

    Multiple gene duplication events in the precursor of the Aculeata (bees, ants, hornets) gave rise to four silk genes. Whilst these homologs encode proteins with similar amino acid composition and coiled coil structure, the retention of all four homologs implies they each are important. In this study we identified, produced and characterized the four silk proteins from Apis dorsata, the giant Asian honeybee. The proteins were readily purified, allowing us to investigate the folding behavior of solutions of individual proteins in comparison to mixtures of all four proteins at concentrations where they assemble into their native coiled coil structure. In contrast to solutions of any one protein type, solutions of a mixture of the four proteins formed coiled coils that were stable against dilution and detergent denaturation. The results are consistent with the formation of a heteromeric coiled coil protein complex. The mechanism of silk protein coiled coil formation and evolution is discussed in light of these results. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana.

    Science.gov (United States)

    McClure, B; Mou, B; Canevascini, S; Bernatzky, R

    1999-11-09

    Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S(C10)-RNase in SI N. alata S(C10)S(C10) and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia x SI N. alata S(C10)S(C10)) hybrids with reduced levels of HT-protein continued to express S(C10)-RNase but failed to reject S(C10)-pollen. Control hybrids expressing both S(C10)-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

  16. Rybp, a polycomb complex-associated protein, is required for mouse eye development

    Directory of Open Access Journals (Sweden)

    Schreiber-Agus Nicole

    2007-04-01

    Full Text Available Abstract Background Rybp (Ring1 and YY1 binding protein is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of Rybp in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for Rybp in ocular development using four in vivo mouse models which resulted in either the ablation or overexpression of Rybp. Results Our results demonstrate that loss of a single Rybp allele in conventional knockout mice often resulted in retinal coloboma, an incomplete closure of the optic fissure, characterized by perturbed localization of Pax6 but not of Pax2. In addition, about one half of Rybp-/- Rybp+/+ chimeric embryos also developed retinal colobomas and malformed lenses. Tissue-specific transgenic overexpression of Rybp in the lens resulted in abnormal fiber cell differentiation and severe lens opacification with increased levels of AP-2α and Sox2, and reduced levels of βA4-crystallin gene expression. Ubiquitous transgenic overexpression of Rybp in the entire eye caused abnormal retinal folds, corneal neovascularization, and lens opacification. Additional changes included defects in anterior eye development. Conclusion These studies establish Rybp as a novel gene that has been associated with coloboma. Other genes linked to coloboma encode various classes of transcription factors such as BCOR, CBP, Chx10, Pax2, Pax6, Six3, Ski, Vax1 and Vax2. We propose that the multiple functions for Rybp in regulating mouse retinal and lens development are mediated by genetic, epigenetic and physical interactions between these genes and proteins.

  17. Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

    International Nuclear Information System (INIS)

    Korzhnev, Dmitry M.; Neculai, Dante; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.; Bezsonova, Irina

    2016-01-01

    HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

  18. Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

    Energy Technology Data Exchange (ETDEWEB)

    Korzhnev, Dmitry M. [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States); Neculai, Dante [Zhejiang University, School of Medicine (China); Dhe-Paganon, Sirano [Dana-Farber Cancer Institute, Department of Cancer Biology (United States); Arrowsmith, Cheryl H. [University of Toronto, Structural Genomics Consortium (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States)

    2016-11-15

    HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

  19. Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

    Science.gov (United States)

    Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

    2016-03-15

    Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

  20. Domain requirements for the Dock adapter protein in growth- cone signaling.

    Science.gov (United States)

    Rao, Y; Zipursky, S L

    1998-03-03

    Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons.

  1. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

    Directory of Open Access Journals (Sweden)

    Berlin Londono-Renteria

    2015-10-01

    Full Text Available Dengue virus (DENV is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379, whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

  2. Digestible protein requirement for Nile tilapia fed with rations based on soybean meal and corn

    Directory of Open Access Journals (Sweden)

    William Franco Carneiro

    2017-09-01

    Full Text Available The objective was to determine the requirement of digestible protein (DP for Nile tilapia fed with diets based on soybean meal and corn. Two hundred Nile tilapia juveniles, sexually reversed, were used for a trial period of 100 days. The animals were distributed in 20 boxes of 1000 L-1 in a random design with five treatments and four replications, the boxes were connected to a water recirculation system. Five isoenergetic diets were tested with different levels of digestible protein (DP: 20, 25, 30, 35 and 40%. At the end of the experimental period were evaluated the zootechnical performance data. A quadratic effect (p < 0.05 was shown to parameters of final weight, weight gain, final length, fillet yield and visceral fat with the best points that ranged from 28.3 to 29.9% of DP. The largest area of hepatocytes was found to the lowest levels of DP in the diet (20 and 25% compared to the other levels, which were similar. Thus, Nile tilapia has adequate performance for the consumption of diets based on soybean and corn meal and the recommended level is 28.3% of PD in these conditions.

  3. Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase

    OpenAIRE

    Wang, Yong-Xiang; Luo, Cheng; Zhao, Dan; Beck, Jürgen; Nassal, Michael

    2012-01-01

    Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their small DNA genomes through protein-primed reverse transcription, mediated by the terminal protein (TP) domain in their P proteins and an RNA stem-loop, ϵ, on the pregenomic RNA (pgRNA). No direct structural data are available for P proteins, but their reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box A to box G), implying a similar architecture; however, experimental support for...

  4. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5′-phosphate-binding protein YggS

    OpenAIRE

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-01-01

    Escherichia coli YggS is a highly conserved pyridoxal 5′-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/M...

  5. Computational analysis of perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus [version 2; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    2016-09-01

    Full Text Available The dramatic transformation of the Zika virus (ZIKV from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV, finally culminating in a vaccine registered for use in endemic regions (CYD-TDV in three countries, provides key insights in developing strategies for tackling ZIKV, which has caused global panic to microcephaly and Guillain-Barre Syndrome. Dengue virus (DENV, a member of the family Flaviviridae, the causal agent of the self-limiting Dengue fever and the potentially fatal hemorrhagic fever/dengue shock syndrome, has been a scourge in tropical countries for many centuries. The recently solved structure of mature ZIKV (PDB ID:5IRE has provided key insights into the structure of the envelope (E and membrane (M proteins, the primary target of neutralizing antibodies. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc. (MEPPitope. These residues are overwhelmingly conserved in ZIKV and all DENV serotypes, and also enumerates residue pairs that undergo significant polarity reversal. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288 that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40, with

  6. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    Directory of Open Access Journals (Sweden)

    Peter eMoffett

    2015-08-01

    Full Text Available Potato cyst nematodes (PCNs, including Globodera rostochiensis (Woll., are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC family. SPRYSEC proteins are unique to members of the genera Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense response in N. tabacum, and tobacco was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

  7. FeatureMap3D - a tool to map protein features and sequence conservation onto homologous structures in the PDB

    DEFF Research Database (Denmark)

    Wernersson, Rasmus; Rapacki, Krzysztof; Stærfeldt, Hans Henrik

    2006-01-01

    FeatureMap3D is a web-based tool that maps protein features onto 3D structures. The user provides sequences annotated with any feature of interest, such as post-translational modifications, protease cleavage sites or exonic structure and FeatureMap3D will then search the Protein Data Bank (PDB) f...

  8. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    Science.gov (United States)

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins that are delivered to the apoplast, as well as...

  9. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    KAUST Repository

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bé lair, Guy; Moffett, Peter

    2015-01-01

    in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic

  10. Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

    KAUST Repository

    Ali, Shawkat

    2015-08-11

    Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

  11. The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1.

    Science.gov (United States)

    Toczyski, D P; Steitz, J A

    1993-01-01

    EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function. Images PMID:8380232

  12. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

    2015-01-01

    Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells ofte...... or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/....

  13. Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa).

    Science.gov (United States)

    Li, Xiao-Jie; Zhang, Ya-Feng; Hou, Mingming; Sun, Feng; Shen, Yun; Xiu, Zhi-Hui; Wang, Xiaomin; Chen, Zong-Liang; Sun, Samuel S M; Small, Ian; Tan, Bao-Cai

    2014-09-01

    RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, altering the amino acid specified by the DNA sequence. Here we report the identification of a critical editing factor of mitochondrial nad7 transcript via molecular characterization of a small kernel 1 (smk1) mutant in Zea mays (maize). Mutations in Smk1 arrest both the embryo and endosperm development. Cloning of Smk1 indicates that it encodes an E-subclass pentatricopeptide repeat (PPR) protein that is targeted to mitochondria. Loss of SMK1 function abolishes the C → U editing at the nad7-836 site, leading to the retention of a proline codon that is edited to encode leucine in the wild type. The smk1 mutant showed dramatically reduced complex-I assembly and NADH dehydrogenase activity, and abnormal biogenesis of the mitochondria. Analysis of the ortholog in Oryza sativa (rice) reveals that rice SMK1 has a conserved function in C → U editing of the mitochondrial nad7-836 site. T-DNA knock-out mutants showed abnormal embryo and endosperm development, resulting in embryo or seedling lethality. The leucine at NAD7-279 is highly conserved from bacteria to flowering plants, and analysis of genome sequences from many plants revealed a molecular coevolution between the requirement for C → U editing at this site and the existence of an SMK1 homolog. These results demonstrate that Smk1 encodes a PPR-E protein that is required for nad7-836 editing, and this editing is critical to NAD7 function in complex-I assembly in mitochondria, and hence to embryo and endosperm development in maize and rice. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  14. EBNA-2 of herpesvirus papio diverges significantly from the type A and type B EBNA-2 proteins of Epstein-Barr virus but retains an efficient transactivation domain with a conserved hydrophobic motif.

    Science.gov (United States)

    Ling, P D; Ryon, J J; Hayward, S D

    1993-01-01

    EBNA-2 contributes to the establishment of Epstein-Barr virus (EBV) latency in B cells and to the resultant alterations in B-cell growth pattern by up-regulating expression from specific viral and cellular promoters. We have taken a comparative approach toward characterizing functional domains within EBNA-2. To this end, we have cloned and sequenced the EBNA-2 gene from the closely related baboon virus herpesvirus papio (HVP). All human EBV isolates have either a type A or type B EBNA-2 gene. However, the HVP EBNA-2 gene falls into neither the type A category nor the type B category, suggesting that the separation into these two subtypes may have been a recent evolutionary event. Comparison of the predicted amino acid sequences indicates 37% amino acid identity with EBV type A EBNA-2 and 35% amino acid identity with type B EBNA-2. To define the domains of EBNA-2 required for transcriptional activation, the DNA binding domain of the GAL4 protein was fused to overlapping segments of EBV EBNA-2. This approach identified a 40-amino-acid (40-aa) EBNA-2 activation domain located between aa 437 and 477. Transactivation ability was completely lost when the amino-terminal boundary of this domain was moved to aa 441, indicating that the motif at aa 437 to 440, Pro-Ile-Leu-Phe, contains residues critical for function. The aa 437 boundary identified in these experiments coincides precisely with a block of conserved sequences in HVP EBNA-2, and the comparable carboxy-terminal region of HVP EBNA-2 also functioned as a strong transcriptional activation domain when fused to the Gal4(1-147) protein. The EBV and HVP EBNA-2 activation domains share a mixed proline-rich, negatively charged character with a striking conservation of positionally equivalent hydrophobic residues. The importance of the individual amino acids making up the Pro-Ile-Leu-Phe motif was examined by mutagenesis. Any alteration of these residues was found to reduce transactivation efficiency, with changes at the

  15. A cellulose synthase-like protein is required for osmotic stress tolerance in Arabidopsis

    KAUST Repository

    Zhu, Jianhua

    2010-04-16

    Osmotic stress imposed by soil salinity and drought stress significantly affects plant growth and development, but osmotic stress sensing and tolerance mechanisms are not well understood. Forward genetic screens using a root-bending assay have previously identified salt overly sensitive (sos) mutants of Arabidopsis that fall into five loci, SOS1 to SOS5. These loci are required for the regulation of ion homeostasis or cell expansion under salt stress, but do not play a major role in plant tolerance to the osmotic stress component of soil salinity or drought. Here we report an additional sos mutant, sos6-1, which defines a locus essential for osmotic stress tolerance. sos6-1 plants are hypersensitive to salt stress and osmotic stress imposed by mannitol or polyethylene glycol in culture media or by water deficit in the soil. SOS6 encodes a cellulose synthase-like protein, AtCSLD5. Only modest differences in cell wall chemical composition could be detected, but we found that sos6-1 mutant plants accumulate high levels of reactive oxygen species (ROS) under osmotic stress and are hypersensitive to the oxidative stress reagent methyl viologen. The results suggest that SOS6/AtCSLD5 is not required for normal plant growth and development but has a critical role in osmotic stress tolerance and this function likely involves its regulation of ROS under stress. © 2010 Blackwell Publishing Ltd.

  16. 40 CFR 174.516 - Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat protein of cucumber mosaic virus; exemption from the requirement of a tolerance. 174.516 Section 174.516 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance...

  17. 40 CFR 174.515 - Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Coat Protein of Papaya Ringspot Virus; exemption from the requirement of a tolerance. 174.515 Section 174.515 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance...

  18. 40 CFR 174.530 - Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry2Ae protein in cotton; temporary exemption from the requirement of a tolerance. 174.530 Section 174.530 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS...

  19. 40 CFR 174.502 - Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1A.105 protein; exemption from the requirement of a tolerance. 174.502 Section 174.502 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and...

  20. 40 CFR 174.504 - Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1F protein in cotton; exemption from the requirement of a tolerance. 174.504 Section 174.504 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and...

  1. 40 CFR 174.509 - Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry3A protein; exemption from the requirement of a tolerance. 174.509 Section 174.509 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance...

  2. 40 CFR 174.517 - Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry9C protein in corn; exemption from the requirement of a tolerance. 174.517 Section 174.517 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and...

  3. 40 CFR 174.520 - Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance. 174.520 Section 174.520 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES AND REQUIREMENTS FOR PLANT-INCORPORATED PROTECTANTS Tolerances and...

  4. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    Science.gov (United States)

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria

    Directory of Open Access Journals (Sweden)

    Muscariello Lidia

    2006-05-01

    Full Text Available Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters. Conclusion We propose that the CscA, CscB, CscC and Csc

  6. Cloning, purification and preliminary crystallographic analysis of a conserved hypothetical protein, SA0961 (YlaN), from Staphylococcus aureus

    International Nuclear Information System (INIS)

    Xu, Ling; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rice, David W.

    2006-01-01

    SA0961 is an unknown hypothetical protein from Staphylococcus aureus that can be identified in the Firmicutes division of Gram-positive bacteria. SA0961 was cloned and the protein was overexpressed in Escherichia coli, purified and subsequently crystallized. SA0961 is an unknown hypothetical protein from Staphylococcus aureus that can be identified in the Firmicutes division of Gram-positive bacteria. The gene for the homologue of SA0961 in Bacillus subtilis, ylaN, has been shown to be essential for cell survival, thus identifying the protein encoded by this gene as a potential target for the development of novel antibiotics. SA0961 was cloned and the protein was overexpressed in Escherichia coli, purified and subsequently crystallized. Crystals of selenomethionine-labelled SA0961 diffract to beyond 2.4 Å resolution and belong to the monoclinic space group P2 1 , with unit-cell parameters a = 31.5, b = 42.7, c = 62.7 Å, β = 92.4° and two molecules in the asymmetric unit. A full structure determination is under way to provide insights into the function of this protein

  7. Cloning, purification and preliminary crystallographic analysis of a conserved hypothetical protein, SA0961 (YlaN), from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ling; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rice, David W., E-mail: d.rice@sheffield.ac.uk [Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN (United Kingdom)

    2006-08-01

    SA0961 is an unknown hypothetical protein from Staphylococcus aureus that can be identified in the Firmicutes division of Gram-positive bacteria. SA0961 was cloned and the protein was overexpressed in Escherichia coli, purified and subsequently crystallized. SA0961 is an unknown hypothetical protein from Staphylococcus aureus that can be identified in the Firmicutes division of Gram-positive bacteria. The gene for the homologue of SA0961 in Bacillus subtilis, ylaN, has been shown to be essential for cell survival, thus identifying the protein encoded by this gene as a potential target for the development of novel antibiotics. SA0961 was cloned and the protein was overexpressed in Escherichia coli, purified and subsequently crystallized. Crystals of selenomethionine-labelled SA0961 diffract to beyond 2.4 Å resolution and belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 31.5, b = 42.7, c = 62.7 Å, β = 92.4° and two molecules in the asymmetric unit. A full structure determination is under way to provide insights into the function of this protein.

  8. Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

    Science.gov (United States)

    Abraham, Anup; Chandler, Douglas E

    2017-10-01

    Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

  9. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The Endocytic Recycling Regulatory Protein EHD1 Is Required for Ocular Lens Development

    Science.gov (United States)

    Arya, Priyanka; Rainey, Mark A.; Bhattacharyya, Sohinee; Mohapatra, Bhopal; George, Manju; Kuracha, Murali R; Storck, Matthew D.; Band, Vimla; Govindarajan, Venkatesh; Band, Hamid

    2015-01-01

    The C-terminal Eps15 homology domain-containing (EHD) proteins play a key role in endocytic recycling, a fundamental cellular process that ensures the return of endocytosed membrane components and receptors back to the cell surface. To define the in vivo biological functions of EHD1, we have generated Ehd1 knockout mice and previously reported a requirement of EHD1 for spermatogenesis. Here, we show that approximately 56% of the Ehd1-null mice displayed gross ocular abnormalities, including anophthalmia, aphakia, microphthalmia and congenital cataracts. Histological characterization of ocular abnormalities showed pleiotropic defects that include a smaller or absent lens, persistence of lens stalk and hyaloid vasculature, and deformed optic cups. To test whether these profound ocular defects resulted from the loss of EHD1 in the lens or in non-lenticular tissues, we deleted the Ehd1 gene selectively in the presumptive lens ectoderm using Le-Cre. Conditional Ehd1 deletion in the lens resulted in developmental defects that included thin epithelial layers, small lenses and absence of corneal endothelium. Ehd1 deletion in the lens also resulted in reduced lens epithelial proliferation, survival and expression of junctional proteins E-cadherin and ZO-1. Finally, Le-Cre-mediated deletion of Ehd1 in the lens led to defects in corneal endothelial differentiation. Taken together, these data reveal a unique role for EHD1 in early lens development and suggest a previously unknown link between the endocytic recycling pathway and regulation of key developmental processes including proliferation, differentiation and morphogenesis. PMID:26455409

  11. ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

    International Nuclear Information System (INIS)

    Zhang Yongfang; Xia Zongqin; Hu Ya'er

    2010-01-01

    Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

  12. Conserved regulatory modules in the Sox9 testis-specific enhancer predict roles for SOX, TCF/LEF, Forkhead, DMRT, and GATA proteins in vertebrate sex determination.

    Science.gov (United States)

    Bagheri-Fam, Stefan; Sinclair, Andrew H; Koopman, Peter; Harley, Vincent R

    2010-03-01

    While the primary sex determining switch varies between vertebrate species, a key downstream event in testicular development, namely the male-specific up-regulation of Sox9, is conserved. To date, only two sex determining switch genes have been identified, Sry in mammals and the Dmrt1-related gene Dmy (Dmrt1bY) in the medaka fish Oryzias latipes. In mice, Sox9 expression is evidently up-regulated by SRY and maintained by SOX9 both of which directly activate the core 1.3 kb testis-specific enhancer of Sox9 (TESCO). How Sox9 expression is up-regulated and maintained in species without Sry (i.e. non-mammalian species) is not understood. In this study, we have undertaken an in-depth comparative genomics approach and show that TESCO contains an evolutionarily conserved region (ECR) of 180 bp which is present in marsupials, monotremes, birds, reptiles and amphibians. The ECR contains highly conserved modules that predict regulatory roles for SOX, TCF/LEF, Forkhead, DMRT, and GATA proteins in vertebrate sex determination/differentiation. Our data suggest that tetrapods share common aspects of Sox9 regulation in the testis, despite having different sex determining switch mechanisms. They also suggest that Sox9 autoregulation is an ancient mechanism shared by all tetrapods, raising the possibility that in mammals, SRY evolved by mimicking this regulation. The validation of ECR regulatory sequences conserved from human to frogs will provide new insights into vertebrate sex determination. Copyright 2009 Elsevier Ltd. All rights reserved.

  13. Fluoxetine requires the endfeet protein aquaporin-4 to enhance plasticity of astrocyte processes

    Directory of Open Access Journals (Sweden)

    Barbara eDi Benedetto

    2016-02-01

    Full Text Available Morphological alterations in astrocytes are characteristic for post mortem brains of patients affected by major depressive disorder (MDD. Recently, a significant reduction in the coverage of blood vessels (BVs by aquaporin-4 (AQP-4-positive astrocyte endfeet has been shown in the prefrontal cortex (PFC of MDD patients, suggesting that either alterations in the morphology of endfeet or in AQP-4 distribution might be responsible for the disease phenotype or constitute a consequence of its progress. Antidepressant drugs (ADs regulate the expression of several proteins, including astrocyte-specific ones. Thus, they may target AQP-4 to induce morphological changes in astrocytes and restore their proper shape or relocate AQP-4 to endfeet. Using an animal model of depression, rats selectively bred for high anxiety-like behavior (HAB, we confirmed a reduced coverage of BVs in the adult PFC by AQP-4-immunoreactive (AQP-4-IR astrocyte processes with respect to nonselected Wistar rats (NAB, thereby validating it for our study. A further evaluation of the morphology of astrocyte in brain slices (ex vivo and in vitro using an antibody against the astrocyte-specific cytoskeletal protein glial fibrillary acidic protein (GFAP revealed that HAB astrocytes extended less processes than NAB cells. Furthermore, short-term drug treatment in vitro with the AD fluoxetine (FLX was sufficient to increase the plasticity of astrocyte processes, enhancing their number in NAB-derived cells and recovering their basal number in HAB-derived cells. This enhanced FLX-dependent plasticity occurred, however, only in the presence of intact AQP-4, as demonstrated by the lack of effect after the downregulation of AQP-4 with RNAi in both NAB and HAB cells. Nonetheless, a similar short-term treatment did neither modulate the coverage of BVs with AQP-4-positive astrocyte endfeet in NAB nor in HAB rats, although dosage and time of treatment were sufficient to fully recover GFAP expression

  14. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    International Nuclear Information System (INIS)

    Parent, Kristin N.; Tang, Jinghua; Cardone, Giovanni; Gilcrease, Eddie B.; Janssen, Mandy E.; Olson, Norman H.; Casjens, Sherwood R.; Baker, Timothy S.

    2014-01-01

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer

  15. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    Energy Technology Data Exchange (ETDEWEB)

    Parent, Kristin N., E-mail: kparent@msu.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Tang, Jinghua; Cardone, Giovanni [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Gilcrease, Eddie B. [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Janssen, Mandy E.; Olson, Norman H. [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); Casjens, Sherwood R., E-mail: sherwood.casjens@path.utah.edu [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Baker, Timothy S., E-mail: tsb@ucsd.edu [Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378 (United States); University of California, San Diego, Division of Biological Sciences, La Jolla, CA, 92093 (United States)

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  16. Protein requirement of young adult Nigerian females on habitual Nigerian diet at the usual level of energy intake.

    Science.gov (United States)

    Egun, G N; Atinmo, T

    1993-09-01

    A short-term N balance study was conducted in twelve healthy female adults aged 21-32 years to determine their protein requirement. Four dietary protein levels (0.3, 0.4, 0.5 and 0.6 g protein/kg per d) were used. Energy intake of the subjects was kept constant at 0.18 MJ/kg per d. All subjects maintained their normal activity throughout the study period. N excretion was determined from the measurements of N in a total collection of urine, faeces, sweat and menstrual fluid for each dietary period. N balance during the four protein levels were -15.15 (SD 5.95), -5.53 (SD 6.71), +6.15 (SD 4.76) and +12.05 (SD 8.63) mg N/kg per d for 0.3, 0.4, 0.5 and 0.6 g protein/kg per d respectively. The calculated average N requirements from regression analysis was 76.0 (SD 3.37) mg N/kg per d (0.48 g protein/kg per d). The estimate of allowance for individual variation to cover the 97.5% population was 95 mg N/kg per d (0.6 g protein/kg per d). The net protein utilization (NPU) of the diet was 0.55. When compared with a similar study with men, there was a significant difference in the protein requirement between sexes. Thus, the unjustifiable sex difference in the protein allowance recommended by the Food and Agriculture Organization/World Health Organization/United Nations University (1985) Expert Consultation group must be reviewed.

  17. Identification of Common Epitopes on a Conserved Region of NSs Proteins Among Tospoviruses of Watermelon silver mottle virus Serogroup.

    Science.gov (United States)

    Chen, Tsung-Chi; Huang, Ching-Wen; Kuo, Yan-Wen; Liu, Fang-Lin; Yuan, Chao-Hsiu Hsuan; Hsu, Hei-Ti; Yeh, Shyi-Dong

    2006-12-01

    ABSTRACT The NSs protein of Watermelon silver mottle virus (WSMoV) was expressed by a Zucchini yellow mosaic virus (ZYMV) vector in squash. The expressed NSs protein with a histidine tag and an additional NIa protease cleavage sequence was isolated by Ni(2+)-NTA resins as a free-form protein and further eluted after sodium dodecyl sulfate-polyacrylamide gel electrophoresis for production of rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum strongly reacted with the NSs crude antigen of WSMoV and weakly reacted with that of a high-temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV), but not with that of Calla lily chlorotic spot virus (CCSV). In contrast, the MAbs reacted strongly with all crude NSs antigens of WSMoV, CaCV, and CCSV. Various deletions of the NSs open reading frame were constructed and expressed by ZYMV vector. Results indicate that all three MAbs target the 89- to 125-amino-acid (aa) region of WSMoV NSs protein. Two indispensable residues of cysteine and lysine were essential for MAbs recognition. Sequence comparison of the deduced MAbs-recognized region with the reported tospoviral NSs proteins revealed the presence of a consensus sequence VRKPGVKNTGCKFTMHNQIFNPN (denoted WNSscon), at the 98- to 120-aa position of NSs proteins, sharing 86 to 100% identities among those of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus. A synthetic WNSscon peptide reacted with the MAbs and verified that the epitopes are present in the 98- to 120-aa region of WSMoV NSs protein. The WSMoV sero-group-specific NSs MAbs provide a means for reliable identification of tospoviruses in this large serogroup.

  18. Carboxy terminal region of the Fanconi anemia protein, FANCG/XRCC9, is required for functional activity.

    Science.gov (United States)

    Kuang, Y; Garcia-Higuera, I; Moran, A; Mondoux, M; Digweed, M; D'Andrea, A D

    2000-09-01

    Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with eight complementation groups. Four of the FA genes have been cloned, and at least three of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a nuclear complex, required for the maintenance of normal chromosome stability. In the current study, mutant forms of the FANCA and FANCG proteins have been generated and analyzed with respect to protein complex formation, nuclear translocation, and functional activity. The results demonstrate that the amino terminal two-thirds of FANCG (FANCG amino acids 1-428) binds to the amino terminal nuclear localization signal (NLS) of the FANCA protein. On the basis of 2-hybrid analysis, the FANCA/FANCG binding is a direct protein-protein interaction. Interestingly, a truncated mutant form of the FANCG protein, lacking the carboxy terminus, binds in a complex with FANCA and translocates to the nucleus; however, this mutant protein fails to bind to FANCC and fails to correct the mitomycin C sensitivity of an FA-G cell line. Taken together, these results demonstrate that binding of FANCG to the amino terminal FANCA NLS sequence is necessary but not sufficient for the functional activity of FANCG. Additional amino acid sequences at the carboxy terminus of FANCG are required for the binding of FANCC in the complex. (Blood. 2000;96:1625-1632)

  19. F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function

    Science.gov (United States)

    Pazhouhandeh, Maghsoud; Dieterle, Monika; Marrocco, Katia; Lechner, Esther; Berry, Bassam; Brault, Véronique; Hemmer, Odile; Kretsch, Thomas; Richards, Kenneth E.; Genschik, Pascal; Ziegler-Graff, Véronique

    2006-01-01

    Plants employ small RNA-mediated posttranscriptional gene silencing as a virus defense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode of action of most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 of two Arabidopsis-infecting poleroviruses interacts by means of a conserved minimal F-box motif with Arabidopsis thaliana orthologs of S-phase kinase-related protein 1 (SKP1), a component of the SCF family of ubiquitin E3 ligases. Point mutations in the F-box-like motif abolished the P0–SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity of P0. Knockdown of expression of a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus infection. Together, the results support a model in which P0 acts as an F-box protein that targets an essential component of the host posttranscriptional gene silencing machinery. PMID:16446454

  20. The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein

    Energy Technology Data Exchange (ETDEWEB)

    Hoenen, Antje [School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane (Australia); Gillespie, Leah [Department of Microbiology, La Trobe University, Melbourne (Australia); Department of Microbiology and Immunology, University of Melbourne, Melbourne (Australia); Morgan, Garry; Heide, Peter van der [Institute for Molecular Bioscience, University of Queensland, Brisbane (Australia); Khromykh, Alexander [School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane (Australia); Australian Infectious Diseases Research Centre, University of Queensland, Brisbane (Australia); Mackenzie, Jason, E-mail: jason.mackenzie@unimelb.edu.au [Department of Microbiology, La Trobe University, Melbourne (Australia); Department of Microbiology and Immunology, University of Melbourne, Melbourne (Australia)

    2014-01-05

    Flaviviruses have evolved means to evade host innate immune responses. Recent evidence suggests this is due to prevention of interferon production and signaling in flavivirus-infected cells. Here we show that the interferon-induced MxA protein can sequester the West Nile virus strain Kunjin virus (WNV{sub KUN}) capsid protein in cytoplasmic tubular structures in an expression-replication system. This sequestering resulted in reduced titers of secreted WNV{sub KUN} particles. We show by electron microscopy, tomography and 3D modeling that these cytoplasmic tubular structures form organized bundles. Additionally we show that recombinant ER-targeted MxA can restrict production of infectious WNV{sub KUN} under conditions of virus infection. Our results indicate a co-ordinated and compartmentalized WNV{sub KUN} assembly process may prevent recognition of viral components by MxA, particularly the capsid protein. This recognition can be exploited if MxA is targeted to intracellular sites of WNV{sub KUN} assembly. This results in further understanding of the mechanisms of flavivirus evasion from the immune system. - Highlights: • We show that the ISG MxA can recognize the West Nile virus capsid protein. • Interaction between WNV C protein and MxA induces cytoplasmic fibrils. • MxA can be retargeted to the ER to restrict WNV particle release. • WNV assembly process is a strategy to avoid MxA recognition.

  1. An effective approach for annotation of protein families with low sequence similarity and conserved motifs: identifying GDSL hydrolases across the plant kingdom.

    Science.gov (United States)

    Vujaklija, Ivan; Bielen, Ana; Paradžik, Tina; Biđin, Siniša; Goldstein, Pavle; Vujaklija, Dušica

    2016-02-18

    The massive accumulation of protein sequences arising from the rapid development of high-throughput sequencing, coupled with automatic annotation, results in high levels of incorrect annotations. In this study, we describe an approach to decrease annotation errors of protein families characterized by low overall sequence similarity. The GDSL lipolytic family comprises proteins with multifunctional properties and high potential for pharmaceutical and industrial applications. The number of proteins assigned to this family has increased rapidly over the last few years. In particular, the natural abundance of GDSL enzymes reported recently in plants indicates that they could be a good source of novel GDSL enzymes. We noticed that a significant proportion of annotated sequences lack specific GDSL motif(s) or catalytic residue(s). Here, we applied motif-based sequence analyses to identify enzymes possessing conserved GDSL motifs in selected proteomes across the plant kingdom. Motif-based HMM scanning (Viterbi decoding-VD and posterior decoding-PD) and the here described PD/VD protocol were successfully applied on 12 selected plant proteomes to identify sequences with GDSL motifs. A significant number of identified GDSL sequences were novel. Moreover, our scanning approach successfully detected protein sequences lacking at least one of the essential motifs (171/820) annotated by Pfam profile search (PfamA) as GDSL. Based on these analyses we provide a curated list of GDSL enzymes from the selected plants. CLANS clustering and phylogenetic analysis helped us to gain a better insight into the evolutionary relationship of all identified GDSL sequences. Three novel GDSL subfamilies as well as unreported variations in GDSL motifs were discovered in this study. In addition, analyses of selected proteomes showed a remarkable expansion of GDSL enzymes in the lycophyte, Selaginella moellendorffii. Finally, we provide a general motif-HMM scanner which is easily accessible through

  2. Normal protein intake is required for body weight loss and weight maintenance, and elevated protein intake for additional preservation of resting energy expenditure and fat free mass.

    Science.gov (United States)

    Soenen, Stijn; Martens, Eveline A P; Hochstenbach-Waelen, Ananda; Lemmens, Sofie G T; Westerterp-Plantenga, Margriet S

    2013-05-01

    Energy-restricted high-protein diets (HPDs) have shown favorable results for body weight (BW) management, yet studies differ in their outcomes depending on the dietary protein content. Our objective was to determine the effects of dietary protein content on BW loss-related variables during a 6-mo energy restriction with the use of diets containing protein at the level of requirement [normal-protein diet (NPD), 0.8 g · kg BW(-1) (.) d(-1)] and above (HPD, 1.2 g · kg BW(-1) (.) d(-1)). In overweight and obese participants (24 men and 48 women), BW, body composition, and metabolic responses were assessed before and after subsequent energy intakes of 100, 33, and 67% of the original individual daily energy requirements. Protein intake was consistent in the NPD (0.8 ± 0.3 g · kg BW(-1) (.) d(-1)) and HPD (1.2 ± 0.3 g · kg BW(-1) (.) d(-1)) groups throughout the study (P body fat mass similarly decreased in the NPD and HPD groups (P initial sparing effect of FFM and lowering of DBP.

  3. Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

    Science.gov (United States)

    Zhang, Limeng; Zhou, Xue; Fan, Ziyao; Tang, Wei; Chen, Liang; Dai, Jian; Wei, Yuhua; Zhang, Jianxin; Yang, Xuan; Yang, Xijing; Liu, Daolong; Yu, Liquan; Zhang, Hua; Wu, Zhijun; Yu, Yongzhong; Sun, Hunan; Cui, Yudong

    2015-01-01

    Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Protein Requirements Are Elevated in Endurance Athletes after Exercise as Determined by the Indicator Amino Acid Oxidation Method.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kato

    Full Text Available A higher protein intake has been recommended for endurance athletes compared with healthy non-exercising individuals based primarily on nitrogen balance methodology. The aim of this study was to determine the estimated average protein requirement and recommended protein intake in endurance athletes during an acute 3-d controlled training period using the indicator amino acid oxidation method. After 2-d of controlled diet (1.4 g protein/kg/d and training (10 and 5km/d, respectively, six male endurance-trained adults (28±4 y of age; Body weight, 64.5±10.0 kg; VO2peak, 60.3±6.7 ml·kg-1·min-1; means±SD performed an acute bout of endurance exercise (20 km treadmill run prior to consuming test diets providing variable amounts of protein (0.2-2.8 g·kg-1·d-1 and sufficient energy. Protein was provided as a crystalline amino acid mixture based on the composition of egg protein with [1-13C]phenylalanine provided to determine whole body phenylalanine flux, 13CO2 excretion, and phenylalanine oxidation. The estimated average protein requirement was determined as the breakpoint after biphasic linear regression analysis with a recommended protein intake defined as the upper 95% confidence interval. Phenylalanine flux (68.8±8.5 μmol·kg-1·h-1 was not affected by protein intake. 13CO2 excretion displayed a robust bi-phase linear relationship (R2 = 0.86 that resulted in an estimated average requirement and a recommended protein intake of 1.65 and 1.83 g protein·kg-1·d-1, respectively, which was similar to values based on phenylalanine oxidation (1.53 and 1.70 g·kg-1·d-1, respectively. We report a recommended protein intake that is greater than the RDA (0.8 g·kg-1·d-1 and current recommendations for endurance athletes (1.2-1.4 g·kg-1·d-1. Our results suggest that the metabolic demand for protein in endurance-trained adults on a higher volume training day is greater than their sedentary peers and current recommendations for athletes based

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

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-01

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

  6. Functional conservation and divergence of four ginger AP1/AGL9 MADS-box genes revealed by analysis of their expression and protein-protein interaction, and ectopic expression of AhFUL gene in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Xiumei Li

    Full Text Available Alpinia genus are known generally as ginger-lilies for showy flowers in the ginger family, Zingiberaceae, and their floral morphology diverges from typical monocotyledon flowers. However, little is known about the functions of ginger MADS-box genes in floral identity. In this study, four AP1/AGL9 MADS-box genes were cloned from Alpinia hainanensis, and protein-protein interactions (PPIs and roles of the four genes in floral homeotic conversion and in floral evolution are surveyed for the first time. AhFUL is clustered to the AP1 lineage, AhSEP4 and AhSEP3b to the SEP lineage, and AhAGL6-like to the AGL6 lineage. The four genes showed conserved and divergent expression patterns, and their encoded proteins were localized in the nucleus. Seven combinations of PPI (AhFUL-AhSEP4, AhFUL-AhAGL6-like, AhFUL-AhSEP3b, AhSEP4-AhAGL6-like, AhSEP4-AhSEP3b, AhAGL6-like-AhSEP3b, and AhSEP3b-AhSEP3b were detected, and the PPI patterns in the AP1/AGL9 lineage revealed that five of the 10 possible combinations are conserved and three are variable, while conclusions cannot yet be made regarding the other two. Ectopic expression of AhFUL in Arabidopsis thaliana led to early flowering and floral organ homeotic conversion to sepal-like or leaf-like. Therefore, we conclude that the four A. hainanensis AP1/AGL9 genes show functional conservation and divergence in the floral identity from other MADS-box genes.

  7. Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

    Directory of Open Access Journals (Sweden)

    Edyta Marcon

    2014-07-01

    Full Text Available Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  8. 14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

    DEFF Research Database (Denmark)

    Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil

    2012-01-01

    Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...... and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate...

  9. ENERGY AND PROTEIN REQUIREMENTS OF GROWING PELIBUEY SHEEP UNDER TROPICAL CONDITIONS ESTIMATED FROM A LITERATURE DATABASE ANALYSES

    Directory of Open Access Journals (Sweden)

    Fernando Duarte

    2012-01-01

    Full Text Available Data from previous studies were used to estimate the metabolizable energy and protein requirements for maintenance and growth and basal metabolism energy requirement of male Pelibuey sheep under tropical conditions were estimated. In addition, empty body weight and mature weight of males and female Pelibuey sheep were also estimated. Basal metabolism energy requirements were estimated with the Cornell Net Carbohydrate and Protein System – Sheep (CNCPS-S model using the a1 factor of the maintenance equation. Mature weight was estimated to be 69 kg for males and 45 kg for females. Empty body weight was estimated to be 81% of live weight. Metabolizable energy and protein requirements for growth were 0.106 Mcal MEm/kg LW0.75 and 2.4 g MP/kg LW0.75 for males. The collected information did not allowed appropriate estimation of female requirements. The basal metabolism energy requirement was estimated to be 0.039 Mcal MEm/kg LW0.75. Energy requirements for basal metabolism were lower in Pelibuey sheep than those reported for wool breeds even though their total requirements were similar.

  10. A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1

    NARCIS (Netherlands)

    Prattes, M.; Loibl, M.; Zisser, G.; Luschnig, D.; Kappel, L.; Rossler, I.; Grassegger, M.; Hromic, A.; Krieger, E.; Gruber, K.; Pertschy, B.; Bergler, H.

    2017-01-01

    AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP

  11. Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

    Czech Academy of Sciences Publication Activity Database

    Beilharz, K.; Nováková, Linda; Fadda, D.; Branny, Pavel; Massida, O.; Veening, J.-W.

    2012-01-01

    Roč. 109, č. 15 (2012), s. 905-913 ISSN 0027-8424 R&D Projects: GA AV ČR IAA600200801; GA MŠk LH12055 Institutional research plan: CEZ:AV0Z50200510 Keywords : PENICILLIN-BINDING PROTEINS * SERINE/THREONINE KINASE * MYCOBACTERIUM- TUBERCULOSIS Subject RIV: EE - Microbiology, Virology Impact factor: 9.737, year: 2012

  12. Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Aashish Srivastava

    Full Text Available Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis, purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis, and an aminoacyl-tRNA synthetase (AARS mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens.

  13. Food and nutritional security requires adequate protein as well as energy, delivered from whole-year crop production.

    Science.gov (United States)

    Coles, Graeme D; Wratten, Stephen D; Porter, John R

    2016-01-01

    Human food security requires the production of sufficient quantities of both high-quality protein and dietary energy. In a series of case-studies from New Zealand, we show that while production of food ingredients from crops on arable land can meet human dietary energy requirements effectively, requirements for high-quality protein are met more efficiently by animal production from such land. We present a model that can be used to assess dietary energy and quality-corrected protein production from various crop and crop/animal production systems, and demonstrate its utility. We extend our analysis with an accompanying economic analysis of commercially-available, pre-prepared or simply-cooked foods that can be produced from our case-study crop and animal products. We calculate the per-person, per-day cost of both quality-corrected protein and dietary energy as provided in the processed foods. We conclude that mixed dairy/cropping systems provide the greatest quantity of high-quality protein per unit price to the consumer, have the highest food energy production and can support the dietary requirements of the highest number of people, when assessed as all-year-round production systems. Global food and nutritional security will largely be an outcome of national or regional agroeconomies addressing their own food needs. We hope that our model will be used for similar analyses of food production systems in other countries, agroecological zones and economies.

  14. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

    2015-01-01

    Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells often...... target diverse regions in highly variable viral pathogens and this diversity may need to be addressed through redefinition of suitable peptide targets. Methods: We have developed a method for antigen assessment and target selection for polyvalent vaccines, with which we identified immune epitopes from...... variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results: We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased...

  15. A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein

    International Nuclear Information System (INIS)

    Staiger, D.; Kaulen, H.; Schell, J.

    1989-01-01

    In the chalcone synthase gene of Antirrhinum majus (snapdragon), 150 base pairs of the 5' flanking region contain cis-acting signals for UV light-induced expression. A nuclear factor, designated CG-1, specifically recognizes a hexameric motif with internal dyad symmetry, CACGTG, located within this light-responsive sequence. Binding of CG-1 is influenced by C-methylation of the CpG dinucleotide in the recognition sequence. CG-1 is a factor found in a variety of dicotyledonous plant species including Nicotiana tabacum, A. majus, Petunia hybrida, Arabidopsis thaliana, and Glycine max. CACGTG motifs contained within trans-acting factor recognition sites in various other plant promoters can interact with CG-1. In addition, the binding site of the human adenovirus major late transcription factor USF can compete for CG-1 binding to the chalcone synthase promoter. This suggests an evolutionary conservation of trans-acting factor recognition sites involved in divergent mechanisms of gene control. (author)

  16. High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71.

    Science.gov (United States)

    Levengood, Jeffrey D; Tolbert, Michele; Li, Mei-Ling; Tolbert, Blanton S

    2013-07-01

    Human Enterovirus 71 (EV71) is an emerging pathogen of infectious disease and a serious threat to public health. Currently, there are no antivirals or vaccines to slow down or prevent EV71 infections, thus underscoring the urgency to better understand mechanisms of host-enterovirus interactions. EV71 uses a type I internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit via a pathway that requires the cytoplasmic localization of hnRNP A1, which acts as an IRES trans-activating factor. The mechanism of how hnRNP A1 trans activates EV71 RNA translation is unknown, however. Here, we report that the UP1 domain of hnRNP A1 interacts specifically with stem loop II (SLII) of the IRES, via a thermodynamically well-defined biphasic transition that involves conserved bulge 5'-AYAGY-3' and hairpin 5'-RY(U/A)CCA-3' loops. Calorimetric titrations of wild-type and mutant SLII constructs reveal these structural elements are essential to form a high-affinity UP1-SLII complex. Mutations that alter the bulge and hairpin primary or secondary structures abrogate the biphasic transition and destabilize the complex. Notably, mutations within the bulge that destabilize the complex correlate with a large reduction in IRES-dependent translational activity and impair EV71 replication. Taken together, this study shows that a conserved SLII structure is necessary to form a functional hnRNP A1-IRES complex, suggesting that small molecules that target this stem loop may have novel antiviral properties.

  17. 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 SA