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Sample records for biotrophy-associated secreted proteins

  1. Unconventional protein secretion.

    Science.gov (United States)

    Ding, Yu; Wang, Juan; Wang, Junqi; Stierhof, York-Dieter; Robinson, David G; Jiang, Liwen

    2012-10-01

    It is generally believed that protein secretion or exocytosis is achieved via a conventional ER (endoplasmic reticulum)-Golgi-TGN (trans-Golgi network)-PM (plasma membrane) pathway in the plant endomembrane system. However, such signal peptide (SP)-dependent protein secretion cannot explain the increasing number of SP-lacking proteins which are found outside of the PM in plant cells. The process by which such leaderless secretory proteins (LSPs) gain access to the cell exterior is termed unconventional protein secretion (UPS) and has been well-studied in animal and yeast cells, but largely ignored by the plant community. Here, we review the evidence for UPS in plants especially in regard to the recently discovered EXPO (exocyst-positive-organelle).

  2. Extracellular secretion of recombinant proteins

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    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  3. Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae

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    Krehenbrink Martin

    2008-01-01

    Full Text Available Abstract Background Proteins secreted by bacteria play an important role in infection of eukaryotic hosts. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. Proteins secreted during the infection process by some rhizobial strains can influence infection and modify the plant defence signalling pathways. The aim of this study was to systematically analyse protein secretion in the recently sequenced strain Rhizobium leguminosarum bv. viciae 3841. Results Similarity searches using defined protein secretion systems from other Gram-negative bacteria as query sequences revealed that R. l. bv. viciae 3841 has ten putative protein secretion systems. These are the general export pathway (GEP, a twin-arginine translocase (TAT secretion system, four separate Type I systems, one putative Type IV system and three Type V autotransporters. Mutations in genes encoding each of these (except the GEP were generated, but only mutations affecting the PrsDE (Type I and TAT systems were observed to affect the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatic analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type I substrates, 12 of which are secreted via the PrsDE, secretion system. The TAT mutant was defective for the symbiosis, forming nodules incapable of nitrogen fixation. Conclusion None of the R. l. bv. viciae 3841 protein secretion systems putatively involved in the secretion of proteins to the extracellular space (Type I, Type IV, Type V is required for establishing the symbiosis with legumes. The PrsDE (Type I system was shown to be the major route of protein secretion in non-symbiotic cells and to secrete proteins of widely varied size and predicted function. This is in contrast to many Type I systems from other bacteria, which typically secrete specific substrates encoded by genes often localised in close proximity to

  4. High throughput recombinant protein production of fungal secreted proteins

    DEFF Research Database (Denmark)

    Vala, Andrea Lages Lino; Roth, Doris; Grell, Morten Nedergaard

    2011-01-01

    a high-throughput protein production system with a special focus on fungal secreted proteins. We use a ligation independent cloning to clone target genes into expression vectors for E. coli and P. pastoris and a small scale test expression to identify constructs producing soluble protein. Expressed...... interaction), between fungi of the order Entomophthorales and aphids (pathogenic interaction), and in the mycoparasitic interaction between the oomycetes Pythium oligandrum and P. ultimum. In general, the high-throughput protein production system can lead to a better understanding of fungal/host interactions...

  5. Identification and characterization of secreted proteins in Eimeria tenella

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    Ramlee, Intan Azlinda; Firdaus-Raih, Mohd; Wan, Kiew-Lian

    2015-09-01

    Eimeria tenella is a protozoan parasite that causes coccidiosis, an economically important disease in the poultry industry. The characterization of proteins that are secreted by parasites have been shown to play important roles in parasite invasion and are considered to be potential control agents. In this study, 775 proteins potentially secreted by E. tenella were identified. These proteins were further filtered to remove mitochondrial proteins. Out of 763 putative secreted proteins, 259 proteins possess transmembrane domains while another 150 proteins have GPI (Glycosylphosphatidylinositol) anchors. Homology search revealed that 315 and 448 proteins have matches with known and hypothetical proteins in the database, respectively. Within this data set, previously characterized secretory proteins such as micronemes, rhoptry kinases and dense granules were detected.

  6. Lipid-independent secretion of a Drosophila Wnt protein.

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    Ching, Wendy; Hang, Howard C; Nusse, Roel

    2008-06-20

    Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt proteins. Interestingly, in this study we find that WntD, a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt proteins, we used a novel labeling method and mass spectrometry to ask whether WntD undergoes lipid modification and found that it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, we find that WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. Our results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt proteins.

  7. Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

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    Lee, Je Min, E-mail: jemin@knu.ac.kr [Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul (Korea, Republic of); Department of Horticultural Science, Kyungpook National University, Daegu (Korea, Republic of); Lee, Sang-Jik [Biotechnology Institute, Nongwoo Bio Co, Ltd, Yeoju (Korea, Republic of); Department of Plant Biology, Cornell University, Ithaca, NY (United States); Rose, Jocelyn K.C. [Department of Plant Biology, Cornell University, Ithaca, NY (United States); Yeam, Inhwa [Department of Horticulture and Breeding, Andong National University, Andong (Korea, Republic of); Kim, Byung-Dong [Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul (Korea, Republic of)

    2014-04-18

    Highlights: • Yeast secretion trap (YST) is a valuable tool for mining secretome. • A total of 80 secreted proteins are newly identified via YST in pepper fruits. • The secreted proteins are differentially regulated during pepper development and ripening. • Transient GFP-fusion assay and in planta secretion trap can effectively validate the secretion of proteins. - Abstract: Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.

  8. CELL-WALL GROWTH AND PROTEIN SECRETION IN FUNGI

    NARCIS (Netherlands)

    SIETSMA, JH; WOSTEN, HAB; WESSELS, JGH

    1995-01-01

    Secretion of proteins is a vital process in fungi. Because hyphal walls form a diffusion barrier for proteins, a mechanism different from diffusion probably exist to transport proteins across the wall. In Schizophyllum commune, evidence has been obtained for synthesis at the hyphal apex of wall comp

  9. The ESX system in Bacillus subtilis mediates protein secretion.

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    Laura A Huppert

    Full Text Available Esat-6 protein secretion systems (ESX or Ess are required for the virulence of several human pathogens, most notably Mycobacterium tuberculosis and Staphylococcus aureus. These secretion systems are defined by a conserved FtsK/SpoIIIE family ATPase and one or more WXG100 family secreted substrates. Gene clusters coding for ESX systems have been identified amongst many organisms including the highly tractable model system, Bacillus subtilis. In this study, we demonstrate that the B. subtilis yuk/yue locus codes for a nonessential ESX secretion system. We develop a functional secretion assay to demonstrate that each of the locus gene products is specifically required for secretion of the WXG100 virulence factor homolog, YukE. We then employ an unbiased approach to search for additional secreted substrates. By quantitative profiling of culture supernatants, we find that YukE may be the sole substrate that depends on the FtsK/SpoIIIE family ATPase for secretion. We discuss potential functional implications for secretion of a unique substrate.

  10. Midgut proteins released by microapocrine secretion in Spodoptera frugiperda.

    Science.gov (United States)

    Silva, Walciane; Cardoso, Christiane; Ribeiro, Alberto F; Terra, Walter R; Ferreira, Clélia

    2013-01-01

    Microapocrine vesicles bud from the lepidopteran midgut microvilli as double membrane vesicles. To identify the proteins secreted by this process, antibodies raised against isolated microapocrine vesicles from Spodoptera frugiperda were used for screening a midgut cDNA expression library. Positive clones were sequenced, assembled and N blasted against S. frugiperda sequences obtained by pyrosequencing midgut mRNA. This procedure led to the extension of microapocrine sequences that were annotated. A similar procedure was used to identify midgut microvillar proteins that necessarily are part of the microapocrine vesicle. Forty-eight proteins were associated with microvillar membranes. They pertain to 8 functional groups: digestive enzymes, peritrophic membrane, protection, transporters, receptors, secretory machinery, cytoskeleton and signaling, and unknown. Twenty-eight proteins are putatively secreted by microapocrine secretion. Most of them are digestive enzymes, but the list also includes proteins involved in protection and in peritrophic membrane formation. Among the identified digestive enzymes, aminopeptidases are typically microvillar and group into the classes 1, 2, 3, 5, and 6. There are two amylases secreted by microapocrine secretion: one is a digestive enzyme and the other is a transporter-like amylase with no clear function. One lipase has a predicted transmembrane loop, whereas the others are supposed to be secreted by microapocrine secretion and be digestive. Trypsin is membrane bound and is delivered by microapocrine secretion, but has no predicted features to bind membranes. It may remain bound through the signal peptide till be delivered into the midgut lumen. Proteins supposed to be involved in the microapocrine secretory machinery were: calmodulin, annexin, myosin 7a, and gelsolin 1. Their putative roles are discussed, but more research is necessary to settle this subject.

  11. Functional genomics to study protein secretion stress in Aspergillus niger

    NARCIS (Netherlands)

    Silva Pinheiro Carvalho, Neuza Daniela

    2011-01-01

    In this thesis we have focused our studies towards a better understanding of different processes involved in the protein secretion pathway that might act as bottlenecks for homologous and heterologous protein production. We have given particular attention to the molecular mechanisms of folding and q

  12. Proteomic analysis of Taenia solium metacestode excretion-secretion proteins.

    Science.gov (United States)

    Victor, Bjorn; Kanobana, Kirezi; Gabriël, Sarah; Polman, Katja; Deckers, Nynke; Dorny, Pierre; Deelder, André M; Palmblad, Magnus

    2012-06-01

    The metacestode larval stage of Taenia solium is the causal agent of a zoonotic disease called cysticercosis. The disease has an important impact on pork trade (due to porcine cysticercosis) and public health (due to human neurocysticercosis). In order to improve the current diagnostic tools and to get a better understanding of the interaction between T. solium metacestodes and their host, there is a need for more information about the proteins that are released by the parasite. In this study, we used protein sequences from different helminths, 1DE, reversed-phase LC, and MS/MS to analyze the excretion-secretion proteins produced by T. solium metacestodes from infected pigs. This is the first report of the T. solium metacestode excretion-secretion proteome. We report 76 proteins including 27 already described T. solium proteins, 17 host proteins and 32 proteins likely to be of T. solium origin, but identified using sequences from other helminths.

  13. Excreted/Secreted Proteins from Trypanosome Procyclic Strains

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    Celestine Michelle Atyame Nten

    2010-01-01

    Full Text Available Trypanosoma secretome was shown to be involved in parasite virulence and is suspected of interfering in parasite life-cycle steps such as establishment in the Glossina midgut, metacyclogenesis. Therefore, we attempted to identify the proteins secreted by procyclic strains of T. brucei gambiense and T. brucei brucei, responsible for human and animal trypanosomiasis, respectively. Using mass spectrometry, 427 and 483 nonredundant proteins were characterized in T. brucei brucei and T. brucei gambiense secretomes, respectively; 35% and 42% of the corresponding secretome proteins were specifically secreted by T. brucei brucei and T. brucei gambiense, respectively, while 279 proteins were common to both subspecies. The proteins were assigned to 12 functional classes. Special attention was paid to the most abundant proteases (14 families because of their potential implication in the infection process and nutrient supply. The presence of proteins usually secreted via an exosome pathway suggests that this type of process is involved in trypanosome ESP secretion. The overall results provide leads for further research to develop novel tools for blocking trypanosome transmission.

  14. Mechanism of Action of Secreted Newt Anterior Gradient Protein

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    Grassme, Kathrin S.; Garza-Garcia, Acely; Delgado, Jean-Paul; Godwin, James W.; Kumar, Anoop; Gates, Phillip B.; Brockes, Jeremy P.

    2016-01-01

    Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family. PMID:27100463

  15. Diversity and subcellular distribution of archaeal secreted proteins

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    Mechthild ePohlschroder

    2012-07-01

    Full Text Available Secreted proteins make up a significant percentage of a prokaryotic proteome and play critical roles in important cellular processes such as polymer degradation, nutrient uptake, signal transduction, cell wall biosynthesis and motility. The majority of archaeal proteins are believed to be secreted either in an unfolded conformation via the universally conserved Sec pathway or in a folded conformation via the Twin arginine transport (Tat pathway. Extensive in vivo and in silico analyses of N-terminal signal peptides that target proteins to these pathways have led to the development of computational tools that not only predict Sec and Tat substrates with high accuracy but also provide information about signal peptide processing and targeting. Predictions therefore include indications as to whether a substrate is a soluble secreted protein, a membrane or cell-wall anchored protein, or a surface structure subunit, and whether it is targeted for post-translational modification such as glycosylation or the addition of a lipid. The use of these in silico tools, in combination with biochemical and genetic analyses of transport pathways and their substrates, has resulted in improved predictions of the subcellular localization of archaeal secreted proteins, allowing for a more accurate annotation of archaeal proteomes, and has led to the identification of potential adaptations to extreme environments, as well as archaeal kingdom-specific pathways. A more comprehensive understanding of the transport pathways and post-translational modifications of secreted archaeal proteins will also generate invaluable insights that will facilitate the identification of commercially valuable archaeal enzymes and the development of heterologous systems in which to efficiently express them.

  16. Differentially expressed protein markers in human submandibular and sublingual secretions.

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    Hu, Shen; Denny, Patricia; Denny, Paul; Xie, Yongming; Loo, Joseph A; Wolinsky, Lawrence E; Li, Yang; McBride, Jim; Ogorzalek Loo, Rachel R; Navazesh, Mavash; Wong, David T

    2004-11-01

    Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

  17. Proteomic mapping of secreted proteins of Trichoderma spp.

    Institute of Scientific and Technical Information of China (English)

    Li S; Bramley P M; Smith J; Cannon P F

    2004-01-01

    @@ A series of highly taxonomically diverse Trichoderma strains were investigated using proteomic approaches, to investigate the utility of protein profiles as taxonomic markers and to identify proteins of potential economic importance. Initial studies have focused on a comparison of single strains of T.aureoviride, T. saturnisporum, T. polysporum, T. longbrachiatum and T. spirale, along with two strains of T. harzianum. All seven strains were grown in synthetic medium supplemented with 2 % (w/v) glycerol, to maximize the diversity of extracellular protein production. Samples of secreted protein were separated by 2D gel electrophoresis and will be characterized by MALDI-TOF peptide fingerprinting.

  18. Proteomic identification of secreted proteins of Propionibacterium acnes

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    Holland Carsten

    2010-08-01

    Full Text Available Abstract Background The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. Results Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS. A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, β-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. Conclusions Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence

  19. Secretion and extracellular space travel of Wnt proteins.

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    Gross, Julia Christina; Boutros, Michael

    2013-08-01

    Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion.

  20. Selection for Genes Encoding Secreted Proteins and Receptors

    Science.gov (United States)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  1. Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST).

    Science.gov (United States)

    Lee, Je Min; Lee, Sang-Jik; Rose, Jocelyn K C; Yeam, Inhwa; Kim, Byung-Dong

    2014-04-18

    Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.

  2. Identification of Anaplasma marginale type IV secretion system effector proteins.

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    Svetlana Lockwood

    Full Text Available BACKGROUND: Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS. The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now. RESULTS: By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141 of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system. CONCLUSIONS: The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.

  3. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Österlund, Tobias; Liu, Zihe

    2013-01-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

  4. Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis

    NARCIS (Netherlands)

    Bolhuis, A; Tjalsma, H; Smith, H.E; Meima, R.; Venema, G; Bron, S; van Dijl, J.M

    1999-01-01

    Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis alpha-amyla

  5. Glycosylation in secreted proteins from yeast Kluyveromyces lactis

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    Santos, A.V.; Passos, F.M.L. [Universidade Federal de Vicosa (UFV), MG (Brazil). Dept. de Microbiologia. Lab. de Fisiologia de Microrganismos; Azevedo, B.R.; Pimenta, A.M.C.; Santoro, M.M. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia. Lab. de Enzimologia e Fisico-Quimica de Proteina

    2008-07-01

    Full text: The nutritional status of a cell culture affects either the expression or the traffic of a number of proteins. The identification of the physiological conditions which favor protein secretion has important biotechnological consequences in designing systems for recombinant extracellular protein industrial production. Yeast Kluyvromyces lactis has been cultured in a continuous stirring tank bioreactor (CSTR) under nitrogen limitation at growth rates (0.03 h{sup -1} and 0.09 h{sup -1}) close to either exponential or stationary batch growth phases, respectively the objective was to investigate the extracellular glycoproteins at these two level of nitrogen limitation. Proteins from free cell extracts were separated by gradient SDS-PAGE (5-15%) and two-dimensional chromatography, and were analyzed by mass spectrometry (MALDI-TOF-TOF-MS). In SDS-PAGE analysis, differences in extracellular proteome were visualized: different proteins profiles at these two growth rates. The 0.09 h-1 growth rate showed larger number of bands using colloidal Coma ssie Blue staining. Different bands were detected at these two growth rates when the PAS assay for glycoprotein detection in polyacrylamide gel was used. The two-dimensional chromatogram profiles were comparatively distinguished between the 0.03 h{sup -1} and 0.09 h{sup -1} growth rate samples. Protein peaks from the second dimension, were subjected to mass spectrometry. The mass spectrums visualized showed glycosylated proteins with N-acetylglucosamine molecules and 8, 9 or 15 hexoses molecules. Comparisons between the proteins averaged mass values with the deduced proteins masses from K. lactis secreted proteins database indicated possible post-translational modifications, such as post-translational proteolysis, acetylation, deamidation and myristoylation.

  6. Unconventional Secretion of Ebola Virus Matrix Protein VP40

    OpenAIRE

    Reynard, Olivier; Reid, St. Patrick; Page, Audrey; Mateo, Mathieu; Alazard-Dany, Nathalie; Raoul, Hervé; Basler, Christopher F.; Volchkov, Viktor E.

    2011-01-01

    The Ebola virus matrix protein VP40 plays an essential role in virus assembly and budding. In this study we reveal that transient VP40 expression results in the release into the culture medium of substantial amounts of soluble monomeric VP40 in addition to the release of virus-like particles containing an oligomeric form of this protein as previously described. We show that VP40 secretion is endoplasmic reticulum/Golgi–independent and is not associated with cell death. Soluble VP40 was observ...

  7. Legionella pneumophila secretes a mitochondrial carrier protein during infection.

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    Pavel Dolezal

    2012-01-01

    Full Text Available The Mitochondrial Carrier Family (MCF is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP, encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

  8. Impact of protein uptake and degradation on recombinant protein secretion in yeast

    DEFF Research Database (Denmark)

    Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva;

    2014-01-01

    Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs and simp......Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs...... and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize...

  9. Infectious Keratitis: Secreted Bacterial Proteins That Mediate Corneal Damage

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    Mary E. Marquart

    2013-01-01

    Full Text Available Ocular bacterial infections are universally treated with antibiotics, which can eliminate the organism but cannot reverse the damage caused by bacterial products already present. The three very common causes of bacterial keratitis—Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae—all produce proteins that directly or indirectly cause damage to the cornea that can result in reduced vision despite antibiotic treatment. Most, but not all, of these proteins are secreted toxins and enzymes that mediate host cell death, degradation of stromal collagen, cleavage of host cell surface molecules, or induction of a damaging inflammatory response. Studies of these bacterial pathogens have determined the proteins of interest that could be targets for future therapeutic options for decreasing corneal damage.

  10. Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

    NARCIS (Netherlands)

    Marciniak, Bogumila C.; Trip, Hein; van-der Veek, Patricia J.; Kuipers, Oscar P.; Marciniak, Bogumiła C.

    2012-01-01

    Background: Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentatio

  11. Computational Analysis of Signal Peptide-Dependent Secreted Proteins in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Computer based software such as the SignalP v3.0, TargetP vl.01, big-PI predictor and TMHMM v2.0 were combined to predict the signal peptides, and the signal peptide-dependent secreted proteins among the 6 700 ORFs in genome of Saccharomyces cerevisiae. The results showed that 163 proteins were the secreted ones containing signal peptides, and they were secreted via Sec pathway. Among the 163 predicted secreted proteins, the signal peptides of 47 secreted proteins included only the H-domain and C-domain, without N-domain, but the signal peptides of other 116 secreted proteins included all the three domains. There were differences in the constitution of signal peptides between the secreted proteins of S. cerevisiae and of Candida albicans, but the length and amino acids types of their signal peptides were similar in general. Few of the same signal peptides occurred in the secreted proteins of S. cerevisiae genome, and the homology could be compared among the secreted proteins with the same signal peptides. The BLAST 2 SEQUENECES and CLUSTAL W were used to align the two protein sequences and multi-protein sequences, respectively. The alignment result indicated that homology of these sequences with the same signal peptide was very highly conservative in amino acid of complete gene. The effect of the signal peptides in S. cerevisia on expression of foreign eukaryotic secreted proteins is discussed in this paper.

  12. Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions.

    Science.gov (United States)

    He, Huan; Liu, Dan; Lin, Hui; Jiang, Shanshan; Ying, Ying; Chun, Shao; Deng, Haiteng; Zaia, Joseph; Wen, Rong; Luo, Zhijun

    2016-07-01

    Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1-4. To determine the functional differences among PEBP1-4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1-3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events.

  13. Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

    Energy Technology Data Exchange (ETDEWEB)

    Klinefelter, G.R.; Hamilton, D.W.

    1985-11-01

    We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-(35S)methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-(35S)methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB(3H)4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis.

  14. Burkholderia cenocepacia type VI secretion system mediates escape of type II secreted proteins into the cytoplasm of infected macrophages.

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    Roberto Rosales-Reyes

    Full Text Available Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS. We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

  15. Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

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    Marciniak Bogumiła C

    2012-05-01

    Full Text Available Abstract Background Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1 β-lactamase of Escherichia coli, membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus and lipoproteins (MntA and YcdH of B. subtilis. Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes. Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 β-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue specifically under membrane proteins overproduction. Conclusions The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.

  16. A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

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    Marino Gennaro

    2006-12-01

    Full Text Available Abstract Background The final aim of recombinant protein production is both to have a high specific production rate and a high product quality. It was already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4°C. Results A novel genetic system for the production and secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 was set up. This system aims at combining the low temperature recombinant product production with the advantages of extra-cellular protein targeting. The psychrophilic α-amylase from Pseudoalteromonas haloplanktis TAB23 was used as secretion carrier. Three chimerical proteins were produced by fusing intra-cellular proteins to C-terminus of the psychrophilic α-amylase and their secretion was analysed. Data reported in this paper demonstrate that all tested chimeras were translocated with a secretion yield always higher than 80%. Conclusion Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the α-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger.

  17. Unconventional protein secretion in plants: a critical assessment.

    Science.gov (United States)

    Robinson, David G; Ding, Yu; Jiang, Liwen

    2016-01-01

    Unconventional protein secretion (UPS) is a collective term for mechanisms by which cytosolic proteins that lack a signal peptide ("leaderless secretory proteins" (LSPs)) can gain access to the cell exterior. Numerous examples of UPS have been well documented in animal and yeast cells. In contrast, our understanding of the mechanism(s) and function of UPS in plants is very limited. This review evaluates the available literature on this subject. The apparent large numbers of LSPs in the plant secretome suggest that UPS also occurs in plants but is not a proof. Although the direct transport of LSPs across the plant plasma membrane (PM) has not yet been described, it is possible that as in other eukaryotes, exosomes may be released from plant cells through fusion of multivesicular bodies (MVBs) with the PM. In this way, LSPs, but also small RNAs (sRNAs), that are passively taken up from the cytosol into the intraluminal vesicles of MVBs, could reach the apoplast. Another possible mechanism is the recently discovered exocyst-positive organelle (EXPO), a double-membrane-bound compartment, distinct from autophagosomes, which appears to sequester LSPs.

  18. Therapeutic Potential of Secreted Amyloid Precursor Protein APPsα

    Science.gov (United States)

    Mockett, Bruce G.; Richter, Max; Abraham, Wickliffe C.; Müller, Ulrike C.

    2017-01-01

    Cleavage of the amyloid precursor protein (APP) by α-secretase generates an extracellularly released fragment termed secreted APP-alpha (APPsα). Not only is this process of interest due to the cleavage of APP within the amyloid-beta sequence, but APPsα itself has many physiological properties that suggest its great potential as a therapeutic target. For example, APPsα is neurotrophic, neuroprotective, neurogenic, a stimulator of protein synthesis and gene expression, and enhances long-term potentiation (LTP) and memory. While most early studies have been conducted in vitro, effectiveness in animal models is now being confirmed. These studies have revealed that either upregulating α-secretase activity, acutely administering APPsα or chronic delivery of APPsα via a gene therapy approach can effectively treat mouse models of Alzheimer’s disease (AD) and other disorders such as traumatic head injury. Together these findings suggest the need for intensifying research efforts to harness the therapeutic potential of this multifunctional protein.

  19. Distribution of Flagella Secreted Protein and Integral Membrane Protein Among Campylobacter jejuni Isolated from Thailand

    Science.gov (United States)

    2011-01-01

    jejuni is a major cause of gastroenteritis worldwide especially in children, travelers, military per- sonnel deployed to developing countries...acidic protein is secreted into the supernatant of broth-grown bacteria . Two alleles of fspA (/spA 1 and fspA2) based on sequence analysis were found in

  20. Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

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    Langella P.

    1999-01-01

    Full Text Available Lactic acid bacteria (LAB are Gram-positive bacteria and are generally regarded as safe (GRAS organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV epitope-protein fusion (BCV-Nuc. BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

  1. Coordination of Pancreatic HCO3- Secretion by Protein-Protein Interaction between Membrane Transporters

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    Lee MG

    2001-07-01

    Full Text Available Increasing evidence suggests that protein-protein interaction is essential in many biological processes including epithelial transport. In this report, we discuss the significance of protein interactions to HCO(3(- secretion in pancreatic duct cells. In pancreatic ducts HCO(3(- secretion is mediated by cystic fibrosis transmembrane conductance regulator (CFTR activated luminal Cl(-/HCO(3(- exchange activity and HCO(3(- absorption is achieved by Na(+-dependent mechanisms including Na(+/H(+ exchanger 3 (NHE3. We found biochemical and functional association between CFTR and NHE3. In addition, protein binding through PDZ modules is needed for this regulatory interaction. CFTR affected NHE3 activities in two ways. Acutely, CFTR augmented the cAMP-dependent inhibition of NHE3. In a chronic mechanism, CFTR increases the luminal expression of Na(+/H(+ exchange in pancreatic duct cells. These findings reveal that protein complexes in the plasma membrane of pancreatic duct cells are highly organized for efficient HCO(3(- secretion.

  2. Maltose-Binding Protein (MBP, a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

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    Raphael Reuten

    Full Text Available Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST, SlyD, and serum albumin (ser alb tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome, which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  3. Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

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    Maaike Kockx

    Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

  4. Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

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    Mattanovich Diethard

    2011-11-01

    Full Text Available Abstract Background The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. Results Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p, we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. Conclusions The current study presents a systems biotechnology-based strategy for the

  5. Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus.

    Science.gov (United States)

    Medina, Martha L; Kiernan, Urban A; Francisco, Wilson A

    2004-03-01

    Few studies have been conducted to identify the extracellular proteins and enzymes secreted by filamentous fungi, particularly with respect to dispensable metabolic pathways. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions. The filamentous fungus Aspergillus flavus can degrade the flavonoid rutin as the only source of carbon via an extracellular enzyme system. In this study, a proteomic analysis was used to differentiate and identify the extracellular rutin-induced and non-induced proteins secreted by A. flavus. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. While 15 rutin-induced proteins and 7 non-induced proteins were identified, more than 90 protein spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced.

  6. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata

    Energy Technology Data Exchange (ETDEWEB)

    Lee, E.Y.H.; Parry, G.; Bissell, M.J.

    1984-01-01

    It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce ..gamma..-casein, a member of the casein gene family, only if they are maintained on floating collagen gels. In this paper we show: (a) Cells on floating collagen gels secrete not only ..gamma..-casein but also ..cap alpha../sub 1/-, ..cap alpha../sub 2/-, and ..beta..-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross-linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane, are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.

  7. Protein-Phospholipid Interactions in Nonclassical Protein Secretion: Problem and Methods of Study

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    David Neivandt

    2013-02-01

    Full Text Available Extracellular proteins devoid of signal peptides use nonclassical secretion mechanisms for their export. These mechanisms are independent of the endoplasmic reticulum and Golgi. Some nonclassically released proteins, particularly fibroblast growth factors (FGF 1 and 2, are exported as a result of their direct translocation through the cell membrane. This process requires specific interactions of released proteins with membrane phospholipids. In this review written by a cell biologist, a structural biologist and two membrane engineers, we discuss the following subjects: (i Phenomenon of nonclassical protein release and its biological significance; (ii Composition of the FGF1 multiprotein release complex (MRC; (iii The relationship between FGF1 export and acidic phospholipid externalization; (iv Interactions of FGF1 MRC components with acidic phospholipids; (v Methods to study the transmembrane translocation of proteins; (vi Membrane models to study nonclassical protein release.

  8. An Experimental Approach for the Identification of Conserved Secreted Proteins in Trypanosomatids

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    Rosa M. Corrales

    2010-01-01

    Full Text Available Extracellular factors produced by Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei are important in the host-parasite relationship. Here, we describe a genome-based approach to identify putative extracellular proteins conserved among trypanosomatids that are likely involved in the classical secretory pathway. Potentially secreted proteins were identified by bioinformatic analysis of the T. cruzi genome. A subset of thirteen genes encoding unknown proteins with orthologs containing a signal peptide sequence in L. infantum, L. major, and T. brucei were transfected into L. infantum. Tagged proteins detected in the extracellular medium confirmed computer predictions in about 25% of the hits. Secretion was confirmed for two L. infantum orthologs proteins using the same experimental system. Infectivity studies of transgenic Leishmania parasites suggest that one of the secreted proteins increases parasite replication inside macrophages. This methodology can identify conserved secreted proteins involved in the classical secretory pathway, and they may represent potential virulence factors in trypanosomatids.

  9. The Evolution of the Secreted Regulatory Protein Progranulin.

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    Roger G E Palfree

    Full Text Available Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i: the origins of metazoan progranulins (ii: the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii: the evolution of granulin module architectures of vertebrate progranulins (iv: the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl

  10. Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization.

    Science.gov (United States)

    Heiss, Silvia; Puxbaum, Verena; Gruber, Clemens; Altmann, Friedrich; Mattanovich, Diethard; Gasser, Brigitte

    2015-07-01

    Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast Pichia pastoris revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the P. pastoris Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

  11. Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

    Science.gov (United States)

    Moua, Pachai S; Gonzalez, Alfonso; Oshiro, Kristin T; Tam, Vivian; Li, Zhiguo Harry; Chang, Jennifer; Leung, Wilson; Yon, Amy; Thor, Der; Venkatram, Sri; Franz, Andreas H; Risser, Douglas D; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2016-08-01

    The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris.

  12. Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus

    Science.gov (United States)

    Parnasa, Rami; Nagar, Elad; Sendersky, Eleonora; Reich, Ziv; Simkovsky, Ryan; Golden, Susan; Schwarz, Rakefet

    2016-01-01

    Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms. PMID:27558743

  13. Mechanical Unfolding of an Autotransporter Passenger Protein Reveals the Secretion Starting Point and Processive Transport Intermediates

    NARCIS (Netherlands)

    Baclayon, Marian; van Ulsen, Peter; Mouhib, Halima; Shabestari, Maryam Hashemi; Verzijden, Timo; Abeln, Sanne; Roos, Wouter H; Wuite, Gijs J L

    2016-01-01

    The backbone of secreted autotransporter passenger proteins generally attains a stable β-helical structure. The secretion of passengers across the outer membrane was proposed to be driven by sequential folding of this structure at the cell surface. This mechanism would require a relatively-stable in

  14. Insulin-degrading enzyme is exported via an unconventional protein secretion pathway

    Directory of Open Access Journals (Sweden)

    Leissring Malcolm A

    2009-01-01

    Full Text Available Abstract Insulin-degrading enzyme (IDE is a ubiquitously expressed zinc-metalloprotease that degrades several pathophysiologically significant extracellular substrates, including insulin and the amyloid β-protein (Aβ, and accumulating evidence suggests that IDE dysfunction may be operative in both type 2 diabetes mellitus and Alzheimer disease (AD. Although IDE is well known to be secreted by a variety of cell types, the underlying trafficking pathway(s remain poorly understood. To address this topic, we investigated the effects of known inhibitors or stimulators of protein secretion on the secretion of IDE from murine hepatocytes and HeLa cells. IDE secretion was found to be unaffected by the classical secretion inhibitors brefeldin A (BFA, monensin, or nocodazole, treatments that readily inhibited the secretion of α1-antitrypsin (AAT overexpressed in the same cells. Using a novel cell-based Aβ-degradation assay, we show further that IDE secretion was similarly unaffected by multiple stimulators of protein secretion, including glyburide and 3'-O-(4-benzoylbenzoyl-ATP (Bz-ATP. The calcium ionophore, A23187, increased extracellular IDE activity, but only under conditions that also elicited cytotoxicity. Our results provide the first biochemical evidence that IDE export is not dependent upon the classical secretion pathway, thereby identifying IDE as a novel member of the select class of unconventionally secreted proteins. Further elucidation of the mechanisms underlying IDE secretion, which would be facilitated by the assays described herein, promises to uncover processes that might be defective in disease or manipulated for therapeutic benefit.

  15. C-terminal domain of hepatitis C virus core protein is essential for secretion

    Institute of Scientific and Technical Information of China (English)

    Soo-Ho Choi; Kyu-Jin Park; So-Yeon Kim; Dong-Hwa Choi; Jung-Min Park; Soon B. Hwang

    2005-01-01

    AIM: We have previously demonstrated that hepatitis C virus (HCV) core protein is efficiently released into the culture medium in insect cells. The objective of this study is to characterize the HCV core secretion in insect cells.METHODS: We constructed recombinant baculoviruses expressing various-length of mutant core proteins, expressed these proteins in insect cells, and examined core protein secretion in insect cells.RESULTS: Only wild type core was efficiently released into the culture medium, although the protein expression level of wild type core was lower than those of other mutant core proteins. We found that the shorter form of the core construct expressed the higher level of protein. However, if more than 18 amino acids of the core were truncated at the C-terminus,core proteins were no longer seareted into the culture medium.Membrane flotation data show that the secreted core proteins are associated with the cellular membrane protein, indicating that HCV core is secreted as a membrane complex.CONCLUSION: The C-terminal 18 amino acids of HCV core were crucial for core secretion into the culture media.Since HCV replication occurs on lipid raft membrane structure,these results suggest that HCV may utilize a unique core release mechanism to escape immune surveillance, thereby potentially representing the feature of HCV morphogenesis.

  16. Protein secretion biotechnology in Gram-positive bacteria with special emphasis on Streptomyces lividans.

    Science.gov (United States)

    Anné, Jozef; Vrancken, Kristof; Van Mellaert, Lieve; Van Impe, Jan; Bernaerts, Kristel

    2014-08-01

    Proteins secreted by Gram-positive bacteria are released into the culture medium with the obvious benefit that they usually retain their native conformation. This property makes these host cells potentially interesting for the production of recombinant proteins, as one can take full profit of established protocols for the purification of active proteins. Several state-of-the-art strategies to increase the yield of the secreted proteins will be discussed, using Streptomyces lividans as an example and compared with approaches used in some other host cells. It will be shown that approaches such as increasing expression and translation levels, choice of secretion pathway and modulation of proteins thereof, avoiding stress responses by changing expression levels of specific (stress) proteins, can be helpful to boost production yield. In addition, the potential of multi-omics approaches as a tool to understand the genetic background and metabolic fluxes in the host cell and to seek for new targets for strain and protein secretion improvement is discussed. It will be shown that S. lividans, along with other Gram-positive host cells, certainly plays a role as a production host for recombinant proteins in an economically viable way. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

  17. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.; Haitjema, Charles H.

    2017-02-21

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  18. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    Science.gov (United States)

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  19. Calcium-dependent activator protein for secretion 2 (CAPS2) promotes BDNF secretion and is critical for the development of GABAergic interneuron network

    OpenAIRE

    Shinoda, Yo; Sadakata, Tetsushi; Nakao, Kazuhito; Katoh-Semba, Ritsuko; Kinameri, Emi; Furuya, Asako; Yanagawa, Yuchio; Hirase, Hajime; Furuichi, Teiichi

    2011-01-01

    Calcium-dependent activator protein for secretion 2 (CAPS2) is a dense-core vesicle-associated protein that is involved in the secretion of BDNF. BDNF has a pivotal role in neuronal survival and development, including the development of inhibitory neurons and their circuits. However, how CAPS2 affects BDNF secretion and its biological significance in inhibitory neurons are largely unknown. Here we reveal the role of CAPS2 in the regulated secretion of BDNF and show the effect of CAPS2 on the ...

  20. Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Schalén, Martin; Anyaogu, Diana Chinyere; Hoof, Jakob Blæsbjerg;

    2016-01-01

    . The background strain was a fluorescent reporter secreting mRFP. The overall effect of the overexpressions could thus be easily monitored through fluorescence measurements, while the effects on physiology were determined in batch cultivations and surface growth studies. Results: Fourteen protein secretion......- and macro-morphology and protein secretion levels. While several constructs exhibited decreased secretion of the model protein, the overexpression of the Rab GTPase RabD resulted in a 40 % increase in secretion in controlled bioreactor cultivations. Fluorescence microscopy revealed alterations of protein...... results indicate that increased expression may be a way for the cell to slow down secretion in order to cope with the increased protein load. By constructing a secretion reporter strain, the study demonstrates a robust way to study the secretion pathway in filamentous fungi....

  1. Protein secretion systems in Pseudomonas aeruginosa: an essay on diversity, evolution and function

    Directory of Open Access Journals (Sweden)

    Alain eFILLOUX

    2011-07-01

    Full Text Available Protein secretion systems are molecular nanomachines used by Gram-negative bacteria to thrive within their environment. They are used to release enzymes that hydrolyze complex carbon sources into usable compounds, or to release proteins that capture essential ions such as iron. They are also used to colonize and survive within eukaryotic hosts, causing acute or chronic infections, subverting the host cell response and escaping the immune system. In this article, the opportunistic human pathogen Pseudomonas aeruginosa is used as a model to review the diversity of secretion systems that bacteria have evolved to achieve these goals. This diversity may result from a progressive transformation of cell envelope complexes that initially may not have been dedicated to secretion. The striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail or efflux pumps is a nice illustration of this evolution. Differences are also needed since various secretion configurations calls for diversity. For example, some proteins are released in the extracellular medium while others are directly injected into the cytosol of eukaryotic cells. Some proteins are folded before being released and transit into the periplasm. Other proteins cross the whole cell envelope at once in an unfolded state. However, the secretion system requires conserved basic elements or features. For example, there is a need for an energy source or for an outer membrane channel. The structure of this review is thus quite unconventional. Instead of listing secretion types one after each other, it presents a melting pot of concepts indicating that secretion types are in constant evolution and use basic principles. In other words, emergence of new secretion systems could be predicted the way Mendeleïev had anticipated characteristics of yet unknown elements.

  2. Bovine peptidoglycan recognition protein-S: antimicrobial activity, localization, secretion, and binding properties.

    Science.gov (United States)

    Tydell, C Chace; Yuan, Jun; Tran, Patti; Selsted, Michael E

    2006-01-15

    Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern recognition molecules of innate immunity that are conserved from insects to humans. Various PGRPs are reported to have diverse functions: they bind bacterial molecules, digest PGN, and are essential to the Toll pathway in Drosophila. One family member, bovine PGN recognition protein-S (bPGRP-S), has been found to bind and kill microorganisms in a PGN-independent manner, raising questions about the identity of the bPGRP-S ligand. Addressing this, we have determined the binding and microbicidal properties of bPGRP-S in a range of solutions approximating physiologic conditions. In this study we show that bPGRP-S interacts with other bacterial components, including LPS and lipoteichoic acid, with higher affinities than for PCP, as determined by their abilities to inhibit bPGRP-S-mediated killing of bacteria. Where and how PGRPs act in vivo is not yet clear. Using Immunogold electron microscopy, PGRP-S was localized to the dense/large granules of naive neutrophils, which contain the oxygen-independent bactericidal proteins of these cells, and to the neutrophil phagolysosome. In addition, Immunogold staining and secretion studies demonstrate that neutrophils secrete PGRP-S when exposed to bacteria. Bovine PGRP-S can mediate direct lysis of heat-killed bacteria; however, PGRP-S-mediated killing of bacteria is independent of this activity. Evidence that bPGRP-S has multiple activities and affinity to several bacterial molecules challenges the assumption that the PGRP family of proteins recapitulates the evolution of TLRs. Mammalian PGRPs do not have a single antimicrobial activity against a narrow range of target organisms; rather, they are generalists in their affinity and activity.

  3. Association among Fibrinolytic Proteins, Metabolic Syndrome Components, Insulin Secretion, and Resistance in Schoolchildren

    Directory of Open Access Journals (Sweden)

    Jin-Shuen Chen

    2015-01-01

    Full Text Available We investigated the role of urokinase plasminogen activator (uPA and its soluble receptors (suPAR and plasminogen activator inhibitor-1 (PAI-1 in metabolic syndrome (MetS components, insulin secretion, and resistance in schoolchildren. We enrolled 387 children, aged 10.3 ± 1.5 years, in Taipei. Anthropometry, fibrinolytic proteins, MetS components, insulin secretion, and resistance were measured. Subjects were divided into normal, overweight, and obese groups. Finally, the relationship between fibrinolytic proteins and metabolic syndrome in boys and girls was analyzed. In boys, PAI-1 was positively associated with body mass index (BMI percentile, hypertriglyceride, insulin secretion, and resistance. In girls, PAI-1 was positively associated with obesity, hypertriglyceridemia, and insulin secretion. In girls, uPA was positively associated with insulin secretion. suPAR was positively associated with high-sensitivity C-reactive protein in both boys and girls, and with BMI percentile and body fat in girls. The obese boys had higher suPAR and PAI-1 levels than the normal group. The obese girls had higher uPA, suPAR, and PAI-1 than the normal group. Boys and girls with MetS had higher PAI-1. Fibrinolytic proteins, especially PAI-1, are associated with MetS components and insulin secretion in children. Fibrinolytic proteins changes were more likely to occur in girls than in boys.

  4. NClassG+: A classifier for non-classically secreted Gram-positive bacterial proteins

    Directory of Open Access Journals (Sweden)

    Pino Camilo

    2011-01-01

    Full Text Available Abstract Background Most predictive methods currently available for the identification of protein secretion mechanisms have focused on classically secreted proteins. In fact, only two methods have been reported for predicting non-classically secreted proteins of Gram-positive bacteria. This study describes the implementation of a sequence-based classifier, denoted as NClassG+, for identifying non-classically secreted Gram-positive bacterial proteins. Results Several feature-based classifiers were trained using different sequence transformation vectors (frequencies, dipeptides, physicochemical factors and PSSM and Support Vector Machines (SVMs with Linear, Polynomial and Gaussian kernel functions. Nested k-fold cross-validation (CV was applied to select the best models, using the inner CV loop to tune the model parameters and the outer CV group to compute the error. The parameters and Kernel functions and the combinations between all possible feature vectors were optimized using grid search. Conclusions The final model was tested against an independent set not previously seen by the model, obtaining better predictive performance compared to SecretomeP V2.0 and SecretPV2.0 for the identification of non-classically secreted proteins. NClassG+ is freely available on the web at http://www.biolisi.unal.edu.co/web-servers/nclassgpositive/

  5. Computational prediction and experimental assessment of secreted/surface proteins from Mycobacterium tuberculosis H37Rv.

    Directory of Open Access Journals (Sweden)

    Carolina Vizcaíno

    2010-06-01

    Full Text Available The mycobacterial cell envelope has been implicated in the pathogenicity of tuberculosis and therefore has been a prime target for the identification and characterization of surface proteins with potential application in drug and vaccine development. In this study, the genome of Mycobacterium tuberculosis H37Rv was screened using Machine Learning tools that included feature-based predictors, general localizers and transmembrane topology predictors to identify proteins that are potentially secreted to the surface of M. tuberculosis, or to the extracellular milieu through different secretory pathways. The subcellular localization of a set of 8 hypothetically secreted/surface candidate proteins was experimentally assessed by cellular fractionation and immunoelectron microscopy (IEM to determine the reliability of the computational methodology proposed here, using 4 secreted/surface proteins with experimental confirmation as positive controls and 2 cytoplasmic proteins as negative controls. Subcellular fractionation and IEM studies provided evidence that the candidate proteins Rv0403c, Rv3630, Rv1022, Rv0835, Rv0361 and Rv0178 are secreted either to the mycobacterial surface or to the extracellular milieu. Surface localization was also confirmed for the positive controls, whereas negative controls were located on the cytoplasm. Based on statistical learning methods, we obtained computational subcellular localization predictions that were experimentally assessed and allowed us to construct a computational protocol with experimental support that allowed us to identify a new set of secreted/surface proteins as potential vaccine candidates.

  6. A protein secretion system linked to bacteroidete gliding motility and pathogenesis.

    Science.gov (United States)

    Sato, Keiko; Naito, Mariko; Yukitake, Hideharu; Hirakawa, Hideki; Shoji, Mikio; McBride, Mark J; Rhodes, Ryan G; Nakayama, Koji

    2010-01-05

    Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum.

  7. Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

    Directory of Open Access Journals (Sweden)

    Cirulli Claudia

    2008-02-01

    Full Text Available Abstract Background In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic α-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity. Results P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS, we have inactivated this secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-β-lactamase chimera. Conclusion Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic α-amylase (the carrier of our recombinant secretion system, while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a

  8. Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein.

    Science.gov (United States)

    Wartenberg, Dirk; Lapp, Katrin; Jacobsen, Ilse D; Dahse, Hans-Martin; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

    2011-11-01

    Surface-associated and secreted proteins represent primarily exposed components of Aspergillus fumigatus during host infection. Several secreted proteins are known to be involved in defense mechanisms or immune evasion, thus, probably contributing to pathogenicity. Furthermore, several secreted antigens were identified as possible biomarkers for the verification of diseases caused by Aspergillus species. Nevertheless, there is only limited knowledge about the composition of the secretome and about molecular functions of particular proteins. To identify secreted proteins potentially essential for virulence, the core secretome of A. fumigatus grown in minimal medium was determined. Two-dimensional gel electrophoretic separation and subsequent MALDI-TOF-MS/MS analyses resulted in the identification of 64 different proteins. Additionally, secretome analyses of A. fumigatus utilizing elastin, collagen or keratin as main carbon and nitrogen source were performed. Thereby, the alkaline serine protease Alp1 was identified as the most abundant protein and hence presumably represents an important protease during host infection. Interestingly, the Asp-hemolysin (Asp-HS), which belongs to the protein family of aegerolysins and which was often suggested to be involved in fungal virulence, was present in the secretome under all growth conditions tested. In addition, a second, non-secreted protein with an aegerolysin domain annotated as Asp-hemolysin-like (HS-like) protein can be found to be encoded in the genome of A. fumigatus. Generation and analysis of Asp-HS and HS-like deletion strains revealed no differences in phenotype compared to the corresponding wild-type strain. Furthermore, hemolysis and cytotoxicity was not altered in both single-deletion and double-deletion mutants lacking both aegerolysin genes. All mutant strains showed no attenuation in virulence in a mouse infection model for invasive pulmonary aspergillosis. Overall, this study provides a comprehensive

  9. The S-layer proteins of Tannerella forsythia are secreted via a type IX secretion system that is decoupled from protein O-glycosylation.

    Science.gov (United States)

    Tomek, M B; Neumann, L; Nimeth, I; Koerdt, A; Andesner, P; Messner, P; Mach, L; Potempa, J S; Schäffer, C

    2014-12-01

    Conserved C-terminal domains (CTD) have been shown to act as a signal for the translocation of certain proteins across the outer membrane of Bacteroidetes via a type IX secretion system (T9SS). The genome sequence of the periodontal pathogen Tannerella forsythia predicts the presence of the components for a T9SS in conjunction with a suite of CTD proteins. T. forsythia is covered with a two-dimensional crystalline surface (S-) layer composed of the glycosylated CTD proteins TfsA and TfsB. To investigate, if T9SS is functional in T. forsythia, T9SS-deficient mutants were generated by targeting either TF0955 (putative C-terminal signal peptidase) or TF2327 (PorK ortholog), and the mutants were analyzed with respect to secretion, assembly and glycosylation of the S-layer proteins as well as proteolytic processing of the CTD and biofilm formation. In either mutant, TfsA and TfsB were incapable of translocation, as evidenced by the absence of the S-layer in transmission electron microscopy of ultrathin-sectioned bacterial cells. Despite being entrapped within the periplasm, mass spectrometry analysis revealed that the S-layer proteins were modified with the complete, mature glycan found on the secreted proteins, indicating that protein translocation and glycosylation are two independent processes. Further, the T9SS mutants showed a denser biofilm with fewer voids compared with the wild-type. This study demonstrates the functionality of T9SS and the requirement of CTD for the outer membrane passage of extracellular proteins in T. forsythia, exemplified by the two S-layer proteins. In addition, T9SS protein translocation is decoupled from O-glycan attachment in T. forsythia.

  10. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins during ex vivo Osteoblast Differentiation of Human Stromal Stem Cells

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Chen, Li; Nielsen, Maria Overbeck;

    2012-01-01

    labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited...

  11. Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis.

    Science.gov (United States)

    Stock, Janpeter; Sarkari, Parveen; Kreibich, Saskia; Brefort, Thomas; Feldbrügge, Michael; Schipper, Kerstin

    2012-10-15

    The demand on the biotechnological production of proteins for pharmaceutical, medical and industrial applications is steadily growing. For the production of challenging proteins, we aim to establish a novel expression platform in the well characterized eukaryotic microorganism Ustilago maydis. In filaments of this fungus, secretion of the endochitinase Cts1 depends on mRNA transport along microtubules, which is mediated by the key RNA-binding protein Rrm4. Here, we report two important findings: (i) Cts1 secretion occurs via a novel unconventional route and (ii) this secretory mechanism can be exploited for the export of active heterologous proteins. Initially, we used β-glucuronidase (Gus) as a reporter for unconventional secretion. This bacterial enzyme is inactivated by N-glycosylation during its passage through the conventional eukaryotic secretory pathway. By contrast, in our system Gus was exported in its active form by fusion to Cts1 confirming its secretion by an unconventional route. As a proof-of-principle for economically important biopharmaceuticals we expressed an active single-chain antibody. Importantly, the novel protein export pathway circumvents N-glycosylation which is advantageous in many applications, e.g., to avoid undesired immune reactions in humans. Thus, the unconventional Cts1 secretion machinery has a high potential for the production of biotechnologically relevant proteins.

  12. Human surfactant protein A2 gene mutations impair dimmer/trimer assembly leading to deficiency in protein sialylation and secretion.

    Directory of Open Access Journals (Sweden)

    Yi Song

    Full Text Available Surfactant protein A2 (SP-A2 plays an essential role in surfactant metabolism and lung host defense. SP-A2 mutations in the carbohydrate recognition domain have been related to familial pulmonary fibrosis and can lead to a recombinant protein secretion deficiency in vitro. In this study, we explored the molecular mechanism of protein secretion deficiency and the subsequent biological effects in CHO-K1 cells expressing both wild-type and several different mutant forms of SP-A2. We demonstrate that the SP-A2 G231V and F198S mutants impair the formation of dimmer/trimer SP-A2 which contributes to the protein secretion defect. A deficiency in sialylation, but not N-linked glycosylation, is critical to the observed dimmer/trimer impairment-induced secretion defect. Furthermore, both mutant forms accumulate in the ER and form NP-40-insoluble aggregates. In addition, the soluble mutant SP-A2 could be partially degraded through the proteasome pathway but not the lysosome or autophagy pathway. Intriguingly, 4-phenylbutyrate acid (4-PBA, a chemical chaperone, alleviates aggregate formation and partially rescued the protein secretion of SP-A2 mutants. In conclusion, SP-A2 G231V and F198S mutants impair the dimmer/trimer assembly, which contributes to the protein sialylation and secretion deficiency. The intracellular protein mutants could be partially degraded through the proteasome pathway and also formed aggregates. The treatment of the cells with 4-PBA resulted in reduced aggregation and rescued the secretion of mutant SP-A2.

  13. Application of native signal sequences for recombinant proteins secretion in Pichia pastoris

    DEFF Research Database (Denmark)

    Borodina, Irina; Do, Duy Duc; Eriksen, Jens C.;

    alpha‐mating factor (MF) prepropeptide from Saccharomyces cerevisiae is most commonly used. Our aim was to test whether signal peptides from P. pastoris native secreted proteins could be used to direct secretion of recombinant proteins. Results Eleven native signal peptides from P. pastoris were tested...... with the promoter and signal peptide of choice (or a mix thereof) and the basic expression vector. The mix is treated with USER enzyme and transformed into E. coli. The plasmids (or plasmid mixes) are further purified, linearized and transformed into P. pastoris. We illustrate the commodity of the system...... by optimization of expression of three different proteins in P. pastoris. Conclusions Native signal peptides from P. pastoris can be used to direct secretion of recombinant proteins. A novel USER‐based P. pastoris system allows easy cloning of protein‐coding gene with the promoter and leader sequence of choice....

  14. Functional genomic analysis of the Bacillus subtilis Tat pathway for protein secretion

    NARCIS (Netherlands)

    van Dijl, JM; Braun, PG; Robinson, C; Quax, WJ; Antelmann, H; Hecker, M; Muller, J; Tjalsma, H; Bron, S; Jongbloed, JDH

    2002-01-01

    Protein secretion from Bacillus species is a major industrial production tool with a market of over $1 billion per year. However, standard export technologies, based on the well-characterised general secretory (See) pathway, are frequently inapplicable for the production of proteins. The recently di

  15. A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Bae, Jung-Hoon; Sung, Bong Hyun; Seo, Jeong-Woo; Kim, Chul Ho; Sohn, Jung-Hoon

    2016-12-01

    Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.

  16. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    Science.gov (United States)

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  17. Human rhinovirus 16 causes Golgi apparatus fragmentation without blocking protein secretion.

    Science.gov (United States)

    Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V; Walton, Ross; Johnston, Sebastian L; Solari, Roberto

    2014-10-01

    The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. Importance: The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be

  18. Interactions between Trypanosoma cruzi secreted proteins and host cell signaling pathways

    Directory of Open Access Journals (Sweden)

    Renata Watanabe Costa

    2016-03-01

    Full Text Available Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6-7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion.

  19. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    Science.gov (United States)

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  20. Protein secretion is required for pregnancy-associated plasma protein-A to promote lung cancer growth in vivo.

    Directory of Open Access Journals (Sweden)

    Hong Pan

    Full Text Available Pregnancy-associated plasma protein-A (PAPPA has been reported to regulate the activity of insulin-like growth factor (IGF signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression.

  1. Campylobacter fetus surface layer proteins are transported by a type I secretion system.

    Science.gov (United States)

    Thompson, S A; Shedd, O L; Ray, K C; Beins, M H; Jorgensen, J P; Blaser, M J

    1998-12-01

    The virulence of Campylobacter fetus, a bacterial pathogen of ungulates and humans, is mediated in part by the presence of a paracrystalline surface layer (S-layer) that confers serum resistance. The subunits of the S-layer are S-layer proteins (SLPs) that are secreted in the absence of an N-terminal signal sequence and attach to either type A or B C. fetus lipopolysaccharide in a serospecific manner. Antigenic variation of multiple SLPs (encoded by sapA homologs) of type A strain 23D occurs by inversion of a promoter-containing DNA element flanked by two sapA homologs. Cloning and sequencing of the entire 6.2-kb invertible region from C. fetus 23D revealed a probable 5.6-kb operon of four overlapping genes (sapCDEF, with sizes of 1,035, 1,752, 1,284, and 1,302 bp, respectively) transcribed in the opposite direction from sapA. The four genes also were present in the invertible region of type B strain 84-107 and were virtually identical to their counterparts in the type A strain. Although SapC had no database homologies, SapD, SapE, and SapF had predicted amino acid homologies with type I protein secretion systems (typified by Escherichia coli HlyBD/TolC or Erwinia chrysanthemi PrtDEF) that utilize C-terminal secretion signals to mediate the secretion of hemolysins, leukotoxins, or proteases from other bacterial species. Analysis of the C termini of four C. fetus SLPs revealed conserved structures that are potential secretion signals. A C. fetus sapD mutant neither produced nor secreted SLPs. E. coli expressing C. fetus sapA and sapCDEF secreted SapA, indicating that the sapCDEF genes are sufficient for SLP secretion. C. fetus SLPs therefore are transported to the cell surface by a type I secretion system.

  2. Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus

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    Chen Yi-Hsing

    2012-09-01

    Full Text Available Abstract Background Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS, a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of Orthologous Groups of protein COG4499 with no known function. Results By generating an internal deletion in essB , we demonstrate that EssB is required for secretion of EsxA. We use a polyclonal antibody to identify EssB and show that the protein fractionates with the plasma membrane of S. aureus . Yet, when produced in Escherichia coli, EssB remains mostly soluble and the purified protein assembles into a highly organized oligomer that can be visualized by electron microscopy. Production of truncated EssB variants in wild-type S. aureus confers a dominant negative phenotype on EsxA secretion. Conclusions The data presented here support the notion that EssB may oligomerize and interact with other membrane components to form the WXG100-specific translocon in S. aureus .

  3. Antibody Array Revealed PRL-3 Affects Protein Phosphorylation and Cytokine Secretion.

    Science.gov (United States)

    Yang, Yongyong; Lian, Shenyi; Meng, Lin; Qu, Like; Shou, Chengchao

    2017-01-01

    Phosphatase of regenerating liver 3 (PRL-3) promotes cancer metastasis and progression via increasing cell motility and invasiveness, however the mechanism is still not fully understood. Previous reports showed that PRL-3 increases the phosphorylation of many important proteins and suspected that PRL-3-enhanced protein phosphorylation may be due to its regulation on cytokines. To investigate PRL-3's impact on protein phosphorylation and cytokine secretion, we performed antibody arrays against protein phosphorylation and cytokines separately. The data showed that PRL-3 could enhance tyrosine phosphorylation and serine/threonine phosphorylation of diverse signaling proteins. Meanwhile, PRL-3 could affect the secretion of a subset of cytokines. Furthermore, we discovered the PRL-3-increased IL-1α secretion was regulated by NF-κB and Jak2-Stat3 pathways and inhibiting IL-1α could reduce PRL-3-enhanced cell migration. Therefore, our result indicated that PRL-3 promotes protein phosphorylation by acting as an 'activator kinase' and consequently regulates cytokine secretion.

  4. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    DEFF Research Database (Denmark)

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.

    2015-01-01

    interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV...... to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could...

  5. High yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Ramos-Martinez, E M; Fimognari, L; Sakuragi, Y

    2017-02-16

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability, and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. In order to increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n=10 or 20]. The yields of the (SP)n-fused Venus were higher than Venus without the glycomodule by up to 12 folds, with the maximum yield of 15 mg L(-1) . Moreover, the presence of the glycomodules confererred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with Brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. This article is protected by copyright. All rights reserved.

  6. Preliminary identification of secreted proteins by Leptospira interrogans serovar Kennewicki strain Pomona Fromm

    Energy Technology Data Exchange (ETDEWEB)

    Ricardi, L.M.P.; Portaro, F.C.; Abreu, P.A.E.; Barbosa, A.S. [Instituto Butantan, Sao Paulo, SP (Brazil); Morais, Z.M.; Vasconcellos, S.A. [Universidade de Sao Paulo (USP), SP (Brazil)

    2012-07-01

    Full text: This project aimed to identify secreted proteins by pathogenic Leptospira interrogans serovar Kennewicki strain Pomona Fromm (LPF) by proteomic analyses. The strain LPF, whose virulence was maintained by passages in hamsters, were cultured in EMJH medium. The supernatants were centrifuged, dialyzed and subjected to lyophilization. Protein samples were resolved first by IEF at pH 3 to 10, immobilized pH gradient 13-cm strips. Strips were then processed for the second-dimension separation on SDS-polyacrylamide gels. Proteins from gel spots were subjected to reduction, cysteine-alkylation, and in-gel tryptic digestion, and analyzed by LC/MS/MS spectrometry. Liquid chromatography-based separation followed by automated tandem mass spectrometry was also used to identify secreted proteins. In silico analyses were performed using the PSORTbV.3.0 program and SignalP server. One major obstacle to secretome studies is the difficulty to obtain extracts of secreted proteins without citoplasmatic contamination. In addition, the extraction of low concentration proteins from large volumes of culture media, which are rich in salts, BSA and other compounds, frequently interfere with most proteomics techniques. For these reasons, several experimental approaches were used to optimize the protocol applied. In spite of this fact, our analysis resulted in the identification of 200 proteins with high confidence. Only 5 of 63 secreted proteins predicted by in silico analysis were found. Other classes identified included proteins that possess signal peptide but whose cellular localization prediction is unknown or may have multiple localization sites, and proteins that lack signal peptide and are thus thought to be secreted via non conventional mechanisms or resulting from cytoplasmic contamination by cell lysis. Many of these are hypothetical proteins with no putative conserved domains detected. To our knowledge, this is the first study to identify secreted proteins by

  7. Gene delivery of therapeutic polypeptides to brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Moos, Torben

    of the proteins. Morphological examination of the protein expression was determined using immunofluorescence detecting FLAG. Additionally, the transfection efficiency were determined by Flow cytometry. Perspective: Our study opens for knowledge on how non-viral gene therapy to BCECs can lead to protein secretion......Background: The potential for treatment of chronic disorders affecting the CNS is complicated by the inability of several drugs to cross the blood-brain barrier (BBB). None-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints....... Results: mRNA expression of proteins with neuroprotective potential in RBEC were enabled. Their expression patters were compared with those of RBE4 and HeLa cells using RT-qPCR analyzes. The evidence for protein synthesis and secretion was obtained by detection of FLAG-tagged to the C-terminal of any...

  8. Secretion of Human Protein C in Mouse Milk

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    Chae-Won Park

    2015-03-01

    Full Text Available To determine the production of recombinant human protein C (rec-hPC in milk, we created two homozygous mice lines for the goat β-casein/hPC transgene. Females and males of both lines (#10 and #11 displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands.

  9. Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation.

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    Ellen Melaleuca Menkhorst

    Full Text Available Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the 'extravillous trophoblast' (EVT invade through the differentiated uterine endometrium (the decidua to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10(-8 M, medroxyprogesterone acetate (10(-7 M and cAMP (0.5 mM for 14 days. Conditioned media (CM was collected on day 2 (non-decidualized CM and 14 (decidualized CM of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1, dipeptidyl peptidase 1 (DPP1/cathepsin C and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro

  10. Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

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    Marizela Delic

    2014-10-01

    Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

  11. The type III secreted protein BspR regulates the virulence genes in Bordetella bronchiseptica.

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    Jun Kurushima

    Full Text Available Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator. The virulence of a BspR mutant (ΔbspR in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.

  12. Proteomic analysis of secreted proteins from Arabidopsis thaliana seedlings: improved recovery following removal of phenolic compounds.

    Science.gov (United States)

    Charmont, Stéphane; Jamet, Elisabeth; Pont-Lezica, Rafael; Canut, Hervé

    2005-02-01

    Arabidopsis thaliana seedlings grown in liquid culture were used to recover proteins secreted from the whole plant. The aim was to identify apoplastic proteins that may be lost during classical extraction procedures such as preparation of cell walls. The inclusion of polyvinyl-polypyrrolidone (PVPP) in the protocol of purification of secreted proteins allowed a more efficient identification of proteins after their separation by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analyses. Improvement of identification was 4-fold. It is related to an increased number of detectable peaks on mass spectra increasing the percentage of sequence coverage, and the identification confidence. The role of PVPP was to trap phenolic compounds and to prevent their unspecific interactions with proteins. These experiments resulted in the identification of 44 secreted proteins, of which 70% were not identified in previous cell wall proteomic studies. This may be due to specific gene regulation in seedlings and/or to a better access to apoplastic proteins not bound to cell walls.

  13. Nuclear Engineering of Microalgae for High Yield Secretion of Recombinant Proteins

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel

    to the glycomodules, accumulation of a fusion protein was dramatically increased by up to 12 folds, with the maximum yield of 15 mg L-1. Characterization of the secreted Venus showed the presence of glycosylations and increased resistance to proteolytic degradation. The results from this thesis demonstrate...

  14. Caught in the act: discovering secreted proteins from fungi and oomycetes in action

    DEFF Research Database (Denmark)

    Roth, Doris; Grell, Morten Nedergaard; Jensen, Annette Bruun;

    -infected host systems. By methods such as TAST (transposon-assisted signal trapping) and PCR with degenerated primers, the libraries are screened for secreted proteins, especially enzymes, which are then further characterized. We investigate a fourth system, the mycorrhizal fungus Paxillus convolutus...

  15. Identification of secreted proteins of Aspergillus oryzae associated with growth on solid cereal substrates

    NARCIS (Netherlands)

    Biesebeke, R. te; Boussier, A.; Biezen, N. van; Hondel, C.A.M.J.J. van den; Punt, P.J.

    2006-01-01

    Filamentous growth of Aspergillus oryzae on solid cereal substrates involves secretion of substrate converting enzymes and a solid substrate specific polarised hyphal growth phenotype. To identify proteins produced under these specific conditions, the extracts of A. oryzae grown on wheat-based media

  16. A novel screening system for secretion of heterologous proteins in Bacillus subtilis

    NARCIS (Netherlands)

    Trip, Hein; van der Veek, Patricia J.; Renniers, Ton C.; Meima, Rob; Sagt, Cees M.; Mohrmann, Lisette; Kuipers, Oscar P.

    2011-01-01

    High-level production of secretory proteins in Bacillus subtilis leads to a stress response involving the two-component system CssRS and its target genes htrA and htrB. Here, we used this sensing system in a reporter strain in which gfp is under control of P(htrA), the secretion stress responsive pr

  17. Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris

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    Valero Francisco

    2007-05-01

    Full Text Available Abstract Background Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a Rhizopus oryzae lipase (ROL in Pichia pastoris by means of 2D-fluorimetric techniques Results In this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in P. pastoris batch cultivations. Furthermore, both the fusion enzyme and the fluorescent protein (GFP S65T mutant retained their biological activity. However, when multiwavelength spectrofluorometry was used for extracellular fusion protein monitoring, riboflavin appeared as a major interfering component with GFP signal. Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity Conclusion P. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in P. pastoris using GFP fusions combined with multiwavelength spectrofluorimetric techniques, riboflavin may interfere with GFP fluorescence signal, thus limiting the application of some GFP variants for on-line extracellular recombinant protein quantification and monitoring purposes.

  18. Temperature-Induced Protein Secretion by Leishmania mexicana Modulates Macrophage Signalling and Function

    OpenAIRE

    Kasra Hassani; Elisabeth Antoniak; Armando Jardim; Martin Olivier

    2011-01-01

    Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 p...

  19. Estrogen stimulates release of secreted amyloid precursor protein from primary rat cortical neurons via protein kinase C pathway

    Institute of Scientific and Technical Information of China (English)

    Sun ZHANG; Ying HUANG; Yi-chun ZHU; Tai YAO

    2005-01-01

    Aim: To investigate the mechanism of the action of estrogen, which stimulates the release of secreted amyloid precursor protein α (sAPPα) and decreases the gen eration of amyloid-β protein (Aβ), a dominant component in senile plaques in the brains of Alzheimer's disease patients. Methods: Experiments were carried out inprimary rat cortical neurons, and Western blot was used to detect sAPPα in aculture medium and the total amount of cellular amyloid precursor protein (APP) in neurons. Results: 17β-Estradiol (but not 17α-estradiol) and β-estradiol 6-(Ocarboxymethyl) oxime: BSA increased the secretion of sAPPα and this effect was blocked by protein kinase C (PKC) inhibitor calphostin C, but not by the classical estrogen receptor antagonist ICI 182,780. Meanwhile, 17β-estradiol did not alter the synthesis of cellular APP. Conclusion: The effect of 17β-estradiol on sAPPα secretion is likely mediated through the membrane binding sites, and needs molecular configuration specificity of the ligand. Furthermore, the action of the PKC dependent pathway might be involved in estrogen-induced sAPPα secretion.

  20. Preconcentrating (within the broth) secreted extracellular proteins during a bakers' yeast fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Effler, W.T. Jr.; Pandey, N.K.; Tanner, R.D.; Malaney, G.W.; Scott, C.D. (ed.)

    1986-01-01

    Proteins secreted by yeast during the fermentation process are spacially fractionated (concentrated at a particular vertical position) within the fermentation vessel due to the phenomenon of bubble fractionation, despite moderately vigorous mixing. The degree of fractionation is influenced by the conditions in which the fermentation takes place. The broth pH strongly influences the extent of fractionation of specific proteins. In addition fractionation is enhanced under anaerobic conditions, presumably because there are more CO2 bubbles present for hydrophobic protein adsorption. The addition of moderate levels of salt to the broth reduces the fractionation for most (but not all) of the proteins.

  1. A Rapid Method for Determining the Concentration of Recombinant Protein Secreted from Pichia pastoris

    Science.gov (United States)

    Sun, L. W.; Zhao, Y.; Niu, L. P.; Jiang, R.; Song, Y.; Feng, H.; feng, K.; Qi, C.

    2011-02-01

    Pichia secretive expression system is one of powerful eukaryotic expression systems in genetic engineering, which is especially suitable for industrial utilization. Because of the low concentration of the target protein in initial experiment, the methods and conditions for expression of the target protein should be optimized according to the protein yield repetitively. It is necessary to set up a rapid, simple and convenient analysis method for protein expression levels instead of the generally used method such as ultrafiltration, purification, dialysis, lyophilization and so on. In this paper, acetone precipitation method was chosen to concentrate the recombinant protein firstly after comparing with four different protein precipitation methods systematically, and then the protein was analyzed by SDS-Polyacrylamide Gel Electrophoresis. The recombinant protein was determined with the feature of protein band by the Automated Image Capture and 1-D Analysis Software directly. With this method, the optimized expression conditions of basic fibroblast growth factor secreted from pichia were obtained, which is as the same as using traditional methods. Hence, a convenient tool to determine the optimized conditions for the expression of recombinant proteins in Pichia was established.

  2. Amyloid precursor protein is trafficked and secreted via synaptic vesicles.

    Directory of Open Access Journals (Sweden)

    Teja W Groemer

    Full Text Available A large body of evidence has implicated amyloid precursor protein (APP and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD. Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling.

  3. A structurally informed autotransporter platform for efficient heterologous protein secretion and display

    Directory of Open Access Journals (Sweden)

    Jong Wouter SP

    2012-06-01

    Full Text Available Abstract Background The self-sufficient autotransporter (AT pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein. Results Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development. Conclusions We developed the first structurally informed AT platform for efficient secretion and

  4. Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus

    Science.gov (United States)

    Rosado, Helena; O'Neill, Alex J.; Blake, Katy L.; Walther, Meik; Long, Paul F.; Hinds, Jason; Taylor, Peter W.

    2012-04-01

    Staphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein-ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

  5. Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

    Directory of Open Access Journals (Sweden)

    Stephens Andrew N

    2011-09-01

    Full Text Available Abstract Background Identification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW proteins. We have optimised a procedure to overcome this limitation. Results Low MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics. Conclusions This technique enables the analysis of secreted proteins of cultured cells low MW and low abundance.

  6. Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva

    Energy Technology Data Exchange (ETDEWEB)

    Mudenda, Lwiindi; Aguilar Pierle, Sebastian; Turse, Joshua E.; Scoles, Glen A.; Purvine, Samuel O.; Nicora, Carrie D.; Clauss, Therese RW; Ueti, Massaro W.; Brown, Wendy C.; Brayton, Kelly A.

    2014-08-07

    Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5 days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5 day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified.

  7. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    Science.gov (United States)

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-10-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

  8. Oit1/Fam3D, a gut-secreted protein displaying nutritional status-dependent regulation.

    Science.gov (United States)

    de Wit, Nicole J W; IJssennagger, Noortje; Oosterink, Els; Keshtkar, Shohreh; Hooiveld, Guido J E J; Mensink, Ronald P; Hammer, Sebastiaan; Smit, Johannes W A; Müller, Michael; van der Meer, Roelof

    2012-11-01

    Oncoprotein-induced transcript 1 (Oit1) was previously identified as a dietary fat-induced gene in the small intestine of C57Bl/6J mice. In this study, we further characterized Oit1 and its human ortholog family with sequence similarity 3, member D (Fam3D), on the messenger RNA as well as the protein level. Oit1 and Fam3D were found to be predominantly expressed in the gastrointestinal tract of mice and humans, respectively. Dietary fat induced a clear and acute up-regulation of Oit1, especially in the jejunum, whereas fasting led to a reduced gene expression in the small intestine. Regarding protein expression, we found a remarkable pattern of Oit1 along the longitudinal axis of the intestine, a predominant villus-restricted expression in the proximal small intestine and a more pronounced crypt expression in the distal parts of the intestine. Using transfection experiments, we confirmed secretion of the Oit1 protein, as was predicted by a signal peptide sequence. Detection of Oit1 and Fam3D in plasma samples indicated that both proteins are secreted to the basolateral site of enterocytes. Moreover, in human plasma samples, we also found an effect of nutritional status on Fam3D levels, with a postprandial elevation and a reduction after fasting. In conclusion, Oit1 and Fam3D are gut-derived proteins that are expressed and secreted in a nutritional status-dependent manner.

  9. Engineering human cells for in vivo secretion of antibody and non-antibody therapeutic proteins.

    Science.gov (United States)

    Sánchez-Martín, David; Sanz, Laura; Álvarez-Vallina, Luis

    2011-12-01

    Purified proteins such as antibodies are widely used as therapeutic agents in clinical medicine. However, clinical-grade proteins for therapeutic use require sophisticated technologies and are extremely expensive to produce. In vivo secretion of therapeutic proteins by genetically engineered human cells may advantageously replace injection of highly purified proteins. The use of gene transfer methods circumvents problems related to large-scale production and purification and offers additional benefits by achieving sustained concentrations of therapeutic protein with a syngenic glycosylation pattern that make the protein potentially less immunogenic. The feasibility of the in vivo production of therapeutic proteins by diverse cells/tissues has now been demonstrated using different techniques, such as ex vivo genetically modified cells and in vivo gene transfer mediated by viral vectors.

  10. Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Christian Konrad

    2010-04-01

    Full Text Available Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM consisting of three paralogous cyst wall proteins (CWP1-3 via organelles termed encystation-specific vesicles (ESVs. Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this "minimal Golgi" hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires

  11. Metformin directly inhibits ghrelin secretion through AMP-activated protein kinase in rat primary gastric cells.

    Science.gov (United States)

    Gagnon, J; Sheppard, E; Anini, Y

    2013-03-01

    The antidiabetic drug Metformin causes weight loss in both diabetic and non-diabetic individuals. Metformin treatment is also associated with lower circulating levels of the orexigenic hormone ghrelin. To test whether Metformin directly affects ghrelin cells, rat primary stomach cells were treated with Metformin and the levels of ghrelin secretion, proghrelin gene expression and activation of adenosine monophosphate-activated protein kinase (AMPK) were examined. Metformin significantly reduced ghrelin secretion and proghrelin mRNA production and both these effects were blocked by co-incubation with the AMPK inhibitor compound C. Furthermore, the AMPK activator 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) significantly inhibited ghrelin secretion. Additionally, ghrelin cells were shown to express AMPK. Finally, Metformin treatment caused a significant increase in the level of phosphorylated (active) AMPK. Our results show that Metformin directly inhibits stomach ghrelin production and secretion through AMPK. This reduction in ghrelin secretion may be one of the key components in Metformin's mechanism of weight loss.

  12. Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana

    Science.gov (United States)

    Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J.; Phu, My L.; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Rodriguez, Raymond L.; Nandi, Somen; McDonald, Karen A.

    2017-01-01

    Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting. PMID:28054967

  13. Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes

    Science.gov (United States)

    2014-01-01

    Background Efficient venom delivery systems are known to occur only in varanoid lizards and advanced colubroidean snakes among squamate reptiles. Although components of these venomous systems might have been present in a common ancestor, the two lineages independently evolved strikingly different venom gland systems. In snakes, venom is produced exclusively by serous glands in the upper jaw. Within the colubroidean radiation, lower jaw seromucous infralabial glands are known only in two distinct lineages–the basal pareatids and the more advanced Neotropical dipsadines known as “goo-eating snakes”. Goo-eaters are a highly diversified, ecologically specialized clade that feeds exclusively on invertebrates (e.g., gastropod molluscs and annelids). Their evolutionary success has been attributed to their peculiar feeding strategies, which remain surprisingly poorly understood. More specifically, it has long been thought that the more derived Dipsadini genera Dipsas and Sibynomorphus use glandular toxins secreted by their infralabial glands to extract snails from their shells. Results Here, we report the presence in the tribe Dipsadini of a novel lower jaw protein-secreting delivery system effected by a gland that is not functionally related to adjacent teeth, but rather opens loosely on the oral epithelium near the tip of the mandible, suggesting that its secretion is not injected into the prey as a form of envenomation but rather helps control the mucus and assists in the ingestion of their highly viscous preys. A similar protein-secreting system is also present in the goo-eating genus Geophis and may share the same adaptive purpose as that hypothesized for Dipsadini. Our phylogenetic hypothesis suggests that the acquisition of a seromucous infralabial gland represents a uniquely derived trait of the goo-eating clade that evolved independently twice within the group as a functionally complex protein-secreting delivery system. Conclusions The acquisition by snail

  14. Pmp-like proteins Pls1 and Pls2 are secreted into the lumen of the Chlamydia trachomatis inclusion.

    Science.gov (United States)

    Jorgensen, Ine; Valdivia, Raphael H

    2008-09-01

    The obligate intracellular pathogen Chlamydia trachomatis secretes effector proteins across the membrane of the pathogen-containing vacuole (inclusion) to modulate host cellular functions. In an immunological screen for secreted chlamydial proteins, we identified CT049 and CT050 as potential inclusion membrane-associated proteins. These acidic, nonglobular proteins are paralogously related to the passenger domain of the polymorphic membrane protein PmpC and, like other Pmp proteins, are highly polymorphic among C. trachomatis ocular and urogenital strains. We generated antibodies to these Pmp-like secreted (Pls) proteins and determined by immunofluorescence microscopy that Pls1 (CT049) and Pls2 (CT050) localized to globular structures within the inclusion lumen and at the inclusion membrane. Fractionation of membranes and cytoplasmic components from infected cells by differential and density gradient centrifugation further indicated that Pls1 and Pls2 associated with membranes distinct from the bulk of bacterial and inclusion membranes. The accumulation of Pls1 and, to a lesser extent, Pls2 in the inclusion lumen was insensitive to the type III secretion inhibitor C1, suggesting that this translocation system is not essential for Pls protein secretion. In contrast, Pls secretion and stability were sensitive to low levels of beta-lactam antibiotics, suggesting that a functional cell wall is required for Pls secretion from the bacterial cell. Finally, we tested the requirement for these proteins in Chlamydia infection by microinjecting anti-Pls1 and anti-Pls2 antibodies into infected cells. Coinjection of anti-Pls1 and -Pls2 antibodies partially inhibited expansion of the inclusion. Because Pls proteins lack classical sec-dependent secretion signals, we propose that Pls proteins are secreted into the inclusion lumen by a novel mechanism to regulate events important for chlamydial replication and inclusion expansion.

  15. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells.

    Science.gov (United States)

    Vogt, Rhonda R; Unda, Richard; Yeh, Lee-Chuan C; Vidro, Eileen K; Lee, John C; Tsin, Andrew T

    2006-08-01

    Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

  16. Heterologous protein secretion in Lactobacilli with modified pSIP vectors.

    Directory of Open Access Journals (Sweden)

    Ingrid Lea Karlskås

    Full Text Available We describe new variants of the modular pSIP-vectors for inducible gene expression and protein secretion in lactobacilli. The basic functionality of the pSIP system was tested in Lactobacillus strains representing 14 species using pSIP411, which harbors the broad-host-range Lactococcus lactis SH71rep replicon and a β-glucuronidase encoding reporter gene. In 10 species, the inducible gene expression system was functional. Based on these results, three pSIP vectors with different signal peptides were modified by replacing their narrow-host-range L. plantarum 256rep replicon with SH71rep and transformed into strains of five different species of Lactobacillus. All recombinant strains secreted the target protein NucA, albeit with varying production levels and secretion efficiencies. The Lp_3050 derived signal peptide generally resulted in the highest levels of secreted NucA. These modified pSIP vectors are useful tools for engineering a wide variety of Lactobacillus species.

  17. The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation

    KAUST Repository

    Zimaro, Tamara

    2014-04-18

    Background: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. Results: The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. Conclusions: Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis. 2014 Zimaro et al.; licensee BioMed Central Ltd.

  18. Novel diagnosis for citrus stubborn disease by detection of a spiroplasma citri-secreted protein.

    Science.gov (United States)

    Shi, Jinxia; Pagliaccia, Deborah; Morgan, Robyn; Qiao, Yongli; Pan, Songqin; Vidalakis, Georgios; Ma, Wenbo

    2014-02-01

    Citrus stubborn disease (CSD), first identified in California, is a widespread bacterial disease found in most arid citrus-producing regions in the United States and the Mediterranean Region. The disease is caused by Spiroplasma citri, an insect-transmitted and phloem-colonizing bacterium. CSD causes significant tree damage resulting in loss of fruit production and quality. Detection of CSD is challenging due to low and fluctuating titer and sporadic distribution of the pathogen in infected trees. In this study, we report the development of a novel diagnostic method for CSD using an S. citri-secreted protein as the detection marker. Microbial pathogens secrete a variety of proteins during infection that can potentially disperse systemically in infected plants with the vascular flow. Therefore, their distribution may not be restricted to the pathogen infection sites and could be used as a biological marker for infection. Using mass spectrometry analysis, we identified a unique secreted protein from S. citri that is highly expressed in the presence of citrus phloem extract. ScCCPP1, an antibody generated against this protein, was able to distinguish S. citri-infected citrus and periwinkle from healthy plants. In addition, the antiserum could be used to detect CSD using a simple direct tissue print assay without the need for sample processing or specialized lab equipment and may be suitable for field surveys. This study provides proof of a novel concept of using pathogen-secreted protein as a marker for diagnosis of a citrus bacterial disease and can probably be applied to other plant diseases.

  19. Calcium-containing phosphopeptides pave the secretory pathway for efficient protein traffic and secretion in fungi.

    Science.gov (United States)

    Martín, Juan F

    2014-01-01

    Casein phosphopeptides (CPPs) containing chelated calcium drastically increase the secretion of extracellular homologous and heterologous proteins in filamentous fungi. Casein phosphopeptides released by digestion of alpha - and beta-casein are rich in phosphoserine residues (SerP). They stimulate enzyme secretion in the gastrointestinal tract and enhance the immune response in mammals, and are used as food supplements. It is well known that casein phosphopeptides transport Ca2+ across the membranes and play an important role in Ca2+ homeostasis in the cells. Addition of CPPs drastically increases the production of heterologous proteins in Aspergillus as host for industrial enzyme production. Recent proteomics studies showed that CPPs alter drastically the vesicle-mediated secretory pathway in filamentous fungi, apparently because they change the calcium concentration in organelles that act as calcium reservoirs. In the organelles calcium homeostasis a major role is played by the pmr1 gene, that encodes a Ca2+/Mn2+ transport ATPase, localized in the Golgi complex; this transporter controls the balance between intra-Golgi and cytoplasmic Ca2+ concentrations. A Golgi-located casein kinase (CkiA) governs the ER to Golgi directionality of the movement of secretory proteins by interacting with the COPII coat of secretory vesicles when they reach the Golgi. Mutants defective in the casein-2 kinase CkiA show abnormal targeting of some secretory proteins, including cytoplasmic membrane amino acid transporters that in ckiA mutants are miss-targeted to vacuolar membranes. Interestingly, addition of CPPs increases a glyceraldehyde-3-phpshate dehydrogenase protein that is known to associate with microtubules and act as a vesicle/membrane fusogenic agent. In summary, CPPs alter the protein secretory pathway in fungi adapting it to a deregulated protein traffic through the organelles and vesicles what results in a drastic increase in secretion of heterologous and also of

  20. Rapid evolution of the sequences and gene repertoires of secreted proteins in bacteria.

    Directory of Open Access Journals (Sweden)

    Teresa Nogueira

    Full Text Available Proteins secreted to the extracellular environment or to the periphery of the cell envelope, the secretome, play essential roles in foraging, antagonistic and mutualistic interactions. We hypothesize that arms races, genetic conflicts and varying selective pressures should lead to the rapid change of sequences and gene repertoires of the secretome. The analysis of 42 bacterial pan-genomes shows that secreted, and especially extracellular proteins, are predominantly encoded in the accessory genome, i.e. among genes not ubiquitous within the clade. Genes encoding outer membrane proteins might engage more frequently in intra-chromosomal gene conversion because they are more often in multi-genic families. The gene sequences encoding the secretome evolve faster than the rest of the genome and in particular at non-synonymous positions. Cell wall proteins in Firmicutes evolve particularly fast when compared with outer membrane proteins of Proteobacteria. Virulence factors are over-represented in the secretome, notably in outer membrane proteins, but cell localization explains more of the variance in substitution rates and gene repertoires than sequence homology to known virulence factors. Accordingly, the repertoires and sequences of the genes encoding the secretome change fast in the clades of obligatory and facultative pathogens and also in the clades of mutualists and free-living bacteria. Our study shows that cell localization shapes genome evolution. In agreement with our hypothesis, the repertoires and the sequences of genes encoding secreted proteins evolve fast. The particularly rapid change of extracellular proteins suggests that these public goods are key players in bacterial adaptation.

  1. Dietary protein-induced hepatic IGF-1 secretion mediated by PPARγ activation

    Science.gov (United States)

    Wan, Xiaojuan; Wang, Songbo; Xu, Jingren; Zhuang, Lu; Xing, Kongping; Zhang, Mengyuan; Zhu, Xiaotong; Wang, Lina; Gao, Ping; Xi, Qianyun; Sun, Jiajie; Zhang, Yongliang; Li, Tiejun; Shu, Gang; Jiang, Qingyan

    2017-01-01

    Dietary protein or amino acid (AA) is a crucial nutritional factor to regulate hepatic insulin-like growth factor-1 (IGF-1) expression and secretion. However, the underlying intracellular mechanism by which dietary protein or AA induces IGF-1 expression remains unknown. We compared the IGF-1 gene expression and plasma IGF-1 level of pigs fed with normal crude protein (CP, 20%) and low-protein levels (LP, 14%). RNA sequencing (RNA-seq) was performed to detect transcript expression in the liver in response to dietary protein. The results showed that serum concentrations and mRNA levels of IGF-1 in the liver were higher in the CP group than in the LP group. RNA-seq analysis identified a total of 1319 differentially expressed transcripts (667 upregulated and 652 downregulated), among which the terms “oxidative phosphorylation”, “ribosome”, “gap junction”, “PPAR signaling pathway”, and “focal adhesion” were enriched. In addition, the porcine primary hepatocyte and HepG2 cell models also demonstrated that the mRNA and protein levels of IGF-1 and PPARγ increased with the increasing AA concentration in the culture. The PPARγ activator troglitazone increased IGF-1 gene expression and secretion in a dose dependent manner. Furthermore, inhibition of PPARγ effectively reversed the effects of the high AA concentration on the mRNA expression of IGF-1 and IGFBP-1 in HepG2 cells. Moreover, the protein levels of IGF-1 and PPARγ, as well as the phosphorylation of mTOR, significantly increased in HepG2 cells under high AA concentrations. mTOR phosphorylation can be decreased by the mTOR antagonist, rapamycin. The immunoprecipitation results also showed that high AA concentrations significantly increased the interaction of mTOR and PPARγ. In summary, PPARγ plays an important role in the regulation of IGF-1 secretion and gene expression in response to dietary protein. PMID:28257428

  2. Identification and quantification of serum proteins secreted into the normal human jejunum

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Hegnhøj, J H

    1990-01-01

    The in vivo transfer of serum proteins to the human intestinal lumen was characterized by crossed immunoelectrophoretic analyses of intestinal perfusates from four healthy volunteers. Serum proteins with molecular masses below 100 kDa and the immunoglobulins were found in human jejunal perfusates....... Larger serum proteins were either absent (alpha and beta lipoproteins) or present in small amounts (alpha 2-macroglobulin, haptoglobulin and ceruloplasmin). These results demonstrate the existence of a selective transfer of serum proteins to the intestinal lumen under physiological conditions....... The intestinal clearance rate was 0.1 ml serum per hour per 10 cm jejunum for albumin, prealbumin, alpha 1-antitrypsin, orosomucoid, transferrin and haemopexin. The rate of secretion of total protein to the jejunal lumen was 100 mg protein per hour per 10 cm jejunum. About 45% was due to immunoglobulins...

  3. Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion

    OpenAIRE

    Jana Krtková; Elizabeth B Thomas; Germain C. M. Alas; Schraner, Elisabeth M.; Behjatnia, Habib R; Hehl, Adrian B.; Paredez, Alexander R.

    2016-01-01

    ABSTRACT Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmi...

  4. Rac regulates giardia lamblia encystation by coordinating cyst wall protein trafficking and secretion

    OpenAIRE

    Krtková, Jana; Elizabeth B Thomas; Germain C. M. Alas; Schraner, Elisabeth M.; Behjatnia, Habib R; Hehl, Adrian B.; Paredez, Alexander R.

    2016-01-01

    UNLABELLED Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia's sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplas...

  5. Secretion of protein and epidermal growth factor (EGF) by transplanted human pancreas.

    Science.gov (United States)

    Konturek, J W; Buesing, M; Hopt, U T; Stachura, J; Becker, H D; Konturek, S J

    1992-08-01

    Epidermal growth factor (EGF) has been localized in human salivary and Brunner's glands and found to stimulate the proliferation of gastrointestinal and pancreatic tissues in animals, but little is known about EGF in human pancreas. This study was designed to determine the distribution and release of EGF in the pancreas and to assess the secretion of EGF and protein by the transplanted human pancreas. The peroxidase antiperoxidase (PAP) immunocytochemical method with anti-hEGF showed that EGF was restricted mainly to the excretory cells lining pancreatic ducts. The EGF immunoreactivity in the pancreatic tissue averaged about 15 +/- 0.5 micrograms/g of tissue wt. The concentration and output of EGF in the pancreatic juice were, respectively, about 3.4 +/- 0.7 ng/mL and 68 + 12 ng/h in basal secretion collected from the whole pancreatic transplant. A significant increase in EGF release from this transplant started about 2 h after its reperfusion and was accompanied by a parallel increase in protein output. Injection of iv secretion (1 U/kg) resulted in a transient rise in EGF output, probably as a result of washout by increased vol flow, whereas HCCK (1 U/kg) caused more prolonged release of EGF accompanied by a marked stimulation of protein secretion. Ingestion of a mixed meal caused an immediate and sustained increment in EGF output, and protein output showed a more protracted increase, reaching its peak in the second postprandial hour. Fractionation of an extract of pancreatic juice on G-5O Sephadex superfine column revealed that EGF immunoreactivity emerged as a major peak in the same position as authentic human EGF (hEGF).(ABSTRACT TRUNCATED AT 250 WORDS)

  6. The rab11 effector protein FIP1 regulates adiponectin trafficking and secretion.

    Science.gov (United States)

    Carson, Brian P; Del Bas, Josep Maria; Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia

    2013-01-01

    Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release.

  7. The rab11 effector protein FIP1 regulates adiponectin trafficking and secretion.

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    Brian P Carson

    Full Text Available Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release.

  8. Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins

    NARCIS (Netherlands)

    Kaba, S.A.; Salcedo, A.M.; Wafula, P.O.; Vlak, J.M.; Oers, van M.M.

    2004-01-01

    The application of the baculovirus-in sect cell expression system for the production of integral membrane and secreted proteins is often more troublesome than for cytoplasmic proteins. One protein expressed at low levels in insect cells is the Theileria parva sporozoite surface protein p67. Theileri

  9. Mutations in the West Nile prM protein affect VLP and virion secretion in vitro.

    Science.gov (United States)

    Calvert, Amanda E; Huang, Claire Y-H; Blair, Carol D; Roehrig, John T

    2012-11-10

    Mutation of the West Nile virus-like particle (WN VLP) prM protein (T20D, K31A, K31V, or K31T) results in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however these proteins remained in the ER and ER-Golgi intermediary compartments of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles.

  10. Cloning and molecular characterization of cDNAs encoding three Ancylostoma ceylanicum secreted proteins.

    Science.gov (United States)

    Siwińska, Anna M; Bąska, Piotr; Daniłowicz-Luebert, Emilia; Januszkiewicz, Kamil; Długosz, Ewa; Wędrychowicz, Halina; Cappello, Michael; Wiśniewski, Marcin

    2013-03-01

    Ancylostoma ceylanicum belongs to a group of soil-transmitted helminths, which infect almost 576 mln people worldwide and are a major cause of anaemia and malnutrition. Upon contact with a permissive host, third-stage larvae (L3) residing in the environment become activated larvae (ssL3), a process associated with changes in the profile of gene expression. Ancylostoma secreted proteins (ASPs) are the major proteins secreted during larvae activation and play a crucial role in hookworm adaptation to parasitism. Here we report the cloning using RACE-PCR technique of three novel ASPs from the hookworm A. ceylanicum (Ace-asp-3, Ace-asp-4, and Ace-asp-5) and computational analysis of the protein sequences. All three proteins contain SCP (Sperm Coating Protein) domain characteristic for previously described ASP proteins. Real-time PCR analysis shows significant up-regulation of Ace-asp-3 and Ace-asp-5 expression in adult worms and correlated down-regulation in ssL3 larvae. On the other hand, expression of Ace-asp-4 was increased in ssL3 stages and decreased in adult parasites.

  11. Temperature-induced protein secretion by Leishmania mexicana modulates macrophage signalling and function.

    Science.gov (United States)

    Hassani, Kasra; Antoniak, Elisabeth; Jardim, Armando; Olivier, Martin

    2011-05-03

    Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.

  12. Temperature-induced protein secretion by Leishmania mexicana modulates macrophage signalling and function.

    Directory of Open Access Journals (Sweden)

    Kasra Hassani

    Full Text Available Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS during transmission from the sandfly vector (ambient temperature, 25-26°C to the mammalian host (37°C. We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.

  13. Have We Overlooked the Importance of Serine/Threonine Protein Phosphatases in Pancreatic Beta-Cells? Role Played by Protein Phosphatase 2A in Insulin Secretion

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    Esser V

    2005-07-01

    Full Text Available Genetic predisposition and environmental influences insidiously converge to cause glucose intolerance and hyperglycemia. Beta-cell compensates by secreting more insulin and when it fails, overt diabetes mellitus ensues. The need to understand the mechanisms involved in insulin secretion cannot be stressed enough. Phosphorylation of proteins plays an important role in regulating insulin secretion. In order to understand how a particular cellular process is regulated by protein phosphorylation the nature of the protein kinases and protein phosphatases involved and the mechanisms that determine when and where these enzymes are active should be investigated. While the actions of protein kinases have been intensely studied within the beta-cell, less emphasis has been placed on protein phosphatases even though they play an important regulatory role. This review focuses on the importance of protein phosphatase 2A in insulin secretion. Most of the present knowledge on protein phosphatase 2A originates from protein phosphatase inhibitor studies on islets and beta-cell lines. The ability of protein phosphatase 2A to change its activity in the presence of glucose and inhibitors provides clues to its role in regulating insulin secretion. An aggressive approach to elucidate the substrates and mechanisms of action of protein phosphatases is crucial to the understanding of phosphorylation events within the beta-cell. Characterizing protein phosphatase 2A within the beta-cell will certainly help us in understanding the mechanisms involved in insulin secretion and provide valuable information for drug development.

  14. Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk.

    Science.gov (United States)

    van Herwaarden, Antonius E; Wagenaar, Els; Merino, Gracia; Jonker, Johan W; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2007-02-01

    The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B(2)) into milk, thus supplying the young with this important nutrient. In Bcrp1(-/-) mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1(-/-) pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B(2) equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters.

  15. P80, the HinT interacting membrane protein, is a secreted antigen of Mycoplasma hominis

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    Henrich Birgit

    2004-12-01

    Full Text Available Abstract Background Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT. HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex. As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT. Results Further characterization of P80 suggested that the 4.7 kDa signal peptide is protected from cleavage only in the membrane bound form. We found several proteins were released into the supernatant of a logarithmic phase mycoplasma culture, including P80, which was reduced in size by 10 kDa. Western blot analysis of recombinant P80 mutants expressed in E. coli and differing in the N-terminal region revealed that mutation of the +1 position of the mature protein (Asn to Pro which is important for signal peptidase I recognition resulted in reduced P80 secretion. All other P80 variants were released into the supernatant, in general as a 74 kDa protein encompassing the helical part of P80. Incubation of M. hominis cells in phosphate buffered saline supplemented with divalent cations revealed that the release of mycoplasma proteins into the supernatant was inhibited by high concentrations of calciumions. Conclusions Our model for secretion of the P80 protein of M. hominis implies a two-step process. In general the P80 protein is transported across the membrane and remains complexed to P60, surface-exposed and membrane anchored via the uncleaved signal sequence. Loss of the 4.7 kDa signal peptide seems to be a pre-requisite for P

  16. Secretion of biologically active interferon-gamma inducible protein-10 (IP-10 by Lactococcus lactis

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    Saucedo-Cardenas Odila

    2008-07-01

    Full Text Available Abstract Background Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10 is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10. Results The IP-10 coding region was isolated from human cDNA and cloned into an L. lactis expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of L. lactis secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of L. lactis. In addition, we demonstrated that the NICE (nisin-controlled gene expression system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by L. lactis was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes. Conclusion Expression and secretion of mature IP-10 was efficiently achieved by L. lactis forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as

  17. Involvement of major facilitator superfamily proteins SfaA and SbnD in staphyloferrin secretion in Staphylococcus aureus.

    Science.gov (United States)

    Hannauer, Mélissa; Sheldon, Jessica R; Heinrichs, David E

    2015-03-12

    A paucity of information exists concerning the mechanism(s) by which bacteria secrete siderophores into the extracellular compartment. We investigated the role of SfaA and SbnD, two major facilitator superfamily (MFS)-type efflux proteins, in the secretion of the Staphylococcus aureus siderophores staphyloferrin A (SA) and staphyloferrin B (SB), respectively. Deletion of sfaA resulted in a drastic reduction of SA secreted into the supernatant with a corresponding accumulation of SA in the cytoplasm and a significant growth defect in cells devoid of SB synthesis. In contrast, sbnD mutants showed transiently lowered levels of secreted SB, suggesting the involvement of additional efflux mechanisms.

  18. Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi.

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    Diane G O Saunders

    Full Text Available Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i contain a secretion signal, (ii are encoded by in planta induced genes, (iii have similarity to haustorial proteins, (iv are small and cysteine rich, (v contain a known effector motif or a nuclear localization signal, (vi are encoded by genes with long intergenic regions, (vii contain internal repeats, and (viii do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.

  19. Using Hierarchical Clustering of Secreted Protein Families to Classify and Rank Candidate Effectors of Rust Fungi

    Science.gov (United States)

    Saunders, Diane G. O.; Win, Joe; Cano, Liliana M.; Szabo, Les J.; Kamoun, Sophien; Raffaele, Sylvain

    2012-01-01

    Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components. PMID:22238666

  20. Correlation of secreted protein acidic and rich in cysteine with diabetic nephropathy.

    Science.gov (United States)

    Li, Lei; Song, Hai-Yan; Liu, Kai; An, Meng-Meng

    2015-01-01

    To detect the serum concentrations of secreted protein acidic and rich in cysteine (SPARC) in patients with diabetic nephropathy and SPARC mRNA and protein expressions in renal tissue of db/db mice (C57BL/KsJ, diabetic nephropathy mice), thus preliminary exploration on the role of secreted protein acidic riches in cysteine in the development of diabetic nephropathy were carried out. Serum SPARC levels in normal subjects, patients with type 2 diabetes mellitus (without diabetic nephropathy), chronic renal failure (without diabetes mellitus), and diabetic nephropathy were determined with enzyme-linked immunosorbent assay. 12-week-old db/db mice (db/db group) and its littermate wild-type control mice (NC group) were selected with 6 from each group, and the kidney tissue were taken. RT-PCR, Western blot, and immunofluorescence were used to detect the mRNA, targeted protein expressions of SPARC and the staining of renal tissue. The serum level of SPARC in diabetic nephropathy group was significantly higher than those in normal group, type 2 diabetes mellitus, and chronic renal failure group (P < 0.05 or P < 0.01). The SPARC level in the type 2 diabetes mellitus group was higher than that in normal group (P < 0.05), but there was no difference between normal group and chronic renal failure. SPARC mRNA and protein levels in renal tissue of db/db mice were higher compared with the normal control group (P < 0.05). The long term hyperglycemic state in patients with diabetic nephropathy causes pathological change of renal tissue. Simultaneously, increased secretion of SPARC from renal tissue results in elevation of serum SPARC level. SPARC correlates with the occurrence and progression of diabetes, and it may play a role in pathological change of diabetic nephropathy.

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

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    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

  2. Structure of a PE–PPE–EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    Science.gov (United States)

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-01-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed “type VII.” How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE–PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways. PMID:25275011

  3. Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion.

    Science.gov (United States)

    Finnie, Christine; Andersen, Birgit; Shahpiri, Azar; Svensson, Birte

    2011-05-01

    The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms.

  4. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Christina Kopp

    2014-11-01

    Full Text Available The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001 and the mRNA abundances of GPR109A (p ≤ 0.05 and chemerin (p ≤ 0.01. Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001. The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

  5. Role of prolyl hydroxylase domain proteins in the regulation of insulin secretion.

    Science.gov (United States)

    Huang, Mei; Paglialunga, Sabina; Wong, Julia M-K; Hoang, Monica; Pillai, Renjitha; Joseph, Jamie W

    2016-03-01

    Type 2 diabetes is associated with impaired nutrient-regulated anaplerosis and insulin secretion in pancreatic β-cells. One key anaplerotic substrate that may be involved in regulating insulin release is α-ketoglutarate (αKG). Since prolyl hydroxylase domain proteins (PHDs) can metabolize cytosolic αKG, we sought to explore the role of this enzyme in the regulation of β-cell function. The oxygen-sensing PHDs regulate the stability of hypoxia-inducible factor 1α (HIF1α) as well as other proline-containing proteins by catalyzing the hydroxylation of proline residues. This reaction is dependent on sufficient levels of oxygen, iron, and αKG. In the present study, we utilized both pharmacological and genetic approaches to assess the impact of inhibiting PHD activity on β-cell function. We demonstrate that ethyl-3,4-dihydroxybenzoate (EDHB), a PHD inhibitor, significantly blunted glucose-stimulated insulin secretion (GSIS) from 832/13 clonal cells, rat, and human islets. EDHB reduced glucose utilization, ATP/ADP ratio, and key TCA cycle intermediates such as pyruvate, citrate, fumarate, and malate. siRNA-mediated knockdown of PHD1 and PHD3 inhibited GSIS, whereas siRNA-mediated knockdown of PHD2 had no effect on GSIS. Taken together, the current results demonstrate an important role for PHDs as mediators of islet insulin secretion.

  6. Sprinter: a novel transmembrane protein required for Wg secretion and signaling.

    Science.gov (United States)

    Goodman, Robyn M; Thombre, Shreya; Firtina, Zeynep; Gray, Dione; Betts, Daniella; Roebuck, Jamie; Spana, Eric P; Selva, Erica M

    2006-12-01

    Wingless (Wg) is a secreted ligand that differentially activates gene expression in target tissues. It belongs to the Wnt family of secreted signaling molecules that regulate cell-to-cell interactions during development. Activation of Wg targets is dependent on the ligand concentration in the extracellular milieu; cellular mechanisms that govern the synthesis, delivery and receipt of Wg are elaborate and complex. We have identified sprinter (srt), which encodes a novel, evolutionarily conserved transmembrane protein required for the transmission of the Wg signal. Mutations in srt cause the accumulation of Wg in cells that express it, and retention of the ligand prevents activation of its target genes in signal-receiving cells. In the absence of Srt activity, levels of Wg targets (including Engrailed in embryos lacking maternal and zygotic srt, and Senseless and Achaete in wing discs) are reduced. Activation of Wg targets in the receiving cells does not require srt. Hence, the function of Srt is restricted to events occurring within the Wg-producing cells. We show that srt is not required for any aspect of Hedgehog (Hh) signal transduction, suggesting specificity of srt for the Wg pathway. We propose that srt encodes a protein required for Wg secretion that regulates maturation, membrane targeting or delivery of Wg. Loss of srt function in turn diminishes Wg-pathway activation in receiving cells.

  7. The Ca(2+)-dependent activator protein for secretion CAPS: do I dock or do I prime?

    Science.gov (United States)

    Stevens, David R; Rettig, Jens

    2009-02-01

    The "Ca(2+)-dependent activator protein for secretion" (CAPS) is a protein which reconstitutes regulated secretion in permeabilized neuroendocrine cells. It is generally accepted that CAPS plays an important role in the release of the contents of dense core vesicles in the nervous system as well as in a variety of other secretory tissues. At which step in the exocytotic process CAPS functions as well as its role in the fusion of synaptic vesicles is still under dispute. A recent growth spurt in the CAPS field has been fueled by genetic approaches in Caenorhabditis elegans and Drosophila as well as the application of knockout and knockdown approaches in mouse cells and in cell lines, respectively. We have attempted to review the body of work that established CAPS as an important regulator of secretion and to describe new information that has furthered our understanding of how CAPS may function. We discuss the conclusions, point out areas where controversy remains, and suggest directions for future experiments.

  8. Insulin Stimulates S100B Secretion and These Proteins Antagonistically Modulate Brain Glucose Metabolism.

    Science.gov (United States)

    Wartchow, Krista Minéia; Tramontina, Ana Carolina; de Souza, Daniela F; Biasibetti, Regina; Bobermin, Larissa D; Gonçalves, Carlos-Alberto

    2016-06-01

    Brain metabolism is highly dependent on glucose, which is derived from the blood circulation and metabolized by the astrocytes and other neural cells via several pathways. Glucose uptake in the brain does not involve insulin-dependent glucose transporters; however, this hormone affects the glucose influx to the brain. Changes in cerebrospinal fluid levels of S100B (an astrocyte-derived protein) have been associated with alterations in glucose metabolism; however, there is no evidence whether insulin modulates glucose metabolism and S100B secretion. Herein, we investigated the effect of S100B on glucose metabolism, measuring D-(3)H-glucose incorporation in two preparations, C6 glioma cells and acute hippocampal slices, and we also investigated the effect of insulin on S100B secretion. Our results showed that: (a) S100B at physiological levels decreases glucose uptake, through the multiligand receptor RAGE and mitogen-activated protein kinase/ERK signaling, and (b) insulin stimulated S100B secretion via PI3K signaling. Our findings indicate the existence of insulin-S100B modulation of glucose utilization in the brain tissue, and may improve our understanding of glucose metabolism in several conditions such as ketosis, streptozotocin-induced dementia and pharmacological exposure to antipsychotics, situations that lead to changes in insulin signaling and extracellular levels of S100B.

  9. Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes.

    Science.gov (United States)

    Park, Jung Eun; Tan, Hon Sen; Datta, Arnab; Lai, Ruenn Chai; Zhang, Huoming; Meng, Wei; Lim, Sai Kiang; Sze, Siu Kwan

    2010-06-01

    Under hypoxia, tumor cells produce a secretion that modulates their microenvironment to facilitate tumor angiogenesis and metastasis. Here, we observed that hypoxic or reoxygenated A431 carcinoma cells exhibited enhanced angiogenic and metastatic potential such as reduced cell-cell and cell-extracellular matrix adhesion, increased invasiveness, and production of a secretion with increased chorioallantoic membrane angiogenic activity. Consistent with these observations, quantitative proteomics revealed that under hypoxia the tumor cells secreted proteins involved in angiogenesis, focal adhesion, extracellular matrix-receptor interaction, and immune cell recruitment. Unexpectedly, the secreted proteins were predominantly cytoplasmic and membrane proteins. Ultracentrifugation at 100,000 x g precipitated 54% of the secreted proteins and enriched for many exosome-associated proteins such as the tetraspanins and Alix and also proteins with the potential to facilitate angiogenesis and metastasis. Two tetraspanins, CD9 and CD81, co-immunoprecipitated. Together, these data suggested that tumor cells secrete proteins and exosomes with the potential to modulate their microenvironment and facilitate angiogenesis and metastasis.

  10. G protein-coupled receptor 120 signaling regulates ghrelin secretion in vivo and in vitro.

    Science.gov (United States)

    Gong, Zhi; Yoshimura, Makoto; Aizawa, Sayaka; Kurotani, Reiko; Zigman, Jeffrey M; Sakai, Takafumi; Sakata, Ichiro

    2014-01-01

    Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced predominantly in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased immediately after feeding and that fasting-induced ghrelin release is controlled by the sympathetic nervous system. However, the mechanisms of plasma ghrelin decrement after feeding are poorly understood. Here, we studied the control of ghrelin secretion using ghrelin-producing cell lines and found that these cells express high levels of mRNA encoding G-protein coupled receptor 120 (GPR120). Addition of GW-9508 (a GPR120 chemical agonist) and α-linolenic acid (a natural ligand for GPR120) inhibited the secretion of ghrelin by ∼50 and 70%, respectively. However, the expression levels of preproghrelin and ghrelin O-acyltransferase (GOAT) mRNAs were not influenced by GW-9508. In contrast, the expression levels of prohormone convertase 1 were decreased significantly by GW-9508 incubation. Moreover, we observed that the inhibitory effect of GW-9508 on ghrelin secretion was blocked by a small interfering RNA (siRNA) targeting the sequence of GPR120. Furthermore, pretreatment with GW-9508 blocked the effect of the norepinephrine (NE)-induced ghrelin elevation in ghrelin cell lines. In addition, we showed that GW-9508 inhibited ghrelin secretion via extracellular signal-regulated kinase activity in ghrelin cell lines. Finally, we found that GW-9508 decreased plasma ghrelin levels in mice. These results suggest that the decrease of ghrelin secretion after feeding is induced partially by long-chain fatty acids that act directly on gastric GPR120-expressing ghrelin cells.

  11. Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion

    Directory of Open Access Journals (Sweden)

    Jana Krtková

    2016-08-01

    Full Text Available Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM. However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER and the Golgi apparatus-like encystation-specific vesicles (ESVs. Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA-tagged GlRac (HA-RacCA revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-RacCA-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis.

  12. A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes

    DEFF Research Database (Denmark)

    Kratchmarova, Irina; Kalume, Dario E; Blagoev, Blagoy;

    2002-01-01

    We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secre...

  13. Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants.

    Science.gov (United States)

    Pradenas, M; Jara, M C; Hernández, N; Zambrano, A; Collins, M T; Kruze, J

    2009-09-18

    Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.

  14. A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.

    Science.gov (United States)

    Grefen, Christopher; Karnik, Rucha; Larson, Emily; Lefoulon, Cécile; Wang, Yizhou; Waghmare, Sakharam; Zhang, Ben; Hills, Adrian; Blatt, Michael R

    2015-01-01

    Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

  15. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

    Science.gov (United States)

    Unterweger, Daniel; Kostiuk, Benjamin; Ötjengerdes, Rina; Wilton, Ashley; Diaz-Satizabal, Laura; Pukatzki, Stefan

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

  16. Absence of repellents in Ustilago maydis induces genes encoding small secreted proteins.

    Science.gov (United States)

    Teertstra, Wieke R; Krijgsheld, Pauline; Wösten, Han A B

    2011-08-01

    The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae.

  17. A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species.

    Science.gov (United States)

    Ryu, Jaewook; Lee, Ukjin; Park, Jiye; Yoo, Do-Hyun; Ahn, Jung Hoon

    2015-03-01

    Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species.

  18. Calcium-dependent activator protein for secretion 2 (CAPS2) promotes BDNF secretion and is critical for the development of GABAergic interneuron network.

    Science.gov (United States)

    Shinoda, Yo; Sadakata, Tetsushi; Nakao, Kazuhito; Katoh-Semba, Ritsuko; Kinameri, Emi; Furuya, Asako; Yanagawa, Yuchio; Hirase, Hajime; Furuichi, Teiichi

    2011-01-04

    Calcium-dependent activator protein for secretion 2 (CAPS2) is a dense-core vesicle-associated protein that is involved in the secretion of BDNF. BDNF has a pivotal role in neuronal survival and development, including the development of inhibitory neurons and their circuits. However, how CAPS2 affects BDNF secretion and its biological significance in inhibitory neurons are largely unknown. Here we reveal the role of CAPS2 in the regulated secretion of BDNF and show the effect of CAPS2 on the development of hippocampal GABAergic systems. We show that CAPS2 is colocalized with BDNF, both synaptically and extrasynaptically in axons of hippocampal neurons. Overexpression of exogenous CAPS2 in hippocampal neurons of CAPS2-KO mice enhanced depolarization-induced BDNF exocytosis events in terms of kinetics, frequency, and amplitude. We also show that in the CAPS2-KO hippocampus, BDNF secretion is reduced, and GABAergic systems are impaired, including a decreased number of GABAergic neurons and their synapses, a decreased number of synaptic vesicles in inhibitory synapses, and a reduced frequency and amplitude of miniature inhibitory postsynaptic currents. Conversely, excitatory neurons in the CAPS2-KO hippocampus were largely unaffected with respect to field excitatory postsynaptic potentials, miniature excitatory postsynaptic currents, and synapse number and morphology. Moreover, CAPS2-KO mice exhibited several GABA system-associated deficits, including reduced late-phase long-term potentiation at CA3-CA1 synapses, decreased hippocampal theta oscillation frequency, and increased anxiety-like behavior. Collectively, these results suggest that CAPS2 promotes activity-dependent BDNF secretion during the postnatal period that is critical for the development of hippocampal GABAergic networks.

  19. Evaluation of Salmonella enterica type III secretion system effector proteins as carriers for heterologous vaccine antigens.

    Science.gov (United States)

    Hegazy, Wael Abdel Halim; Xu, Xin; Metelitsa, Leonid; Hensel, Michael

    2012-03-01

    Live attenuated strains of Salmonella enterica have a high potential as carriers of recombinant vaccines. The type III secretion system (T3SS)-dependent translocation of S. enterica can be deployed for delivery of heterologous antigens to antigen-presenting cells. Here we investigated the efficacy of various effector proteins of the Salmonella pathogenicity island (SPI2)-encoded T3SS for the translocation of model antigens and elicitation of immune responses. The SPI2 T3SS effector proteins SifA, SteC, SseL, SseJ, and SseF share an endosomal membrane-associated subcellular localization after translocation. We observed that all effector proteins could be used to translocate fusion proteins with the model antigens ovalbumin and listeriolysin into the cytosol of host cells. Under in vitro conditions, fusion proteins with SseJ and SteC stimulated T-cell responses that were superior to those triggered by fusion proteins with SseF. However, in mice vaccinated with Salmonella carrier strains, only fusion proteins based on SseJ or SifA elicited potent T-cell responses. These data demonstrate that the selection of an optimal SPI2 effector protein for T3SS-mediated translocation is a critical parameter for the rational design of effective Salmonella-based recombinant vaccines.

  20. A dynamic study of protein secretion and aggregation in the secretory pathway.

    Science.gov (United States)

    Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; Sitia, Roberto; Anelli, Tiziana

    2014-01-01

    Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

  1. A dynamic study of protein secretion and aggregation in the secretory pathway.

    Directory of Open Access Journals (Sweden)

    Maria Francesca Mossuto

    Full Text Available Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.

  2. Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and beta-cell apoptosis.

    Science.gov (United States)

    Berchtold, Lukas Adrian; Størling, Zenia Marian; Ortis, Fernanda; Lage, Kasper; Bang-Berthelsen, Claus; Bergholdt, Regine; Hald, Jacob; Brorsson, Caroline Anna; Eizirik, Decio Laks; Pociot, Flemming; Brunak, Søren; Størling, Joachim

    2011-09-13

    Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing genes in T1D, we performed an in silico "phenome-interactome analysis" on a genome-wide linkage scan dataset. This method prioritizes candidates according to their physical interactions at the protein level with other proteins involved in diabetes. A total of 11 genes were predicted to be likely disease genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17. Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi knockdown experiments established that HIP14 is an antiapoptotic protein required for β-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1β and IFN-γ) that mediate β-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated rat and human islets. Overexpression of HIP14 was associated with a decrease in IL-1β-induced NF-κB activity and protection against IL-1β-mediated apoptosis. Our study demonstrates that the current network biology approach is a valid method to identify genes of importance for T1D and may therefore embody the basis for more rational and targeted therapeutic approaches.

  3. Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

    Directory of Open Access Journals (Sweden)

    Jeong Y

    2015-11-01

    Full Text Available Yoon Jeong,1,2 Kwan Hong Lee,1,2 Hansoo Park,3 Jonghoon Choi1,2 1Department of Bionano Technology, Graduate School, Hanyang University, Seoul, 2Department of Bionano Engineering, Hanyang University ERICA, Ansan, 3School of Integrative Engineering, Chung-Ang University, Seoul, South Korea Abstract: We present an evaluation of protein-G-terminated glass slides that may contain a suitable substrate for aligning the orientation of antibodies to obtain better binding moiety to the target antigen. The results of the protein-G-terminated slides were compared with those obtained with epoxy-based slides to evaluate signal enhancement for human immunoglobulin G (IgG targets, and an increase in the average fluorescence intensity was observed for the lowest measurable amount of IgG target in the assay using protein-G-terminated slides. Applying this strategy for signal amplification to single-cell assays improves the limits of detection for human IgG protein and cytokines (interleukin-2 and interferon-γ captured from hybridomas. Our data indicate that protein-G-terminated slides have a higher binding capacity for antigens and have better spot-to-spot consistency than that of traditional epoxy-based slides. These properties would be beneficial in the detection of fine amounts of single-cell-secreted proteins, which may provide key insights into cell–cell communication and immune responses. Keywords: microwell array, antibody’s orientation, single cell analysis, secreted cytokine, protein-G-terminated surface

  4. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

    NARCIS (Netherlands)

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi - Pol, Magda; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    2014-01-01

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but r

  5. Phospholipase C-related catalytically inactive protein (PRIP controls KIF5B-mediated insulin secretion

    Directory of Open Access Journals (Sweden)

    Satoshi Asano

    2014-05-01

    Full Text Available We previously reported that phospholipase C-related catalytically inactive protein (PRIP-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6 cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrin-labeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA-receptor-associated protein (GABARAP, a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.

  6. Hierarchical effector protein transport by the Salmonella Typhimurium SPI-1 type III secretion system.

    Directory of Open Access Journals (Sweden)

    Brit Winnen

    Full Text Available BACKGROUND: Type III secretion systems (TTSS are employed by numerous pathogenic and symbiotic bacteria to inject a cocktail of different "effector proteins" into host cells. These effectors subvert host cell signaling to establish symbiosis or disease. METHODOLOGY/PRINCIPAL FINDINGS: We have studied the injection of SipA and SptP, two effector proteins of the invasion-associated Salmonella type III secretion system (TTSS-1. SipA and SptP trigger different host cell responses. SipA contributes to triggering actin rearrangements and invasion while SptP reverses the actin rearrangements after the invasion has been completed. Nevertheless, SipA and SptP were both pre-formed and stored in the bacterial cytosol before host cell encounter. By time lapse microscopy, we observed that SipA was injected earlier than SptP. Computer modeling revealed that two assumptions were sufficient to explain this injection hierarchy: a large number of SipA and SptP molecules compete for transport via a limiting number of TTSS; and the TTSS recognize SipA more efficiently than SptP. CONCLUSIONS/SIGNIFICANCE: This novel mechanism of hierarchical effector protein injection may serve to avoid functional interference between SipA and SptP. An injection hierarchy of this type may be of general importance, allowing bacteria to precisely time the host cell manipulation by type III effectors.

  7. The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG

    Directory of Open Access Journals (Sweden)

    Lebeer Sarah

    2012-02-01

    Full Text Available Abstract Background Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes. Results Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108 bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein. Conclusions In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with Con

  8. Secreted Protein Acidic and Rich in Cysteine (SPARC) Mediates Metastatic Dormancy of Prostate Cancer in Bone.

    Science.gov (United States)

    Sharma, Sambad; Xing, Fei; Liu, Yin; Wu, Kerui; Said, Neveen; Pochampally, Radhika; Shiozawa, Yusuke; Lin, Hui-Kuan; Balaji, K C; Watabe, Kounosuke

    2016-09-09

    Prostate cancer is known to frequently recur in bone; however, how dormant cells switch its phenotype leading to recurrent tumor remains poorly understood. We have isolated two syngeneic cell lines (indolent and aggressive) through in vivo selection by implanting PC3mm stem-like cells into tibial bones. We found that indolent cells retained the dormant phenotype, whereas aggressive cells grew rapidly in bone in vivo, and the growth rates of both cells in culture were similar, suggesting a role of the tumor microenvironment in the regulation of dormancy and recurrence. Indolent cells were found to secrete a high level of secreted protein acidic and rich in cysteine (SPARC), which significantly stimulated the expression of BMP7 in bone marrow stromal cells. The secreted BMP7 then kept cancer cells in a dormant state by inducing senescence, reducing "stemness," and activating dormancy-associated p38 MAPK signaling and p21 expression in cancer cells. Importantly, we found that SPARC was epigenetically silenced in aggressive cells by promoter methylation, but 5-azacytidine treatment reactivated the expression. Furthermore, high SPARC promoter methylation negatively correlated with disease-free survival of prostate cancer patients. We also found that the COX2 inhibitor NS398 down-regulated DNMTs and increased expression of SPARC, which led to tumor growth suppression in bone in vivo These findings suggest that SPARC plays a key role in maintaining the dormancy of prostate cancer cells in the bone microenvironment.

  9. Macroautophagy and Cell Responses Related to Mitochondrial Dysfunction, Lipid Metabolism and Unconventional Secretion of Proteins

    Science.gov (United States)

    Demine, Stéphane; Michel, Sébastien; Vannuvel, Kayleen; Wanet, Anaïs; Renard, Patricia; Arnould, Thierry

    2012-01-01

    Macroautophagy has important physiological roles and its cytoprotective or detrimental function is compromised in various diseases such as many cancers and metabolic diseases. However, the importance of autophagy for cell responses has also been demonstrated in many other physiological and pathological situations. In this review, we discuss some of the recently discovered mechanisms involved in specific and unspecific autophagy related to mitochondrial dysfunction and organelle degradation, lipid metabolism and lipophagy as well as recent findings and evidence that link autophagy to unconventional protein secretion. PMID:24710422

  10. Macroautophagy and Cell Responses Related to Mitochondrial Dysfunction, Lipid Metabolism and Unconventional Secretion of Proteins

    Directory of Open Access Journals (Sweden)

    Thierry Arnould

    2012-06-01

    Full Text Available Macroautophagy has important physiological roles and its cytoprotective or detrimental function is compromised in various diseases such as many cancers and metabolic diseases. However, the importance of autophagy for cell responses has also been demonstrated in many other physiological and pathological situations. In this review, we discuss some of the recently discovered mechanisms involved in specific and unspecific autophagy related to mitochondrial dysfunction and organelle degradation, lipid metabolism and lipophagy as well as recent findings and evidence that link autophagy to unconventional protein secretion.

  11. Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion

    Directory of Open Access Journals (Sweden)

    Voigt Christopher A

    2010-10-01

    Full Text Available Abstract Background The type III secretion system (T3SS is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1 can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein. Results To test how the timing and level of protein expression affects secretion, we designed a hybrid promoter that combines an IPTG-inducible system with a natural genetic circuit that controls effector expression in Salmonella (psicA. LacO operators are placed in various locations in the psicA promoter and the optimal induction occurs when a single operator is placed at the +5nt (234-fold and a lower basal level of expression is achieved when a second operator is placed at -63nt to take advantage of DNA looping. Using this tool, we find that the secretion efficiency (protein secreted divided by total expressed is constant as a function of total expressed. We also demonstrate that the secretion flux peaks at 8 hours. We then use whole gene DNA synthesis to construct codon optimized spider silk genes for full-length (3129 amino acids Latrodectus hesperus dragline silk, Bombyx mori cocoon silk, and Nephila clavipes flagelliform silk and PCR is used to create eight truncations of these genes. These proteins are all unfolded polypeptides and they encompass a variety of length, charge, and amino acid compositions. We find those proteins fewer than 550 amino acids reliably secrete and the probability declines significantly after ~700 amino acids. There also is a charge optimum at -2.4, and secretion efficiency declines for very positively or negatively charged proteins. There is no significant correlation with hydrophobicity

  12. Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation.

    Science.gov (United States)

    White, Michael J V; Roife, David; Gomer, Richard H

    2015-08-15

    To metastasize, tumor cells often need to migrate through a layer of collagen-containing scar tissue which encapsulates the tumor. A key component of scar tissue and fibrosing diseases is the monocyte-derived fibrocyte, a collagen-secreting profibrotic cell. To test the hypothesis that invasive tumor cells may block the formation of the fibrous sheath, we determined whether tumor cells secrete factors that inhibit monocyte-derived fibrocyte differentiation. We found that the human metastatic breast cancer cell line MDA-MB-231 secretes activity that inhibits human monocyte-derived fibrocyte differentiation, whereas less aggressive breast cancer cell lines secrete less of this activity. Purification indicated that Galectin-3 binding protein (LGALS3BP) is the active factor. Recombinant LGALS3BP inhibits monocyte-derived fibrocyte differentiation, and immunodepletion of LGALS3BP from MDA-MB 231 conditioned media removes the monocyte-derived fibrocyte differentiation-inhibiting activity. LGALS3BP inhibits the differentiation of monocyte-derived fibrocytes from wild-type mouse spleen cells, but not from SIGN-R1(-/-) mouse spleen cells, suggesting that CD209/SIGN-R1 is required for the LGALS3BP effect. Galectin-3 and galectin-1, binding partners of LGALS3BP, potentiate monocyte-derived fibrocyte differentiation. In breast cancer biopsies, increased levels of tumor cell-associated LGALS3BP were observed in regions of the tumor that were invading the surrounding stroma. These findings suggest LGALS3BP and galectin-3 as new targets to treat metastatic cancer and fibrosing diseases.

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

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    Jacqueline Schmuckli-Maurer

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

  14. Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation.

    Science.gov (United States)

    Rigg, Rachel A; Healy, Laura D; Nowak, Marie S; Mallet, Jérémy; Thierheimer, Marisa L D; Pang, Jiaqing; McCarty, Owen J T; Aslan, Joseph E

    2016-04-01

    Molecular chaperones that support protein quality control, including heat shock protein 70 (Hsp70), participate in diverse aspects of cellular and physiological function. Recent studies have reported roles for specific chaperone activities in blood platelets in maintaining hemostasis; however, the functions of Hsp70 in platelet physiology remain uninvestigated. Here we characterize roles for Hsp70 activity in platelet activation and function. In vitro biochemical, microscopy, flow cytometry, and aggregometry assays of platelet function, as well as ex vivo analyses of platelet aggregate formation in whole blood under shear, were carried out under Hsp70-inhibited conditions. Inhibition of platelet Hsp70 blocked platelet aggregation and granule secretion in response to collagen-related peptide (CRP), which engages the immunoreceptor tyrosine-based activation motif-bearing collagen receptor glycoprotein VI (GPVI)-Fc receptor-γ chain complex. Hsp70 inhibition also reduced platelet integrin-αIIbβ3 activation downstream of GPVI, as Hsp70-inhibited platelets showed reduced PAC-1 and fibrinogen binding. Ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear. Biochemical studies supported a role for Hsp70 in maintaining the assembly of the linker for activation of T cells signalosome, which couples GPVI-initiated signaling to integrin activation, secretion, and platelet function. Together, our results suggest that Hsp70 regulates platelet activation and function by supporting linker for activation of T cells-associated signaling events downstream of platelet GPVI engagement, suggesting a role for Hsp70 in the intracellular organization of signaling systems that mediate platelet secretion, "inside-out" activation of platelet integrin-αIIbβ3, platelet-platelet aggregation, and, ultimately, hemostatic plug and thrombus formation.

  15. Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis.

    Science.gov (United States)

    Greening, David W; Ji, Hong; Kapp, Eugene A; Simpson, Richard J

    2013-11-01

    Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433-451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3-30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1-3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS-MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6

  16. Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus

    OpenAIRE

    Chen Yi-Hsing; Anderson Mark; Hendrickx Antoni PA; Missiakas Dominique

    2012-01-01

    Abstract Background Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS), a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of ...

  17. Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation.

    Science.gov (United States)

    Scheidegger, F; Ellner, Y; Guye, P; Rhomberg, T A; Weber, H; Augustin, H G; Dehio, C

    2009-07-01

    The zoonotic pathogen Bartonella henselae (Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.

  18. Chronic effects of a Salmonella type III secretion effector protein AvrA in vivo.

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    Rong Lu

    Full Text Available BACKGROUND: Salmonella infection is a common public health problem that can become chronic and increase the risk of inflammatory bowel diseases and cancer. AvrA is a Salmonella bacterial type III secretion effector protein. Increasing evidence demonstrates that AvrA is a multi-functional enzyme with critical roles in inhibiting inflammation, regulating apoptosis, and enhancing proliferation. However, the chronic effects of Salmonella and effector AvrA in vivo are still unknown. Moreover, alive, mutated, non-invasive Salmonella is used as a vector to specifically target cancer cells. However, studies are lacking on chronic infection with non-pathogenic or mutated Salmonella in the host. METHODS/PRINCIPAL FINDINGS: We infected mice with Salmonella Typhimurium for 27 weeks and investigated the physiological effects as well as the role of AvrA in intestinal inflammation. We found altered body weight, intestinal pathology, and bacterial translocation in spleen, liver, and gallbladder in chronically Salmonella-infected mice. Moreover, AvrA suppressed intestinal inflammation and inhibited the secretion of cytokines IL-12, IFN-gamma, and TNF-alpha. AvrA expression in Salmonella enhanced its invasion ability. Liver abscess and Salmonella translocation in the gallbladder were observed and may be associated with AvrA expression in Salmonella. CONCLUSION/SIGNIFICANCE: We created a mouse model with persistent Salmonella infection in vivo. Our study further emphasizes the importance of the Salmonella effector protein AvrA in intestinal inflammation, bacterial translocation, and chronic infection in vivo.

  19. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bip

  20. Engineering the supply chain for protein production/secretion in yeasts and mammalian cells.

    Science.gov (United States)

    Klein, Tobias; Niklas, Jens; Heinzle, Elmar

    2015-03-01

    Metabolic bottlenecks play an increasing role in yeasts and mammalian cells applied for high-performance production of proteins, particularly of pharmaceutical ones that require complex posttranslational modifications. We review the present status and developments focusing on the rational metabolic engineering of such cells to optimize the supply chain for building blocks and energy. Methods comprise selection of beneficial genetic modifications, rational design of media and feeding strategies. Design of better producer cells based on whole genome-wide metabolic network analysis becomes increasingly possible. High-resolution methods of metabolic flux analysis for the complex networks in these compartmented cells are increasingly available. We discuss phenomena that are common to both types of organisms but also those that are different with respect to the supply chain for the production and secretion of pharmaceutical proteins.

  1. Secreted Protein Acidic and Rich in Cysteine (SPARC) in Human Skeletal Muscle

    DEFF Research Database (Denmark)

    Jørgensen, Louise H; Petersson, Stine J; Sellathurai, Jeeva

    2009-01-01

    Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further......, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15-16 weeks. SPARC protein was detected...... indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis...

  2. Partially Protective Immunity Induced by a 20 kDa Protein Secreted by Trichinella spiralis Stichocytes.

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    Kuo Bi

    Full Text Available Trichinella spiralis infection induces protective immunity against re-infection in animal models. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Trichinella infection and immunodiagnosis.The T. spiralis adult cDNA library was immunoscreened with sera from pigs experimentally infected with 20,000 infective T. spiralis larvae. Total 43 positive clones encoding for 28 proteins were identified; one of the immunodominant proteins was 20 kDa Ts-ES-1 secreted by Trichinella stichocytes and existing in the excretory/secretory (ES products of T. spiralis adult and muscle larval worms. Ts-ES-1 contains 172 amino acids with a typical signal peptide in the first 20 amino acids. The expression of Ts-ES-1 was detected in both the adult and muscle larval stages at the mRNA and protein expression levels. Mice immunized with recombinant Ts-ES-1 (rTs-ES-1 formulated with ISA50v2 adjuvant exhibited a significant worm reduction in both the adult worm (27% and muscle larvae burden (42.1% after a challenge with T. spiralis compared to the adjuvant control group (p<0.01. The rTs-ES-1-induced protection was associated with a high level of specific anti-Ts-ES-1 IgG antibodies and a Th1/Th2 mixed immune response.The newly identified rTs-ES-1 is an immunodominant protein secreted by Trichinella stichocytes during natural infection and enables to the induction of partial protective immunity in vaccinated mice against Trichinella infection. Therefore, rTs-ES-1 is a potential candidate for vaccine development against trichinellosis.

  3. Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures

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    Jordà Joel

    2012-05-01

    Full Text Available Abstract Background The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. Results The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Conclusions Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic

  4. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins.

    Science.gov (United States)

    Helliwell, Emily E; Vega-Arreguín, Julio; Shi, Zi; Bailey, Bryan; Xiao, Shunyuan; Maximova, Siela N; Tyler, Brett M; Guiltinan, Mark J

    2016-03-01

    The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.

  5. Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

    Science.gov (United States)

    Knobloch, Denise; Ostermann, Kai; Rödel, Gerhard

    2012-01-01

    Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

  6. A type II protein secretory pathway required for levansucrase secretion by Gluconacetobacter diazotrophicus.

    Science.gov (United States)

    Arrieta, Juan G; Sotolongo, Mailin; Menéndez, Carmen; Alfonso, Dubiel; Trujillo, Luis E; Soto, Melvis; Ramírez, Ricardo; Hernández, Lázaro

    2004-08-01

    The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G. diazotrophicus structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys(162) by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G. diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae.

  7. The inner rod protein controls substrate switching and needle length in a Salmonella type III secretion system.

    Science.gov (United States)

    Lefebre, Matthew D; Galán, Jorge E

    2014-01-14

    Type III secretion machines are essential for the biology of many bacteria that are pathogenic or symbiotic for animals, plants, or insects. They exert their function by delivering bacterial effector proteins into target eukaryotic cells. The core component of these machines is the needle complex, a multiprotein structure that spans the bacterial envelope and serves as a conduit for proteins that transit this secretion pathway. The needle complex is composed of a multiring base embedded in the bacterial envelope and a filament-like structure, the needle, that projects from the bacterial surface and is linked to the base by the inner rod. Assembly of the needle complex proceeds in a step-wise fashion that is initiated by the assembly of the base and is followed by the export of the building subunits for the needle and inner rod substructures. Once assembled, the needle complex reprograms its specificity and becomes competent for the secretion of effector proteins. Here through genetic, biochemical, and electron microscopy analyses of the Salmonella inner rod protein subunit PrgJ we present evidence that the assembly of the inner rod dictates the timing of substrate switching and needle length. Furthermore, the identification of mutations in PrgJ that specifically alter the hierarchy of protein secretion provides additional support for a complex role of the inner rod substructure in type III secretion.

  8. Unfolded Protein Response (UPR Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Martin Hampel

    Full Text Available The unfolded protein response (UPR, a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER, coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

  9. Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

    Science.gov (United States)

    Hampel, Martin; Jakobi, Mareike; Schmitz, Lara; Meyer, Ute; Finkernagel, Florian; Doehlemann, Gunther; Heimel, Kai

    2016-01-01

    The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

  10. Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae

    Directory of Open Access Journals (Sweden)

    Thomas eBadet

    2015-09-01

    Full Text Available Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases.

  11. The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells.

    Science.gov (United States)

    Satchidanandam, Vijaya; Kumar, Naveen; Biswas, Sunetra; Jumani, Rajiv S; Jain, Chandni; Rani, Rajni; Aggarwal, Bharti; Singh, Jaya; Kotnur, Mohan Rao; Sridharan, Anand

    2016-04-01

    We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+)and CD8(+)T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

  12. Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A (sSpAD as a secretion - tag

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    Paal Michael

    2010-11-01

    Full Text Available Abstract Background Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous. Results We discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation. The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD. Conclusions In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.

  13. Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe--a quantitative approach using 13C-based metabolic flux analysis.

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    Klein, Tobias; Lange, Sabrina; Wilhelm, Nadine; Bureik, Matthias; Yang, Tae-Hoon; Heinzle, Elmar; Schneider, Konstantin

    2014-01-01

    Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying (13)C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.

  14. The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

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    Heesemann Jürgen

    2007-07-01

    Full Text Available Abstract Background Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica share a virulence plasmid encoding a type three secretion system (T3SS. This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins, the specific Yop chaperones (Sycs, and the Ysc (Yop secretion proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study. Results We have established the large-scale production of recombinant SycO in its outright form. We confirm that Y. enterocolitica SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in Y. enterocolitica decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that in vitro SycO interacts with YscM1, a negative regulator of Yop expression in Y. enterocolitica. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants. Conclusion We present evidence that SycO is integrated into the regulatory network of the Yersinia T3SS. Our picture of the Yersinia T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified.

  15. PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

    Science.gov (United States)

    Veith, Paul D.; Butler, Catherine A.; Nor Muhammad, Nor A.; Chen, Yu-Yen; Slakeski, Nada; Peng, Benjamin; Zhang, Lianyi; Dashper, Stuart G.; Cross, Keith J.; Cleal, Steven M.; Moore, Caroline; Reynolds, Eric C.

    2016-01-01

    Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS. PMID:27711252

  16. Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD.

    Science.gov (United States)

    Chatterjee, Abhishek; Caballero-Franco, Celia; Bakker, Dannika; Totten, Stephanie; Jardim, Armando

    2015-10-16

    Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280-320-kDa oligomeric structure consisting of ∼6-7 subunits.

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

    Science.gov (United States)

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

    2011-01-01

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

  18. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

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    Kosalková Katarina

    2012-01-01

    Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  19. Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis.

    Science.gov (United States)

    Robinson, Mark W; Massie, Diane H; Connolly, Bernadette

    2007-01-01

    The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections.

  20. Paracrine Secreted Frizzled-Related Protein 4 Inhibits Melanocytes Differentiation in Hair Follicle

    Science.gov (United States)

    Guo, Haiying; Lei, Mingxing; Li, Yuhong; Liu, Yingxin; Tang, Yinhong; Xing, Yizhan; Deng, Fang

    2017-01-01

    Wnt signaling plays crucial role in regulating melanocyte stem cells/melanocyte differentiation in the hair follicle. However, how the Wnt signaling is balanced to be overactivated to control follicular melanocytes behavior remains unknown. Here, by using immunofluorescence staining, we showed that secreted frizzled-related protein 4 (sFRP4) is preferentially expressed in the skin epidermal cells rather than in melanocytes. By overexpression of sFRP4 in skin cells in vivo and in vitro, we found that sFRP4 attenuates activation of Wnt signaling, resulting in decrease of melanocytes differentiation in the regenerating hair follicle. Our findings unveiled a new regulator that involves modulating melanocytes differentiation through a paracrine mechanism in hair follicle, supplying a hope for potential therapeutic application to treat skin pigmentation disorders. PMID:28337220

  1. A Case with Significant Proteinuria Caused by Secreted Protein from Urothelial Carcinoma

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    Masanori Sakakima

    2011-01-01

    Full Text Available 58-year-old female was admitted to our hospital complaining isolated proteinuria of 1.7 g/day. Abdominal echography showed right-sided unilateral hydronephrosis, and computed tomography pointed out a tumor of the right renal pelvis, suggesting cancer of renal pelvis. The right nephroureterectomy was carried out. Pathological diagnosis was urothelial carcinoma. Renal tissue revealed no apparent glomerulopathy with tubular atrophy, interstitial fibrosis, and mildly-to-moderately interstitial mononuclear cell infiltration. Immunofluorescence study showed no deposition of immunoreactanct, and electron microscopy showed almost normal glomerulus without electron dense deposit. Proteinuria disappeared within 6 days after the operation. Moderate amount of proteinuria in our patient was probably caused by secreted protein from urothelial carcinoma. This condition is rare but should be taken into account in patients with even moderate amount of proteinuria.

  2. Characterization of the ectodomain of the envelope protein of dengue virus type 4: expression, membrane association, secretion and particle formation in the absence of precursor membrane protein.

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    Szu-Chia Hsieh

    Full Text Available The envelope (E of dengue virus (DENV is the major target of neutralizing antibodies and vaccine development. After biosynthesis E protein forms a heterodimer with precursor membrane (prM protein. Recent reports of infection enhancement by anti-prM monoclonal antibodies (mAbs suggest anti-prM responses could be potentially harmful. Previously, we studied a series of C-terminal truncation constructs expressing DENV type 4 prM/E or E proteins and found the ectodomain of E protein alone could be recognized by all 12 mAbs tested, suggesting E protein ectodomain as a potential subunit immunogen without inducing anti-prM response. The characteristics of DENV E protein ectodomain in the absence of prM protein remains largely unknown.In this study, we investigated the expression, membrane association, glycosylation pattern, secretion and particle formation of E protein ectodomain of DENV4 in the presence or absence of prM protein. E protein ectodomain associated with membrane in or beyond trans-Golgi and contained primarily complex glycans, whereas full-length E protein associated with ER membrane and contained high mannose glycans. In the absence of prM protein, E protein ectodomain can secrete as well as form particles of approximately 49 nm in diameter, as revealed by sucrose gradient ultracentrifugation with or without detergent and electron microscopy. Mutational analysis revealed that the secretion of E protein ectodomain was affected by N-linked glycosylation and could be restored by treatment with ammonia chloride.Considering the enhancement of DENV infectivity by anti-prM antibodies, our findings provide new insights into the expression and secretion of E protein ectodomain in the absence of prM protein and contribute to future subunit vaccine design.

  3. Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion

    OpenAIRE

    Yoshii, Kentarou; Konno, Akihiro; Goto, Akiko; Nio, Junko; Obara, Mayumi; Ueki, Tomotaka; Hayasaka, Daisuke; Mizutani, Tetsuya; Kariwa, Hiroaki; Takashima, Ikuo

    2004-01-01

    Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway. Virus envelope proteins play important roles in this process. In this study, the effect of mutations in the envelope proteins of tick-borne encephalitis (TBE) virus on secretion of virus-like particles (VLPs), using a recombinant plasmid expression system was analysed. It was found that a single point mutation at position 63 in prM induces a reduction in s...

  4. The Wnt secretion protein Evi/Gpr177 promotes glioma tumourigenesis.

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    Augustin, Iris; Goidts, Violaine; Bongers, Angelika; Kerr, Grainne; Vollert, Gordon; Radlwimmer, Bernhard; Hartmann, Christian; Herold-Mende, Christel; Reifenberger, Guido; von Deimling, Andreas; Boutros, Michael

    2012-01-01

    Malignant astrocytomas are highly aggressive brain tumours with poor prognosis. While a number of structural genomic changes and dysregulation of signalling pathways in gliomas have been described, the identification of biomarkers and druggable targets remains an important task for novel diagnostic and therapeutic approaches. Here, we show that the Wnt-specific secretory protein Evi (also known as GPR177/Wntless/Sprinter) is overexpressed in astrocytic gliomas. Evi/Wls is a core Wnt signalling component and a specific regulator of pan-Wnt protein secretion, affecting both canonical and non-canonical signalling. We demonstrate that its depletion in glioma and glioma-derived stem-like cells led to decreased cell proliferation and apoptosis. Furthermore, Evi/Wls silencing in glioma cells reduced cell migration and the capacity to form tumours in vivo. We further show that Evi/Wls overexpression is sufficient to promote downstream Wnt signalling. Taken together, our study identifies Evi/Wls as an essential regulator of glioma tumourigenesis, identifying a pathway-specific protein trafficking factor as an oncogene and offering novel therapeutic options to interfere with the aberrant regulation of growth factors at the site of production.

  5. A new method to extract matrix proteins directly from the secretion of the mollusk mantle and the role of these proteins in shell biomineralization.

    Science.gov (United States)

    Liu, Xiaojun; Liu, Chang; Chen, Lei; Sun, Juan; Zhou, Yujuan; Li, Qi; Zheng, Guilan; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2011-10-01

    Considering the continuous and substantive secretory ability of the mantle in vitro, we report a new technique to produce shell-matrix proteins by inducing the mantle, after removal from the organism's body, to secrete soluble-matrix proteins into phosphate buffer. By this method, a large amount of matrix proteins could be obtained in 2 h. Experiments involving in vitro calcium carbonate crystallization and organic framework calcium carbonate crystallization indicated that these proteins retain high bioactivity and play key roles in shell biomineralization. Phosphate buffer-soluble proteins secreted by the margin of the mantles (MSPs) were used to reconstruct the stages in the growth of the prismatic layer of the decalcified organic frameworks. The MSPs were observed to aggregate calcites in vitro, and this ability enabled the mollusk to form big calcites in the prismatic layer. During shell biomineralization, an important stage after the self-assembly of the biomacromolecules and the formation of crystals is the assembly of the two parts to form a firm structure. Moreover, a new type of matrix protein, functioning as the binding factor between the crystals and the organic frameworks, was shown to exist in the phosphate buffer-soluble proteins secreted by the central part of mantles (CSPs). Nanoscale-sized bowl-like aragonites, with heights of ∼800 nm, were induced by CSPs in vitro. This method is a successful example of obtaining functional proteins through secretion by animal tissues.

  6. Genome-wide analysis of small secreted cysteine-rich proteins identifies candidate effector proteins potentially involved in Fusarium graminearum-wheat interactions

    Science.gov (United States)

    Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight ...

  7. Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

    NARCIS (Netherlands)

    Meissner, Daniel; Vollstedt, Angela; van Dijl, Jan Maarten; Freudl, Roland

    2007-01-01

    In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the sec

  8. The Pseudomonas aeruginosa patatin-like protein PlpD is the archetype of a novel Type V secretion system.

    Science.gov (United States)

    Salacha, Richard; Kovacić, Filip; Brochier-Armanet, Céline; Wilhelm, Susanne; Tommassen, Jan; Filloux, Alain; Voulhoux, Romé; Bleves, Sophie

    2010-06-01

    We discovered a novel secreted protein by Pseudomonas aeruginosa, PlpD, as a member of the bacterial lipolytic enzyme family of patatin-like proteins (PLPs). PlpD is synthesized as a single molecule consisting of a secreted domain fused to a transporter domain. The N-terminus of PlpD includes a classical signal peptide followed by the four PLP conserved blocks that account for its lipase activity. The C-terminus consists of a POTRA (polypeptide transport-associated) motif preceding a putative 16-stranded beta-barrel similar to those of TpsB transporters of Type Vb secretion system. We showed that the C-terminus remains inserted into the outer membrane while the patatin moiety is secreted. The association between a TpsB component and a passenger protein is a unique hybrid organization that we propose to classify as Type Vd. More than 200 PlpD orthologues exist among pathogenic and environmental bacteria, which suggests that bacteria secrete numerous PLPs using this newly defined mechanism.

  9. Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis

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    González Mauricio

    2009-09-01

    Full Text Available Abstract Background Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis. Results Microarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction. Conclusion Our data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we

  10. Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

    Science.gov (United States)

    Chaudhury, Sukanya; Nordhues, Bryce A; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N

    2015-11-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion.

  11. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

    Science.gov (United States)

    Waldera Lupa, Daniel M; Kalfalah, Faiza; Safferling, Kai; Boukamp, Petra; Poschmann, Gereon; Volpi, Elena; Götz-Rösch, Christine; Bernerd, Francoise; Haag, Laura; Huebenthal, Ulrike; Fritsche, Ellen; Boege, Fritz; Grabe, Niels; Tigges, Julia; Stühler, Kai; Krutmann, Jean

    2015-08-01

    Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

  12. Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC-homologue HgdD in protein secretion.

    Science.gov (United States)

    Hahn, Alexander; Stevanovic, Mara; Brouwer, Eva; Bublak, Daniela; Tripp, Joanna; Schorge, Tobias; Karas, Michael; Schleiff, Enrico

    2015-03-01

    Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC-homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst-forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss-of-function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild-type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin-tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD-mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD-dependent pathway.

  13. Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles.

    Science.gov (United States)

    Cahill, Bethaney K; Seeley, Kent W; Gutel, Dedra; Ellis, Terri N

    2015-11-01

    Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC-MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane.

  14. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    Science.gov (United States)

    Jung, Woongsic; Campbell, Robert L; Gwak, Yunho; Kim, Jong Im; Davies, Peter L; Jin, EonSeon

    2016-01-01

    Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

  15. Expression of PPARα modifies fatty acid effects on insulin secretion in uncoupling protein-2 knockout mice

    Directory of Open Access Journals (Sweden)

    Chan Catherine B

    2007-03-01

    Full Text Available Abstract Aims/hypothesis In uncoupling protein-2 (UCP2 knockout (KO mice, protection of beta cells from fatty acid exposure is dependent upon transcriptional events mediated by peroxisome proliferator-activated receptor-α (PPARα. Methods PPARα expression was reduced in isolated islets from UCP2KO and wild-type (WT mice with siRNA for PPARα (siPPARα overnight. Some islets were also cultured with oleic or palmitic acid, then glucose stimulated insulin secretion (GSIS was measured. Expression of genes was examined by quantitative RT-PCR or immunoblotting. PPARα activation was assessed by oligonucleotide consensus sequence binding. Results siPPARα treatment reduced PPARα protein expression in KO and WT islets by >85%. In siPPARα-treated UCP2KO islets, PA but not OA treatment significantly decreased the insulin response to 16.5 mM glucose. In WT islets, siPPARα treatment did not modify GSIS in PA and OA exposed groups. In WT islets, PA treatment significantly increased UCP2 mRNA and protein expression. Both PA and OA treatment significantly increased PPARα expression in UCP2KO and WT islets but OA treatment augmented PPARα protein expression only in UCP2KO islets (p Conclusion These data show that the negative effect of saturated fatty acid on GSIS is mediated by PPARα/UCP2. Knockout of UCP2 protects beta-cells from PA exposure. However, in the absence of both UCP2 and PPARα even a short exposure (24 h to PA significantly impairs GSIS.

  16. Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone.

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    Alexandre Chlenski

    Full Text Available Secreted Protein Acidic and Rich in Cysteine (SPARC is one of the major non-structural proteins of the extracellular matrix (ECM in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+ concentrations are low, high extracellular concentrations of Ca(2+ activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+ concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide

  17. Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

    Science.gov (United States)

    Nicholas, K.R.; Fisher, J.A.; Muths, E.; Trott, J.; Janssens, P.A.; Reich, C.; Shaw, D.C.

    2001-01-01

    Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of ??-lactalbumin, ??-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young. ?? 2001 Elsevier Science Inc.

  18. A yeast-based model system for cloning secreted and membrane proteins

    Directory of Open Access Journals (Sweden)

    SURPILI MARCELO J.

    2002-01-01

    Full Text Available The targeting of proteins to cell organelles and membranes, or of proteins destined to secretion, is coordinated by signal sequences located at the 5´-end of their respective genes. A signal sequence trap system was envisaged in which a truncated version of the yeast acid phosphatase pho5 gene lacking the start codon and signal sequence could serve as a reporter gene. A fraction enriched in 5´-end fragments obtained by PCR from a potato guard-cell cDNA library was cloned in frame to the acid phosphatase gene and the acid phosphatase activity was assayed directly in yeast colonies grown on selective medium. Putative signal sequences targeting the acid phosphatase to the membrane or to the outside of the cell were used to screen the cDNA bank in order to recover the original full-size sequence which gave rise to the signal sequence. Two unknown sequences displaying marked tissue-specific expression were retrieved, one of them (YE139 with a higher expression level in green buds and stem cells, and the other one (YE290 with a higher expression level in androceum, gyneceum, and roots. The limitations of the system are further analyzed using other sequences as control.

  19. Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Jamaine; Wang, Jiawei; Tropea, Joseph E.; Zhang, Di; Dauter, Zbigniew; Waugh, David S.; Wlodawer, Alexander (SAIC); (NCI)

    2009-01-28

    VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave {alpha}-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 {angstrom} resolution. The shape of the molecule resembles the letter 'V,' with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

  20. Aberrant Expression and Secretion of Heat Shock Protein 90 in Patients with Bullous Pemphigoid

    Science.gov (United States)

    Tukaj, Stefan; Kleszczyński, Konrad; Vafia, Katerina; Groth, Stephanie; Meyersburg, Damian; Trzonkowski, Piotr; Ludwig, Ralf J.; Zillikens, Detlef; Schmidt, Enno; Fischer, Tobias W.; Kasperkiewicz, Michael

    2013-01-01

    The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses and its inhibition has proven successful in different mouse models of autoimmune diseases, including epidermolysis bullosa acquisita. Here, we investigated expression levels and secretory responses of Hsp90 in patients with bullous pemphigoid (BP), the most common subepidermal autoimmune blistering skin disease. In comparison to healthy controls, the following observations were made: (i) Hsp90 was highly expressed in the skin of BP patients, whereas its serum levels were decreased and inversely associated with IgG autoantibody levels against the NC16A immunodominant region of the BP180 autoantigen, (ii) in contrast, neither aberrant levels of circulating Hsp90 nor any correlation of this protein with serum autoantibodies was found in a control cohort of autoimmune bullous disease patients with pemphigus vulgaris, (iii) Hsp90 was highly expressed in and restrictedly released from peripheral blood mononuclear cells of BP patients, and (iv) Hsp90 was potently induced in and restrictedly secreted from human keratinocyte (HaCaT) cells by BP serum and isolated anti-BP180 NC16A IgG autoantibodies, respectively. Our results reveal an upregulated Hsp90 expression at the site of inflammation and an autoantibody-mediated dysregulation of the intracellular and extracellular distribution of this chaperone in BP patients. These findings suggest that Hsp90 may play a pathophysiological role and represent a novel potential treatment target in BP. PMID:23936217

  1. Aberrant expression and secretion of heat shock protein 90 in patients with bullous pemphigoid.

    Directory of Open Access Journals (Sweden)

    Stefan Tukaj

    Full Text Available The cell stress chaperone heat shock protein 90 (Hsp90 has been implicated in inflammatory responses and its inhibition has proven successful in different mouse models of autoimmune diseases, including epidermolysis bullosa acquisita. Here, we investigated expression levels and secretory responses of Hsp90 in patients with bullous pemphigoid (BP, the most common subepidermal autoimmune blistering skin disease. In comparison to healthy controls, the following observations were made: (i Hsp90 was highly expressed in the skin of BP patients, whereas its serum levels were decreased and inversely associated with IgG autoantibody levels against the NC16A immunodominant region of the BP180 autoantigen, (ii in contrast, neither aberrant levels of circulating Hsp90 nor any correlation of this protein with serum autoantibodies was found in a control cohort of autoimmune bullous disease patients with pemphigus vulgaris, (iii Hsp90 was highly expressed in and restrictedly released from peripheral blood mononuclear cells of BP patients, and (iv Hsp90 was potently induced in and restrictedly secreted from human keratinocyte (HaCaT cells by BP serum and isolated anti-BP180 NC16A IgG autoantibodies, respectively. Our results reveal an upregulated Hsp90 expression at the site of inflammation and an autoantibody-mediated dysregulation of the intracellular and extracellular distribution of this chaperone in BP patients. These findings suggest that Hsp90 may play a pathophysiological role and represent a novel potential treatment target in BP.

  2. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    Directory of Open Access Journals (Sweden)

    Fernando Navarro-Garcia

    2013-01-01

    Full Text Available The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology.

  3. Erwinia amylovora type three-secreted proteins trigger cell death and defense responses in Arabidopsis thaliana.

    Science.gov (United States)

    Degrave, A; Fagard, M; Perino, C; Brisset, M N; Gaubert, S; Laroche, S; Patrit, O; Barny, M-A

    2008-08-01

    Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting plants of the rosaceous family. E. amylovora pathogenicity requires a functional type three secretion system (T3SS). We show here that E. amylovora triggers a T3SS-dependent cell death on Arabidopsis thaliana. The plants respond by inducing T3SS-dependent defense responses, including salicylic acid (SA)-independent callose deposition, activation of the SA defense pathway, reactive oxygen species (ROS) accumulation, and part of the jasmonic acid/ethylene defense pathway. Several of these reactions are similar to what is observed in host plants. We show that the cell death triggered by E. amylovora on A. thaliana could not be simply explained by the recognition of AvrRpt2 ea by the resistance gene product RPS2. We then analyzed the role of type three-secreted proteins (T3SPs) DspA/E, HrpN, and HrpW in the induction of cell death and defense reactions in A. thaliana following infection with the corresponding E. amylovora mutant strains. HrpN and DspA/E were found to play an important role in the induction of cell death, activation of defense pathways, and ROS accumulation. None of the T3SPs tested played a major role in the induction of SA-independent callose deposition. The relative importance of T3SPs in A. thaliana is correlated with their relative importance in the disease process on host plants, indicating that A. thaliana can be used as a model to study their role.

  4. Blood outgrowth endothelial cells as a vehicle for transgene expression of hepatocyte-secreted proteins via Sleeping Beauty.

    Science.gov (United States)

    Kren, Betsy T; Yin, Wenxan; Key, Nigel S; Hebbel, Robert P; Steer, Clifford J

    2007-01-01

    The therapeutic use of autologous cells with the capacity for extensive in vitro expansion and manipulation prior to host administration has been an area of significant investigation over the last decade. Blood outgrowth endothelial cells (BOECs) are derived from the circulation and exhibit proliferative growth, in vivo engraftment, and survival characteristics for long-term expression of endogenously secreted proteins, such as factor VIII (FVIII). The authors describe a modified method for the isolation, culture, and expansion of these cells that is readily accomplished using standard laboratory methods. Using a commercially available transfection reagent, approximately 30% of these primary cells can be routinely transfected with the nonviral Sleeping Beauty transposon for long-term, stable transgene expression. Moreover, the results indicate that these cells have the ability to secrete functionally active proteins that are synthesized endogenously by hepatocytes and require post-translational modification including alpha1-antitrypsin and clotting factors VII and IX. This, coupled with their notably long half-life of years, suggests that these cells may provide an appropriate vehicle for secretion of a variety of proteins produced by different cell types in vivo. Thus, BOECs have the potential to provide clinically relevant secreted proteins for diseases other than those of endothelial origin.

  5. The type 3 protein secretion system of Cupriavidus taiwanensis strain LMG19424 compromises symbiosis with Leucaena leucocephala.

    Science.gov (United States)

    Saad, Maged M; Crèvecoeur, Michèle; Masson-Boivin, Catherine; Perret, Xavier

    2012-10-01

    Cupriavidus taiwanensis forms proficient symbioses with a few Mimosa species. Inactivation of a type III protein secretion system (T3SS) had no effect on Mimosa pudica but allowed C. taiwanensis to establish chronic infections and fix nitrogen in Leucaena leucocephala. Unlike what was observed for other rhizobia, glutamate rather than plant flavonoids mediated transcriptional activation of this atypical T3SS.

  6. The breast cancer resistance protein (BCRP/ABCG2) affects pharmacokinetics, hepatobiliary excretion, and milk secretion of the antibiotic nitrofurantoin

    NARCIS (Netherlands)

    Merino, G; Jonker, JW; Wagenaar, E; van Herwaarden, AE; Schinkel, AH

    2005-01-01

    Nitrofurantoin is a commonly used urinary tract antibiotic prescribed to lactating woman. It is actively transported into human and rat milk by an unknown mechanism. Our group has demonstrated an important role of the breast cancer resistance protein (BCRP/ABCG2) in the secretion of xenotoxins into

  7. Role of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretion.

    Science.gov (United States)

    Goto, Akiko; Yoshii, Kentarou; Obara, Mayumi; Ueki, Tomotaka; Mizutani, Tetsuya; Kariwa, Hiroaki; Takashima, Ikuo

    2005-04-27

    The tick-borne encephalitis (TBE) virus has two membrane glycoproteins (prM and E), which each has one N-linked glycan. Constructs that express prM and E proteins of TBE virus have been shown to produce virus-like particles (VLPs), which have surface properties that are similar to those of infectious viruses. To reveal the function of glycosylation of the TBE virus prM and E proteins in the secretion of VLPs, we expressed glycosylation-mutated prM and E proteins and compared the secretion levels and biological properties of the VLPs. In the prM protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 60% of the wild-type level. On the other hand, in the E or prM-E protein glycosylation-deficient mutant, the level of secreted E protein was reduced to 10% of the wild-type level. Furthermore, the mutant which was glycosylated at positions 66 and 154 in protein E, the level of secreted E protein was four-fold higher than that of the wild-type. However, in the mutant which was glycosylated at position 66 only, E protein secretion was reduced to only 10% of the wild-type level. These data suggest that the glycan associated with the N-linked glycosylation site at position 154 in protein E plays an important role in VLP secretion.

  8. Cloning and expression of Mycobacterium bovis secreted protein MPB51 in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    JIANG Xiuyun; WANG Chunfeng; WANG Chunfang; HE Zhaoyang

    2007-01-01

    The purpose of this study is to clone,identify,and express the mature secreted protein MPB51 from Mycobacterium bovis and to lay a good foundation for the diagnosis of M.bovis,for applying M.bovis vaccine into clinical practice,and for detection of immunity effectiveness.The gene encoding MPB51 was amplified from M.bovis Valleel 11 chromosomal DNA by using PCR technique,PCR product was approximately 800 bp DNA segment.Clone vector pGEM-T-51 was successfully constructed by the PCR product that was cloned into pGEM-T vector by using T-A clone technique,pGEM-T-51 and pET28a(+)were digested by BamH I and EcoR I double enzymes.The prokaryotic expression vector pET28a-51 was constructed by using the purified MPB51 gene that was subcloned into the expression vector pET28a(+).Plasmid containing pET28a-51 was transformed into competence E.coli BL21(DE3).The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE.An approximately 30 kDa exogenous protein was observed on the SDS-PAGE.The protein was analyzed by using Western-blotting and it had the antigenic activity ofM.bovis.These results could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.

  9. Stimulation of Hepatoma Cell Invasiveness and Metastatic Potential by Proteins Secreted From Irradiated Nonparenchymal Cells

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    Zhou Leyuan [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Wang Zhiming [Department of Medical Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Gao Yabo [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Wang Lingyan [Experimental Research Center, Zhongshan Hospital, Fudan University, Shanghai (China); Zeng Zhaochong, E-mail: zeng.zhaochong@zs-hospital.sh.cn [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China)

    2012-11-01

    Purpose: To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect. Methods and Materials: Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA). Results: In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 {+-} 4.74) than in RH10Gy-SnonR (30.6 {+-} 3.85) cells, and lowest in McA-RH7777 (11.4 {+-} 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 {+-} 5.38), RH10Gy-SnonR (22.17 {+-} 4.26), and McA-RH7777 (8.3 {+-} 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-{alpha} and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR. Conclusions: Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of

  10. Comparative proteomic analysis of extracellular secreted proteins expressed by two pathogenic Acanthamoeba castellanii clinical isolates and a non-pathogenic ATCC strain.

    Science.gov (United States)

    Huang, Jian-Ming; Lin, Wei-Chen; Li, Sung-Chou; Shih, Min-Hsiu; Chan, Wen-Ching; Shin, Jyh-Wei; Huang, Fu-Chin

    2016-07-01

    Acanthamoeba keratitis (AK) is a serious ocular disease caused by pathogenic Acanthamoeba gaining entry through wounds in the corneal injury; generally, patients at risk for contracting AK wear contact lenses, usually over a long period of time. Moreover, pathogenic Acanthamoeba causes serious consequences: it makes the cornea turbid and difficult to operate on, including procedures such as enucleation of the eyeball. At present, diagnosis of this disease is not straightforward, and treatment is very demanding. We have established the comparative transcriptome and extracellular secreted proteomic database according to the non-pathogenic strain ATCC 30010 and the pathogenic strains NCKU_B and NCKU_D. We identified 44 secreted proteins successfully, 10 consensus secreted proteins and 34 strain-specific secreted proteins. These proteins may provide targets for therapy and immuno-diagnosis of Acanthamoeba infections. This study shows a suitable approach to identify secreted proteins in Acanthamoeba and provides new perspectives for the study of molecules potentially involved in the AK.

  11. Ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae.

    Science.gov (United States)

    Guo, Wei; Cai, Lu-Lu; Zou, Hua-Song; Ma, Wen-Xiu; Liu, Xi-Ling; Zou, Li-Fang; Li, Yu-Rong; Chen, Xiao-Bin; Chen, Gong-You

    2012-08-01

    The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99(A) and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice.

  12. The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" encodes an afp-like prophage possibly used for protein secretion.

    Science.gov (United States)

    Penz, Thomas; Horn, Matthias; Schmitz-Esser, Stephan

    2010-01-01

    The recently sequenced genome of the obligate intracellular amoeba symbiont 'Candidatus Amoebophilus asiaticus' is unique among prokaryotic genomes due to its extremely large fraction of genes encoding proteins harboring eukaryotic domains such as ankyrin-repeats, TPR/SEL1 repeats, leucine-rich repeats, as well as F- and U-box domains, most of which likely serve in the interaction with the amoeba host. Here we provide evidence for the presence of additional proteins which are presumably presented extracellularly and should thus also be important for host cell interaction. Surprisingly, we did not find homologues of any of the well-known protein secretion systems required to translocate effector proteins into the host cell in the A. asiaticus genome, and the type six secretion systems seems to be incomplete. Here we describe the presence of a putative prophage in the A. asiaticus genome, which shows similarity to the antifeeding prophage from the insect pathogen Serratia entomophila. In S. entomophila this system is used to deliver toxins into insect hosts. This putative antifeeding-like prophage might thus represent the missing protein secretion apparatus in A. asiaticus.

  13. Recent advances in recombinant protein expression by Corynebacterium, Brevibacterium, and Streptomyces: from transcription and translation regulation to secretion pathway selection.

    Science.gov (United States)

    Liu, Long; Yang, Haiquan; Shin, Hyun-Dong; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-11-01

    Gram-positive bacteria are widely used to produce recombinant proteins, amino acids, organic acids, higher alcohols, and polymers. Many proteins have been expressed in Gram-positive hosts such as Corynebacterium, Brevibacterium, and Streptomyces. The favorable and advantageous characteristics (e.g., high secretion capacity and efficient production of metabolic products) of these species have increased the biotechnological applications of bacteria. However, owing to multiplicity from genes encoding the proteins and expression hosts, the expression of recombinant proteins is limited in Gram-positive bacteria. Because there is a very recent review about protein expression in Bacillus subtilis, here we summarize recent strategies for efficient expression of recombinant proteins in the other three typical Gram-positive bacteria (Corynebacterium, Brevibacterium, and Streptomyces) and discuss future prospects. We hope that this review will contribute to the development of recombinant protein expression in Corynebacterium, Brevibacterium, and Streptomyces.

  14. Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion

    DEFF Research Database (Denmark)

    Finnie, Christine; Andersen, Birgit; Shahpiri, Azar

    2011-01-01

    The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberel......The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response...... to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling...... molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted...

  15. Iron-regulated biofilm formation in Staphylococcus aureus Newman requires ica and the secreted protein Emp.

    Science.gov (United States)

    Johnson, Miranda; Cockayne, Alan; Morrissey, Julie A

    2008-04-01

    Staphylococcus aureus biofilm formation is induced in iron-restricted growth conditions in vitro. In this study, we showed that Emp and Eap play important roles in low-iron-induced biofilm formation of S. aureus Newman. Eap and Emp are secreted proteins which are non-covalently attached to the S. aureus cell surface and have previously been implicated in a number of aspects of S. aureus pathogenesis. We showed here that the transcription of these important virulence factors is induced by growth in low-iron medium, reflective of the in vivo environment. Our results show that iron regulation of Eap and Emp is Fur independent. However, Fur is required for full induction of eap and emp expression in low-iron conditions. In this study, we demonstrated that in addition to Fur, low-iron-induced biofilm formation requires Sae, Agr, and SarA. In iron-restricted growth conditions, Sae and Agr are essential for Emp and Eap expression and hence for biofilm formation, whereas SarA appears to have a less-significant role. We also showed that expression of the ica operon is required for biofilm formation in iron-restricted growth conditions. We demonstrated that in fact, ica is required for the expression of the important multifunctional virulence determinants eap and emp.

  16. The role of secreted frizzled-related protein 2 expression in prostate cancer.

    LENUS (Irish Health Repository)

    O'Hurley, Gillian

    2012-02-01

    AIMS: Improved prostate cancer (PCa)-specific biomarkers are urgently required to distinguish between indolent and aggressive disease, in order to avoid overtreatment. In this study, we investigated the prostatic tissue expression of secreted frizzled-related protein (SFRP)-2. METHODS AND RESULTS: Following immunohistochemical analysis on PCa tissue microarrays with samples from 216 patients, strong\\/moderate SFRP-2 expression was observed in epithelial cells of benign prostatic hyperplasia, and negative\\/weak SFRP-2 expression was observed in the majority of tumour epithelia. However, among Gleason grade 5 carcinomas, 40% showed strong\\/moderate SFRP-2 expression and 60% showed negative SFRP-2 expression in epithelial cells. Further microscopic evaluation of Gleason grade 5 tumours revealed different morphological patterns, corresponding with differential SFRP-2 expression. The first subgroup (referred to as Type A) appeared to have a morphologically solid growth pattern, whereas the second subgroup (referred to as Type B) appeared to have a more diffuse pattern. Furthermore, 100% (4\\/4) of Type A patients experienced biochemical recurrence, as compared with 0% (0\\/6) of Type B patients. CONCLUSIONS: These results imply: (i) that there is a loss of SFRP-2 expression from benign to malignant prostate glands; and (ii) differential SFRP-2 expression among two possible subgroups of Gleason grade 5 tumours.

  17. Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

    DEFF Research Database (Denmark)

    Barba Espin, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per;

    2014-01-01

    The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro...... respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping...... shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions...

  18. Expression patterns of Wnt signaling component, secreted frizzled-related protein 3 in astrocytoma and glioblastoma

    Science.gov (United States)

    PEĆINA-ŠLAUS, NIVES; KAFKA, ANJA; VAROŠANEC, ANA MARIA; MARKOVIĆ, LEON; KRSNIK, ŽELJKA; NJIRIĆ, NIKO; MRAK, GORAN

    2016-01-01

    Secreted frizzled-related protein 3 (SFRP3) is a member of the family of soluble proteins, which modulate the Wnt signaling cascade. Novel research has identified aberrant expression of SFRPs in different types of cancer. In the present study the expression intensities and localizations of the SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. The results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Tumors were classified into four malignancy grades according to the World Health Organization guidelines. Moderate (P=0.014) and strong (P=0.028) nuclear expression levels were significantly different in pilocytic (grade I) and diffuse (grade II) astrocytomas demonstrating higher expression values, as compared with anaplastic astrocytoma (grade III) and glioblastoma (grade IV). When the sample was divided into two groups, the moderate and high cytoplasmic expression levels were observed to be significantly higher in glioblastomas than in the group comprising astrocytoma II and III. Furthermore, the results indicated that high grade tumors were associated with lower values of moderate (P=0.002) and strong (P=0.018) nuclear expression in comparison to low grade tumors. Analysis of cytoplasmic staining demonstrated that strong cytoplasmic expression was significantly higher in the astrocytoma III and IV group than in the astrocytoma I and II group (P=0.048). Furthermore, lower grade astrocytomas exhibited reduced membranous SFRP3 staining when compared with higher grade astrocytomas and this difference was statistically significant (P=0.036). The present results demonstrated that SFRP3 protein expression levels were decreased in the nucleus in higher grade astrocytoma (indicating the expected behavior of an antagonist of Wnt signaling), whereas when the SFRP3 was located in the cytoplasm an

  19. Expression patterns of Wnt signaling component, secreted frizzled‑related protein 3 in astrocytoma and glioblastoma.

    Science.gov (United States)

    Pećina-Šlaus, Nives; Kafka, Anja; Varošanec, Ana Maria; Marković, Leon; Krsnik, Željka; Njirić, Niko; Mrak, Goran

    2016-05-01

    Secreted frizzled-related protein 3 (SFRP3) is a member of the family of soluble proteins, which modulate the Wnt signaling cascade. Novel research has identified aberrant expression of SFRPs in different types of cancer. In the present study the expression intensities and localizations of the SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. The results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Tumors were classified into four malignancy grades according to the World Health Organization guidelines. Moderate (P=0.014) and strong (P=0.028) nuclear expression levels were significantly different in pilocytic (grade I) and diffuse (grade II) astrocytomas demonstrating higher expression values, as compared with anaplastic astrocytoma (grade III) and glioblastoma (grade IV). When the sample was divided into two groups, the moderate and high cytoplasmic expression levels were observed to be significantly higher in glioblastomas than in the group comprising astrocytoma II and III. Furthermore, the results indicated that high grade tumors were associated with lower values of moderate (P=0.002) and strong (P=0.018) nuclear expression in comparison to low grade tumors. Analysis of cytoplasmic staining demonstrated that strong cytoplasmic expression was significantly higher in the astrocytoma III and IV group than in the astrocytoma I and II group (P=0.048). Furthermore, lower grade astrocytomas exhibited reduced membranous SFRP3 staining when compared with higher grade astrocytomas and this difference was statistically significant (P=0.036). The present results demonstrated that SFRP3 protein expression levels were decreased in the nucleus in higher grade astrocytoma (indicating the expected behavior of an antagonist of Wnt signaling), whereas when the SFRP3 was located in the

  20. Gibberellic acid-induced aleurone layers responding to heat shock or tunicamycin provide insight into the N-glycoproteome, protein secretion, and endoplasmic reticulum stress.

    Science.gov (United States)

    Barba-Espín, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per; Svensson, Birte; Finnie, Christine

    2014-02-01

    The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion.

  1. Developmentally regulated Ca2+-dependent activator protein for secretion 2 (CAPS2) is involved in BDNF secretion and is associated with autism susceptibility.

    Science.gov (United States)

    Sadakata, Tetsushi; Furuichi, Teiichi

    2009-09-01

    The postnatal development of the cerebellum is accomplished via a series of cytogenetic and morphogenetic events encoded in the genome. To decipher the underlying genetic basis of these events we have systematized the spatio-temporal gene expression profiles during mouse cerebellar development in the Cerebellar Development Transcriptome Database (CDT-DB). Using the CDT-DB, Ca(2+)-dependent activator protein for secretion 2 (CAPS2 or CADPS2) was identified as a developmentally regulated gene that is predominantly expressed in cerebellar granule cells (GCs) with an expression peak around the first or second postnatal week. CAPS2 protein is concentrated in parallel fiber (PF) terminals and is associated with secretory vesicles containing brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). CAPS2 enhances release of BDNF and NT-3, both of which are essential for normal cerebellar development. CAPS2-deficient (CAPS2(-/-)) mice show reduced secretion of BDNF and NT-3; consequently, the cerebella of these mice exhibit developmental deficits, such as delayed development and increased cell death in GCs, fewer branched dendrites on Purkinje cells (PCs), and loss of the intercrural fissure. The PF-PC synapses have aberrant cytoarchitectures and electrophysiological properties. These abnormal cellular and morphological phenotypes are more severe around the cerebellar vermis, in which hypoplasia has been reported in autism patients. Moreover, CAPS2(-/-) mice had fewer cortical and hippocampal parvalbumin-positive interneurons and some autistic-like behavioral phenotypes. In the CAPS2 genes of some autistic patients an aberrant splicing variant and non-synonymous SNPs have been identified. These recent studies implicate CAPS2 in autism susceptibility. Therefore, CAPS2(-/-) mice will be a useful model animal in which to study aspects of the neuropathology and behaviors characteristic of developmental disorders.

  2. Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence.

    Science.gov (United States)

    Zhang, Zhanquan; Qin, Guozheng; Li, Boqiang; Tian, Shiping

    2014-06-01

    Pathogenic fungi usually secrete a series of virulence factors to the extracellular environment to facilitate infection. Rab GTPases play a central role in the secretory pathway. To explore the function of Rab/GTPase in filamentous fungi, we knocked out a Rab/GTPase family gene, Bcsas1, in Botrytis cinerea, an aggressive fungal pathogen that infects more than 200 plant species. A detailed analysis was conducted on the virulence and the secretory capability of the mutants. The results indicated that knockout of Bcsas1 inhibited hyphal development and reduced sporulation of B. cinerea on potato dextrose agar plates resulting in reduced virulence on various fruit hosts. Knocking out the Bcsas1 gene led to an accumulation of transport vesicles at the hyphal tip, significantly reduced extracellular protein content, and lowered the activity of polygalacturonase and xylanase in the extracellular medium. However, mutation of Bcsas1 did not affect the expression of genes encoding polygalacturonase and xylanase, suggesting the secretion of these two family enzymes was suppressed in the mutant. Moreover, a comparative analysis of the secretome provided further evidence that the disruption of Bcsas1 in mutant strains significantly depressed the secretion of polysaccharide hydrolases and proteases. The results indicate that Bcsas1, the Rab8/SEC4-like gene, plays a crucial role in development, protein secretion, and virulence of B. cinerea.

  3. Effects of Monoclonal Antibody Against Porcine 40-kDa Adipocyte-Specific Membrane Protein on Endocrine Secretion in Pigs

    Institute of Scientific and Technical Information of China (English)

    LIU Ling-yun; HU Hong-mei; ZHAO Su-mei; ZHANG Xi; DUAN Gang; GAO Shi-zheng

    2009-01-01

    The present study was to investigate the effect of monocional antibody against porcine 40-kDa adipocyte-specific membrane protein on endocrine secretion in pigs, in order to provide the evidence for application of this antibody to reduce excessive fat deposition in pig production. 40 Landrace × Saba pigs were randomly divided into 8 groups: 2 control groups were given saline with 10 mL, respectively, and the 6 treatment groups were given monoclonal antibody against porcine 40-kDa adipocyte-specific membrane protein with 0.1,0.5, and 1.0 mg kg-1 body weight at 15 or 60 kg body weight,respectively, all treatments were performed by intraperitoneal injection. The results showed that this monoclonal antibody could significantly reduce serum insulin level and increase levels of serum growth hormone (GH), insulin-like growth factor-1 (IGF-1), triiodothyronine (T3), and tetraiodothyronine (T4) either at 15 or 60 kg body weight injection. However,more marked effect was observed at 15 kg body weight treatment. Moreover, the dose-dependent effect of this monoclonal antibody on endocrine secretion was also observed. This result revealed that this monoclonal antibody increased secretion of hormones regulating fat lysis and reduced secretion of hormones regulating fat synthesis, suggests the reduction of porcine excessive fat deposition by this monoclonal antibody was carried out through affecting hormones regulating fat metabolism.

  4. Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion.

    Science.gov (United States)

    Yoshii, Kentarou; Konno, Akihiro; Goto, Akiko; Nio, Junko; Obara, Mayumi; Ueki, Tomotaka; Hayasaka, Daisuke; Mizutani, Tetsuya; Kariwa, Hiroaki; Takashima, Ikuo

    2004-10-01

    Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway. Virus envelope proteins play important roles in this process. In this study, the effect of mutations in the envelope proteins of tick-borne encephalitis (TBE) virus on secretion of virus-like particles (VLPs), using a recombinant plasmid expression system was analysed. It was found that a single point mutation at position 63 in prM induces a reduction in secretion of VLPs. The mutation in prM did not affect the folding of the envelope proteins, and chaperone-like activity of prM was maintained. As observed by immunofluorescence microscopy, viral envelope proteins with the mutation in prM were scarce in the Golgi complex, and accumulated in the ER. Electron microscopic analysis of cells expressing the mutated prM revealed that many tubular structures were present in the lumen. The insertion of the prM mutation at aa 63 into the viral genome reduced the production of infectious virus particles. This data suggest that prM plays a crucial role in the virus budding process.

  5. Serum concentrations and peripheral secretion of the beta chemokines monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α in alcoholic liver disease

    OpenAIRE

    Fisher, N; Neil, D.; Williams, A.; Adams, D.

    1999-01-01

    BACKGROUND—Alcoholic liver disease is associated with increased hepatic expression of monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1α (MIP-1α).
AIMS—To determine whether concentrations of chemokines in the peripheral circulation reflect disease activity, and whether chemokine secretion is restricted to the liver or is part of a systemic inflammatory response in alcoholic liver disease.
PATIENTS—Fifty one patients with alcoholic liver disease and 12 healthy co...

  6. Quantification of a Non-conventional Protein Secretion: The Low-Molecular-Weight FGF-2 Example.

    Science.gov (United States)

    Arcondéguy, Tania; Touriol, Christian; Lacazette, Eric

    2016-01-01

    Quantification of secreted factors is most often measured with enzyme-linked immunosorbent assay (ELISA), Western Blot, or more recently with antibody arrays. However, some of these, like low-molecular-weight fibroblast growth factor-2 (LMW FGF-2; the 18 kDa form), exemplify a set of secreted but almost non-diffusible molecular actors. It has been proposed that phosphorylated FGF-2 is secreted via a non-vesicular mechanism and that heparan sulfate proteoglycans function as extracellular reservoir but also as actors for its secretion. Heparan sulfate is a linear sulfated polysaccharide present on proteoglycans found in the extracellular matrix or anchored in the plasma membrane (syndecan). Moreover the LMW FGF-2 secretion appears to be activated upon FGF-1 treatment. In order to estimate quantification of such factor export across the plasma membrane, technical approaches are presented (evaluation of LMW FGF-2: (1) secretion, (2) extracellular matrix reservoir, and (3) secretion modulation by surrounding factors) and the importance of such procedures in the comprehension of the biology of these growth factors is underlined.

  7. A candidate approach implicates the secreted Salmonella effector protein SpvB in P-body disassembly.

    Directory of Open Access Journals (Sweden)

    Ana Eulalio

    Full Text Available P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. P-bodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.

  8. Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Meshcheryakov, Vladimir A. [Okinawa Instiute of Science and Technology, Okinawa 904-0495 (Japan); Kitao, Akio [University of Tokyo, Tokyo 113-0032 (Japan); Core Research for Evolutionary Science and Technology, Tokyo 113-0032 (Japan); Matsunami, Hideyuki; Samatey, Fadel A., E-mail: f.a.samatey@oist.jp [Okinawa Instiute of Science and Technology, Okinawa 904-0495 (Japan)

    2013-05-01

    Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, the crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.

  9. Hypermethylation and aberrant expression of secreted fizzled-related protein genes in pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Xian-Min Bu; Cheng-Hai Zhao; Ning Zhang; Feng Gao; Shuai Lin; Xian-Wei Dai

    2008-01-01

    AIM:To determine the methylation status and aberrant expression of some secreted frizzled-related protein (SFRP) genes in pancreatic cancer and explore their role in pancreatic carcinogenesis. METHODS:Methylation status and expression of SFRP genes were detected by methylation-specific PCR (MSPCR) and reverse-transcription PCR (RT-PCR) respectively. RESULTS:The frequencies of methylation for SFRP genes 1,2,4,5 were 70%, 48.3%,60% and 76.7% in pancreatic cancer samples, and 21.7%, 20%,10% and 36.7% in matched cancer adjacent normal tissue samples,respectively (χ2=28.23,P<0.0001 for SFRP gene 1; χ2=10.71,P=0.001 for SFRP gene 2;χ2=32.97,P<0.0001 for SFRP gene 4;χ2=19.55,P<0.0001 for SFRP gene 5). Expression loss of SFRP genes 1,2,4 and 5 was found in 65%,40%,55% and 71.7% of 60 pancreatic cancer samples, and 25%,15%,18.3% and 31.7% of matched cancer adjacent normal tissue samples,respectively (χ2=19.39,P<0.0001 for SFRP gene 1;χ2=9.40,P=0.002 for SFRP gene 2;χ2=17.37,P<0.0001 for SFRP gene 4;χ2=19.22,P<0.0001 for SFRP gene 5).SFRP gene 1 was methylated but not expressed in PC-3 and PANC-1,SFRP gene 2 was methylated but not expressed in PANC-1 and CFPAC-1,SFRP gene 4 was methylated but not expressed in PC-3,and SFRP gene 5 was methylated but not expressed in CFPAC-1. CONCLUSION:Hypermethylation and aberrant expression of SFRP genes are common in pancreatic cancer,which may be involved in pancreatic carcinogenesis.

  10. A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.

    Science.gov (United States)

    Rindler, M J; Traber, M G

    1988-08-01

    Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.

  11. Kisspeptin regulates gonadotropin-releasing hormone secretion in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats

    Institute of Scientific and Technical Information of China (English)

    Haogang Xue; Chunying Yang; Xiaodong Ge; Weiqi Sun; Chun Li; Mingyu Qi

    2013-01-01

    Kisspeptin is essential for activation of the hypothalamo-pituitary-gonadal axis. In this study, we established gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. Rats were injected with 1, 10, or 100 pM kisspeptin-10, a peptide derived from full-length kisspeptin, into the arcuate nucleus and medial preoptic area, and with the kisspeptin antagonist peptide 234 into the lateral cerebral ventricle. The results of immunohistochemical staining revealed that pulsatile luteinizing hormone secretion was suppressed after injection of antagonist peptide 234 into the lateral cerebral ventricle, and a significant increase in luteinizing hormone level was observed after kisspeptin-10 injection into the arcuate nucleus and medial preoptic area. The results of an enzyme-linked immunosorbent assay showed that luteinizing hormone levels during the first hour of kisspeptin-10 infusion into the arcuate nucleus were significantly greater in the 100 pM kisspeptin-10 group than in the 10 pM kisspeptin-10 group. These findings indicate that kisspeptin directly promotes gonadotropin-releasing hormone secretion and luteinizing hormone release in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. The arcuate nucleus is a key component of the kisspeptin-G protein-coupled receptor 54 signaling pathway underlying regulating luteinizing hormone pulse secretion.

  12. Low molecular mass GTP-binding proteins are secreted from mammary epithelial cells in association with lipid globules.

    Science.gov (United States)

    Ghosal, D; Ankrapp, D; Keenan, T W

    1993-07-01

    Secretion of milk lipid globules is achieved through encapsulation of triacylglycerol-rich lipid droplets in a specialized region of apical plasma membrane of mammary epithelial cells. A class of low molecular mass GTP-binding proteins were associated tightly with the lipid globule membrane, and these proteins appeared to change from peripheral to integral membrane proteins during intracellular growth and transit of lipid globule precursors. Inclusion of GTP or GTP gamma S in incubation medium stimulated secretion of lipids from primary cultures of permeabilized rat mammary epithelial cells. Six polypeptides with molecular masses between 28 and 21 kDa were detected by ability to bind GTP gamma S following separation of lipid-globule-associated proteins by SDS-PAGE and transblotting onto nitrocellulose. That all of these polypeptides were distinct immunologically from the archetype ras was evident from lack of immunoreactivity with p21 ras G-protein monoclonal antibody in Western blots. This monoclonal antibody bound to a 23 kDa polypeptide of lipid droplets that was not detected with the GTP gamma S binding assay. A 25 kDa component of milk lipid globules was a potent substrate for ADP-ribosylation by botulinum toxin C3, but cholera toxin was much less effective, suggesting that this component may belong to the rac class of G-proteins. The 21 kDa component was related immunologically to ADP ribosylation factor.

  13. Label-free detection of protein molecules secreted from an organ-on-a-chip model for drug toxicity assays

    Science.gov (United States)

    Morales, Andres W.; Zhang, Yu S.; Aleman, Julio; Alerasool, Parissa; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

    2016-03-01

    Clinical attrition is about 30% from failure of drug candidates due to toxic side effects, increasing the drug development costs significantly and slowing down the drug discovery process. This partly originates from the fact that the animal models do not accurately represent human physiology. Hence there is a clear unmet need for developing drug toxicity assays using human-based models that are complementary to traditional animal models before starting expensive clinical trials. Organ-on-a-chip techniques developed in recent years have generated a variety of human organ models mimicking different human physiological conditions. However, it is extremely challenging to monitor the transient and long-term response of the organ models to drug treatments during drug toxicity tests. First, when an organ-on-a-chip model interacts with drugs, a certain amount of protein molecules may be released into the medium due to certain drug effects, but the amount of the protein molecules is limited, since the organ tissue grown inside microfluidic bioreactors have minimum volume. Second, traditional fluorescence techniques cannot be utilized for real-time monitoring of the concentration of the protein molecules, because the protein molecules are continuously secreted from the tissue and it is practically impossible to achieve fluorescence labeling in the dynamically changing environment. Therefore, direct measurements of the secreted protein molecules with a label-free approach is strongly desired for organs-on-a-chip applications. In this paper, we report the development of a photonic crystal-based biosensor for label-free assays of secreted protein molecules from a liver-on-a-chip model. Ultrahigh detection sensitivity and specificity have been demonstrated.

  14. Impaired Insulin Secretion in Perfused Pancreases Isolated from Offspring of Female Rats Fed a Low Protein Whey-Based Diet

    Directory of Open Access Journals (Sweden)

    Matthew PG Barnett

    2008-07-01

    Full Text Available Context Insufficient maternal protein intake has been postulated to cause impaired fuel metabolism and diabetes mellitus in adult mammalian progeny, but the mechanism remains unclear. Objective To investigate the effect of a maternal low protein whey-based diet during pregnancy and lactation on pancreatic function and skeletal muscle glucose metabolism in the offspring. Animals Sprague-Dawley rats: 8 mothers and 46 offspring. Design Female rats were fed throughout pregnancy and lactation with otherwisecomplete isoenergetic diets sufficient (20% whey protein; control: n=3 or insufficient (5% whey protein; low-protein: n=5 in whey protein. From weaning all offspring ate control diet. Main outcome measures Food intake and weight gain were measured for both mothers and offspring, and in vitro functional studies of endocrine pancreas and skeletal muscle were performed on offspring at 40 and 50 days of age, respectively. Results Food intake (P=0.004 and weight gain (P=0.006 were lower in low protein than control mothers during early gestation. Offspring of low protein mothers had significant lower body weight from 5 to 15 days of age, although there was no significant difference in food consumption. Glucose, arginine- and glucose/arginine-stimulated insulin secretion from perfused pancreases isolated from low protein offspring were decreased by between 55 and 65% compared with control values. Studies in skeletal muscle demonstrated no difference in insulin sensitivity between the two groups. Conclusions Dietary whey protein insufficiency in female rats during pregnancy and lactation can evoke major changes in insulin secretion in progeny, and these changes represent a persistent functional abnormality in the endocrine pancreas.

  15. Lack of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine attenuates liver fibrogenesis in mice.

    Directory of Open Access Journals (Sweden)

    Catalina Atorrasagasti

    Full Text Available INTRODUCTION: Secreted Protein, Acidic and Rich in Cysteine (SPARC is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC. METHODS: Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC(+/+ and knock-out (SPARC(-/- mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC(-/- and SPARC(+/+ mice using Affymetrix Mouse Gene ST 1.0 array. RESULTS: SPARC expression was found induced in fibrotic livers of mouse and human. SPARC(-/- mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/- mice when compared to SPARC(+/+ mice; in addition, MMP-2 expression was increased in SPARC(-/- mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-β1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli. CONCLUSIONS: Overall our data suggest that

  16. Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein.

    Science.gov (United States)

    Yurgel, Svetlana N; Rice, Jennifer; Domreis, Elizabeth; Lynch, Joseph; Sa, Na; Qamar, Zeeshan; Rajamani, Sathish; Gao, Mengsheng; Roje, Sanja; Bauer, Wolfgang D

    2014-05-01

    Sinorhizobium meliloti, the nitrogen-fixing bacterial symbiont of Medicago spp. and other legumes, secretes a considerable amount of riboflavin. This precursor of the cofactors flavin mononucleotide and flavin adenine dinucleotide is a bioactive molecule that has a beneficial effect on plant growth. The ribBA gene of S. meliloti codes for a putative bifunctional enzyme with dihydroxybutanone phosphate synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps of the riboflavin biosynthesis pathway. We show here that an in-frame deletion of ribBA does not cause riboflavin auxotrophy or affect the ability of S. meliloti to establish an effective symbiosis with the host plant but does affect the ability of the bacteria to secrete flavins, colonize host-plant roots, and compete for nodulation. A strain missing the RibBA protein retains considerable GTP cyclohydrolase II activity. Based on these results, we hypothesize that S. meliloti has two partly interchangeable modules for biosynthesis of riboflavin, one fulfilling the internal need for flavins in bacterial metabolism and the other producing riboflavin for secretion. Our data also indicate that bacteria-derived flavins play a role in communication between rhizobia and the legume host and that the RibBA protein is important in this communication process even though it is not essential for riboflavin biosynthesis and symbiosis.

  17. Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K

    Science.gov (United States)

    Ohmae, Saori; Noma, Naruto; Toyomoto, Masayasu; Shinohara, Masahiro; Takeiri, Masatoshi; Fuji, Hiroaki; Takemoto, Kenji; Iwaisako, Keiko; Fujita, Tomoko; Takeda, Norihiko; Kawatani, Makoto; Aoyama, Mineyoshi; Hagiwara, Masatoshi; Ishihama, Yasushi; Asagiri, Masataka

    2017-01-01

    Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome–plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases. PMID:28300073

  18. Roles of sulfonylurea receptor 1 and multidrug resistance protein 1 in modulating insulin secretion in human insulinoma

    Institute of Scientific and Technical Information of China (English)

    Cheng-Jiang Li; Hua-Li Zhou; Jun Li; Hong-TianYao; Rong Su; Wen-Peng Li

    2011-01-01

    BACKGROUND: Sulfonylurea receptor 1 (SUR1) and multidrug resistance protein 1 (MRP1) are two prominent members of multidrug resistance proteins associated with insulin secretion. The aims of this study were to investigate their expression in insulinomas and their sole and synergistic effects in modulating abnormalinsulinsecretion. METHODS: Fasting glucose, insulin and C-peptide were measured in 11 insulinoma patients and 11 healthy controls. Prolonged oral glucose tolerance tests were performed in 6 insulinoma patients. Insulin content, SUR1 and MRP1 were detected in 11 insulinoma patients by immunohistochemistry. SUR1 and MRP1 were also detected in 6 insulinoma patients by immunofluorescence. RESULTS: Insulinoma patients presented the typical demons-trations of Whipple's triad. Fasting glucose of each insulinoma patient was lower than 2.8 mmol/L, and simultaneous insulin and C-peptide were increased in insulinoma patients. Prolonged oral glucose tolerance tests showed that insulin secretion in insulinoma patients were also stimulated by high glucose. Immunohistochemistry and immunofluorescence staining showed that SUR1 increased, but MRP1 decreased in insulinoma compared with the adjacent islets. CONCLUSIONS: The hypersecretion of insulin in insulinomas might be, at least partially, due to the enrichment of SUR1. In contrast, MRP1, which is down-regulated in insulinomas, might reflect a negative feedback in insulin secretion.

  19. Role of secreted conjunctival mucosal cytokine and chemokine proteins in different stages of trachomatous disease.

    Directory of Open Access Journals (Sweden)

    Troy A Skwor

    Full Text Available BACKGROUND: Chlamydia trachomatis is responsible for trachoma, the primary cause of preventable blindness worldwide. Plans to eradicate trachoma using the World Health Organization's SAFE program (Surgery, Antibiotics, Facial Cleanliness and Environment Improvement have resulted in recurrence of infection and disease following cessation of treatment in many endemic countries, suggesting the need for a vaccine to control infection and trachomatous disease. Vaccine development requires, in part, knowledge of the mucosal host immune responses in both healthy and trachomatous conjuctivae-an area of research that remains insufficiently studied. METHODOLOGY/PRINCIPAL FINDINGS: We characterized 25 secreted cytokines and chemokines from the conjunctival mucosa of individuals residing in a trachoma endemic region of Nepal using Luminex X100 multiplexing technology. Immunomodulating effects of concurrent C. trachomatis infection were also examined. We found that proinflammatory cytokines IL-1beta (r = 0.259, P = 0.001 and TNFalpha (r = 0.168, P<0.05 were significantly associated with trachomatous disease and concurrent C. trachomatis infection compared with age and sex matched controls from the same region who did not have trachoma. In support of these findings, anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra was negatively associated with chronic scarring trachoma (r = -0.249, P = 0.001. Additional cytokines (Th1, IL-12p40 [r = -0.212, P<0.01], and Th2, IL-4 and IL-13 [r = -0.165 and -0.189, respectively, P<0.05 for both] were negatively associated with chronic scarring trachoma, suggesting a protective role. Conversely, a pathogenic role for the Th3/Tr1 cytokine IL-10 (r = 0.180, P<0.05 was evident with increased levels for all trachoma grades. New risk factors for chronic scarring trachoma included IL-6 and IL-15 (r = 0.259 and 0.292, respectively, P<0.005 for both with increased levels for concurrent C. trachomatis infections (r = 0.206, P

  20. The Structure of the Toxin and Type Six Secretion System Substrate Tse2 in Complex with Its Immunity Protein.

    Science.gov (United States)

    Robb, Craig S; Robb, Melissa; Nano, Francis E; Boraston, Alisdair B

    2016-02-01

    Tse2 is a cytoactive toxin secreted by a type six secretion apparatus of Pseudomonas aeruginosa. The Tse2 toxin naturally attacks a target in the cytoplasm of bacterial cells, and can cause toxicity if artificially introduced into eukaryotic cells. The X-ray crystal structure of the complex of Tse2 and its cognate immunity protein Tsi2 revealed a heterotetrameric structure with an extensive binding interface. Structural identity was found between Tse2 and NAD-dependent enzymes, especially ADP-ribosylating toxins, which facilitated the identification of the Tse2 active site and revealed it to be occluded upon binding the inhibitor Tsi2. The structural identity shared with NAD-dependent enzymes, including conserved catalytic residues, suggests that the mechanism of Tse2 toxicity may be NAD dependent.

  1. Molecular characterization and polyclonal antibody generation against core component CagX protein of Helicobacter pylori type IV secretion system

    Science.gov (United States)

    Gopal, Gopal Jee; Kumar, Awanish; Pal, Jagannath; Mukhopadhyay, Gauranga

    2014-01-01

    Gram-negative bacteria Helicobacter pylori cause gastric ulcer, duodenal cancer, and found in almost half of the world’s residents. The protein responsible for this disease is secreted through type IV secretion system (TFSS) of H. pylori. TFSS is encoded by 40-kb region of chromosomal DNA known as cag-pathogenicity island (PAI). TFSS comprises of three major components: cytoplasmic/inner membrane ATPase, transmembrane core-complex and outer membranous pilli, and associated subunits. Core complex consists of CagX, CagT, CagM, and Cag3(δ) proteins as per existing knowledge. In this study, we have characterized one of the important component of core-complex forming sub-unit protein, i.e., CagX. Complete ORF of CagX except signal peptide coding region was cloned and expressed in pET28a vector. Purification of CagX protein was performed, and polyclonal anti-sera against full-length recombinant CagX were raised in rabbit model. We obtained a very specific and high titer, CagX anti-sera that were utilized to characterize endogenous CagX. Surface localization of CagX was also seen by immunofluorescence microscopy. In short for the first time a full-length CagX was characterized, and we showed that CagX is the part of high molecular weight core complex, which is important for assembly and function of H. pylori TFSS. PMID:24637488

  2. Increased blood levels of IgG reactive with secreted Streptococcus pyogenes proteins in chronic plaque psoriasis.

    Science.gov (United States)

    El-Rachkidy, Rana G; Hales, Jonathan M; Freestone, Primrose P E; Young, Helen S; Griffiths, Christopher E M; Camp, Richard D R

    2007-06-01

    A pathogenic role for Streptococcus (S) pyogenes infections in chronic plaque psoriasis is suspected but poorly defined. We separated cellular and supernatant proteins from S. pyogenes cultures by high-resolution two-dimensional gel electrophoresis, and used immunoblotting to demonstrate the diversity of serum or plasma IgGs that react with elements of the proteome of this bacterium. We have shown that a substantial proportion of IgG-reactive proteins from cultured S. pyogenes are secreted. The total secreted protein fraction, including diverse IgG-binding elements, was subsequently used in an ELISA to measure blood titers of reactive IgG. This ELISA showed that blood samples from patients with chronic plaque psoriasis contained significantly higher titers of reactive IgG than samples from age- and sex-matched healthy controls (P=0.0009). In contrast, neither a standard assay measuring antistreptolysin O titers nor ELISAs measuring titers of IgG reactive with protein fractions from Staphylococcus aureus and Staphylococcus epidermidis, were able to distinguish between blood samples from the two groups. These findings justify the hypothesis that S. pyogenes infections are more important in the pathogenesis of chronic plaque psoriasis than has previously been recognized, and indicate the need for further controlled therapeutic trials of antibacterial measures in this common skin disease.

  3. A novel role of protein tyrosine kinase2 in mediating chloride secretion in human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Lihua Liang

    Full Text Available Ca(2+ activated Cl(- channels (CaCC are up-regulated in cystic fibrosis (CF airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl(- secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca(2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na(+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl(- secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2 is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca(2+ and Cl(- secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

  4. Secretion of organic anions by hepatocytes : Involvement of homologues of the multidrug resistance protein

    NARCIS (Netherlands)

    Muller, M; Roelofsen, H; Jansen, PLM

    1996-01-01

    The canalicular multispecific organic anion transporter (cMOAT) is one of at least four ATP-dependent transport systems identified so far in the canalicular membrane domain. Using mutant rat strains that lack organic anion secretion, the substrate specificity of cMOAT has been characterized. cMOAT a

  5. Secreted and immunogenic proteins produced by the honey bee bacterial pathogen, Paenibacillus larvae

    Science.gov (United States)

    American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae dur...

  6. Sensing of Bacterial Type IV Secretion via the Unfolded Protein Response

    NARCIS (Netherlands)

    de Jong, Maarten F.; Starr, Tregei; Winter, Maria G.; den Hartigh, Andreas B.; Child, Robert; Knodler, Leigh A.; van Dijl, Jan Maarten; Celli, Jean; Tsolis, Renee M.

    2013-01-01

    Host cytokine responses to Brucella abortus infection are elicited predominantly by the deployment of a type IV secretion system (T4SS). However, the mechanism by which the T4SS elicits inflammation remains unknown. Here we show that translocation of the T4SS substrate VceC into host cells induces p

  7. Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

    Science.gov (United States)

    Kneuper, Holger; Cao, Zhen Ping; Twomey, Kate B; Zoltner, Martin; Jäger, Franziska; Cargill, James S; Chalmers, James; van der Kooi-Pol, Magdalena M; van Dijl, Jan Maarten; Ryan, Robert P; Hunter, William N; Palmer, Tracy

    2014-09-01

    The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

  8. Pertussis toxin non-sensitive G protein mediates cholinergic stimulation for secretion of pancreastatin and somatostatin from QGP-1N cells.

    Science.gov (United States)

    Funakoshi, A; Tateishi, K; Tsuru, M; Kono, A

    1992-01-02

    To clarify the possible role of a guanine nucleotide-binding protein (G-protein) in the signal transducing system activated by carbachol, actions of carbachol on human pancreastatin producing cell line (QGP-1N) were compared with those of fluoride, a well-known activator of stimulatory (Gs) or inhibitory (Gi) G protein. 10(-5) M of carbachol as well as 20 mM of NaF stimulated secretion of pancreastatin and somatostatin and intracellular Ca2+ mobilization. These secretion and Ca2+ mobilization were not modified by pertussis toxin, an inhibitor of Gi protein. These results suggest that pancreastatin and somatostatin secretions from QGP-1N are regulated by acetylcholine through a muscarinic receptor coupled to the activation of polyphosphoinositide breakdown by a G protein, which appears to be fluoride sensitive but is other than a Gi-like protein.

  9. JNK mitogen-activated protein kinase limits calcium-dependent chloride secretion across colonic epithelial cells.

    LENUS (Irish Health Repository)

    Donnellan, Fergal

    2010-01-01

    Neuroimmune agonists induce epithelial Cl(-) secretion through elevations in intracellular Ca2+ or cAMP. Previously, we demonstrated that epidermal growth factor receptor (EGFR) transactivation and subsequent ERK MAPK activation limits secretory responses to Ca2+-dependent, but not cAMP-dependent, agonists. Although JNK MAPKs are also expressed in epithelial cells, their role in regulating transport function is unknown. Here, we investigated the potential role for JNK in regulating Cl(-) secretion in T(84) colonic epithelial cells. Western blot analysis revealed that a prototypical Ca2+-dependent secretagogue, carbachol (CCh; 100 microM), induced phosphorylation of both the 46-kDa and 54-kDa isoforms of JNK. This effect was mimicked by thapsigargin (TG), which specifically elevates intracellular Ca2+, but not by forskolin (FSK; 10 microM), which elevates cAMP. CCh-induced JNK phosphorylation was attenuated by the EGFR inhibitor, tyrphostin-AG1478 (1 microM). Pretreatment of voltage-clamped T(84) cells with SP600125 (2 microM), a specific JNK inhibitor, potentiated secretory responses to both CCh and TG but not to FSK. The effects of SP600125 on CCh-induced secretion were not additive with those of the ERK inhibitor, PD98059. Finally, in apically permeabilized T(84) cell monolayers, SP600125 potentiated CCh-induced K+ conductances but not Na+\\/K+ATPase activity. These data demonstrate a novel role for JNK MAPK in regulating Ca2+ but not cAMP-dependent epithelial Cl(-) secretion. JNK activation is mediated by EGFR transactivation and exerts its antisecretory effects through inhibition of basolateral K+ channels. These data further our understanding of mechanisms regulating epithelial secretion and underscore the potential for exploitation of MAPK-dependent signaling in treatment of intestinal transport disorders.

  10. Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and β-cell apoptosis

    DEFF Research Database (Denmark)

    Berchtold, Lukas Adrian; Størling, Zenia Marian; Ortis, Fernanda

    2011-01-01

    Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease-causing...

  11. Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots.

    Science.gov (United States)

    Huet, Yoann; Ekouna, Jean-Pierre Ele; Caron, Aurore; Mezreb, Katiba; Boitel-Conti, Michèle; Guerineau, François

    2014-01-01

    A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots.

  12. Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose

    Energy Technology Data Exchange (ETDEWEB)

    Currie, Devin [Dartmouth College; Guss, Adam M [ORNL; Herring, Christopher [Mascoma Corporation; Giannone, Richard J [ORNL; Johnson, Courtney M [ORNL; Lankford, Patricia K [ORNL; Brown, Steven D [ORNL; Hettich, Robert {Bob} L [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    2014-01-01

    Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars

  13. The LspB protein is involved in the secretion of the LspA1 and LspA2 proteins by Haemophilus ducreyi.

    Science.gov (United States)

    Ward, Christine K; Mock, Jason R; Hansen, Eric J

    2004-04-01

    The LspA1 and LspA2 proteins of Haemophilus ducreyi 35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of the Bordetella pertussis filamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. The lspA2 open reading frame is flanked upstream by a gene, lspB, that encodes a predicted protein with homology to the B. pertussis FhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. The H. ducreyi lspB gene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that the lspB gene was transcribed together with the lspA2 gene on a single mRNA transcript. Polyclonal H. ducreyi LspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction of H. ducreyi 35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids from H. ducreyi lspB and lspA1 lspB mutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of the lspB mutant with the wild-type lspB gene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, the lspB mutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that the H. ducreyi LspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.

  14. Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao-Wen Hao; Sheng-Tao Zhu; Yuan-Long He; Peng Li; Yong-Jun Wang; Shu-Tian Zhang

    2012-01-01

    AIM: To investigate the expression and methylation status of the secreted frizzled-related protein 2 (SFRP2) in esophageal squamous cell carcinoma (ESCC) and explore its role in ESCC carcinogenesis.METHODS: Seven ESCC cell lines (KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1) and one immortalized human esophageal epithelial cell line (Het-1A), 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2'-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro.RESULTS: SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2'-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue (0.939 ± 0.398 vs 1.51 ± 0.399, P < 0.01). SFRP2 methylation was higher in tumor tissue than in paired normal tissue (95% vs 65%, P < 0.05). The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3.1/v5 his

  15. Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling.

    Directory of Open Access Journals (Sweden)

    Sasisekhar Bennuru

    Full Text Available Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf, L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs in the available databases. Moreover, this analysis was able to confirm the presence of 274 "hypothetical" proteins inferred from gene prediction algorithms applied to the B. malayi (Bm genome. Not surprisingly, the majority (160/274 of these "hypothetical" proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase, MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host-parasite interaction.

  16. Vesicle-associated membrane protein 3 (VAMP-3) and VAMP-8 are present in human platelets and are required for granule secretion.

    Science.gov (United States)

    Polgár, János; Chung, Sul-Hee; Reed, Guy L

    2002-08-01

    Secretion of platelet granules is necessary for normal hemostasis. Platelet secretion requires soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex formation between different members of the syntaxin, SNAP-25, and vesicle-associated membrane protein (VAMP) gene families. Using microcapillary reverse-phase high-performance liquid chromatography-nano-electrospray tandem mass spectrometry, we identified VAMP-3 and VAMP-8 as VAMP isoforms coimmunoprecipitated from platelets with syntaxin 4. Immunoblotting experiments confirmed the presence of VAMP-3 and VAMP-8 but not VAMP-1 or VAMP-2 in platelets. To examine the effect of VAMP proteins on platelet secretion, soluble recombinant (r) VAMP-2, rVAMP-3, and rVAMP-8 were incubated with streptolysin O-permeabilized platelets. Secretion of alpha granules (monitored by flow cytometric measurement of P-selectin) was blocked, and dense-granule secretion (assessed by release of carbon 14-serotonin) was almost completely inhibited by rVAMP-3, whereas rVAMP-8 inhibited secretion of dense granules but not alpha granules. In contrast, rVAMP-2, which formed SNARE complexes in vitro, had no effect on platelet exocytosis. We conclude that VAMP-3 and VAMP-8 form SNARE complexes with platelet syntaxin 4 and are required for platelet granule secretion.

  17. Functional interaction between type III-secreted protein IncA of Chlamydophila psittaci and human G3BP1.

    Science.gov (United States)

    Borth, Nicole; Litsche, Katrin; Franke, Claudia; Sachse, Konrad; Saluz, Hans Peter; Hänel, Frank

    2011-01-31

    Chlamydophila (Cp.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. These obligate intracellular bacteria are distinguished by a unique biphasic developmental cycle, which includes proliferation in a membrane-bound compartment termed inclusion. All Chlamydiaceae spp. possess a coding capacity for core components of a Type III secretion apparatus, which mediates specific delivery of anti-host effector proteins either into the chlamydial inclusion membrane or into the cytoplasm of target eukaryotic cells. Here we describe the interaction between Type III-secreted protein IncA of Cp. psittaci and host protein G3BP1 in a yeast two-hybrid system. In GST-pull down and co-immunoprecipitation experiments both in vitro and in vivo interaction between full-length IncA and G3BP1 were shown. Using fluorescence microscopy, the localization of G3BP1 near the inclusion membrane of Cp. psittaci-infected Hep-2 cells was demonstrated. Notably, infection of Hep-2 cells with Cp. psittaci and overexpression of IncA in HEK293 cells led to a decrease in c-Myc protein concentration. This effect could be ascribed to the interaction between IncA and G3BP1 since overexpression of an IncA mutant construct disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesize that lowering the host cell c-Myc protein concentration may be part of a strategy employed by Cp. psittaci to avoid apoptosis and scale down host cell proliferation.

  18. Identification of a secreted casein kinase 1 in Leishmania donovani: effect of protein over expression on parasite growth and virulence.

    Directory of Open Access Journals (Sweden)

    Mary Dan-Goor

    Full Text Available Casein kinase 1 (CK1 plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780 is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001. Analysis by flow cytometry showed a higher percentage, ∼4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005. These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.

  19. Association of the Cytoplasmic Membrane Protein XpsN with the Outer Membrane Protein XpsD in the Type II Protein Secretion Apparatus of Xanthomonas campestris pv. Campestris

    Science.gov (United States)

    Lee, Hsien-Ming; Wang, Kuan-Cheng; Liu, Yi-Ling; Yew, Hsin-Yan; Chen, Ling-Yun; Leu, Wei-Ming; Chen, David Chanhen; Hu, Nien-Tai

    2000-01-01

    An xps gene cluster composed of 11 open reading frames is required for the type II protein secretion in Xanthomonas campestris pv. campestris. Immediately upstream of the xpsD gene, which encodes an outer membrane protein that serves as the secretion channel by forming multimers, there exists an open reading frame (previously designated ORF2) that could encode a protein of 261 amino acid residues. Its N-terminal hydrophobic region is a likely membrane-anchoring sequence. Antibody raised against this protein could detect in the wild-type strain of X. campestris pv. campestris a protein band with an apparent molecular mass of 36 kDa by Western blotting. Its aberrant slow migration in sodium dodecyl sulfate-polyacrylamide gels might be due to its high proline content. We designated this protein XpsN. By constructing a mutant strain with an in-frame deletion of the chromosomal xpsN gene, we demonstrated that it is required for the secretion of extracellular enzyme by X. campestris pv. campestris. Subcellular fractionation studies indicated that the XpsN protein was tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis revealed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated that the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)6-tagged XpsD protein from the metal affinity chromatography column. All observations suggested that the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with various truncated XpsD proteins revealed that the C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved in complex formation between the two. PMID:10692359

  20. Mucin-like glycoprotein secretion is mediated by cyclic-AMP and protein kinase C signal transduction pathways in rat corneal epithelium.

    Science.gov (United States)

    Nakamura, M; Endo, K; Nakata, K

    1998-05-01

    Ocular surface mucin is secreted from both goblet cells in the conjunctival epithelium and corneal epithelial cells. To clarify its mechanism of secretion in corneal epithelial cells, a rat cornea organ culture system was used to evaluate the second messenger roles of cyclic-AMP (cAMP), cyclic-GMP (cGMP) and protein kinase C (PKC) in modulating mucin-like glycoprotein secretion. Rat cornea sections (3 mm diameter) were cultured in TC-199 medium, and radiolabeled with sodium sulfate for 18 hr. After washing, the corneas were treated with various second messenger modulating agents for 30 min. The culture media were reacted with Dolichos biflorus (DBA)-lectin, and mucin-like glycoprotein was isolated. Then the radioactivity of DBA-binding mucin-like glycoprotein was isolated. Then the radioactivity of DBA-binding mucin-like glycoprotein was measured. There was a time-dependent increase in mucin-like glycoprotein was measured. There was a time-dependent increase in mucin-like glycoprotein secretion, whereas after corneal epithelial debridement the secretion was markedly inhibited by 81%. Mucin-like glycoprotein secretion was stimulated in a dose-dependent manner following elevation of cAMP levels by exposure to either forskolin, dibutyryl cAMP or 3-isobutyl-1-methylxanthine. Concomitant exposure to the cAMP dependent protein kinase inhibitor, KT5720 completely inhibited their stimulatory effects. Neither exposure to dibutyryl cGMP nor nitroprusside affected mucin-like glycoprotein secretion. Stimulation by PKC, phorbol 12, 13-dibutyrate (PDBu) also increased mucin-like glycoprotein secretion in a dose-dependent fashion. The PKC inhibitor, calphostin C completely inhibited the stimulation by PDBu of mucine-like glycoprotein secretion. These results demonstrate that corneal epithelial cells secrete mucin-like glycoprotein, which is mediated by cAMP and PKC signal transduction pathways.

  1. Purification and Characteriztion of the Type III Secretion System Protein from Burkholderia mallei

    Science.gov (United States)

    2013-08-01

    Piscataway, NJ). The final construct included a 10× histidine (10× His) affinity tag at the carboxyl (C)-terminus and a cleavable (enterokinase...maltose-binding protein ( MBP ) fusion at the amino (N)-terminus. 2.2 Protein Expression The construct was transformed into E. coli T7 Express...expression, and all cystine residues were replaced with serine to minimize protein aggregation . Model building of the BsaS protein suggested that none of

  2. Impact of neutrophil-secreted myeloid related proteins 8 and 14 (MRP 8/14) on leishmaniasis progression.

    Science.gov (United States)

    Contreras, Irazú; Shio, Marina T; Cesaro, Annabelle; Tessier, Philippe A; Olivier, Martin

    2013-01-01

    The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.

  3. Proteomic analysis of pig (Sus scrofa olfactory soluble proteome reveals O-GlcNAcylation of secreted odorant-binding proteins

    Directory of Open Access Journals (Sweden)

    Patricia eNAGNAN-LE MEILLOUR

    2014-12-01

    Full Text Available The diversity of olfactory binding proteins (OBPs is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

  4. Over-expression of the cercosporin facilitator protein, CFP, in Cercospora kikuchii up-regulates production and secretion of cercosporin.

    Science.gov (United States)

    Upchurch, R G; Rose, M S; Eweida, M

    2001-10-16

    CFP (cercosporin facilitator protein), a light-regulated gene from the soybean fungal pathogen Cercospora kikuchii, encodes the putative major facilitator transporter of the fungal polyketide cercosporin. Gene disruption of CFP in C. kikuchii strain Gus-3 resulted in dramatically reduced cercosporin production and virulence, and increased sensitivity to the toxin. Two C. kikuchii transformant strains (10-1 and 10-11) that over-produce cercosporin were recovered from the complementation of CFP gene-disrupted strain Gus-3. Southern analysis revealed that these strains contained multiple genomic copies of CFP and over-expressed CFP transcript and protein. Although 10-1 and 10-11 produce and secrete significantly elevated levels of cercosporin, they exhibit wild-type resistance to cercosporin, and maintain a wild-type pattern of light-regulated toxin accumulation. Restoration of wild-type cercosporin resistance in 10-1 and 10-11 suggests that CFP does contribute substantially to cercosporin resistance via toxin secretion. The three-fold increase in toxin accumulation, predominantly associated with the mycelium fraction of these CFP multi-copy strains, suggests that CFP may also have a significant, but unknown, role in regulating toxin production.

  5. Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

    Science.gov (United States)

    Bumba, Ladislav; Masin, Jiri; Macek, Pavel; Wald, Tomas; Motlova, Lucia; Bibova, Ilona; Klimova, Nela; Bednarova, Lucie; Veverka, Vaclav; Kachala, Michael; Svergun, Dmitri I; Barinka, Cyril; Sebo, Peter

    2016-04-07

    Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.

  6. Neudesin as a unique secreted protein with multi-functional roles in neural functions, energy metabolism, and tumorigenesis

    Directory of Open Access Journals (Sweden)

    Hiroya eOhta

    2015-05-01

    Full Text Available Neudesin was originally identified as a secreted protein with neurotrophic activity, and, thereafter, was also termed neuron-derived neurotrophic factor (NENF or the candidate oncogene GIG47. Neudesin with a conserved cytochrome 5-like heme/steroid-binding domain activates intracellular signaling pathways possibly through the activation of G protein-coupled receptors. In the brain, hypothalamic Neudesin decreases food intake. Neudesin knockout mice also exhibit anxiety-like behavior, indicating its roles in the hippocampal anxiety circuitry. Neudesin is also expressed in various peripheral tissues. Neudesin knockout mice are strongly resistant to high-fat diet-induced obesity due to elevated systemic sympathetic activity, heat production, and adipocytic lipolysis. Neudesin, which is over-expressed or induced by DNA hypomethylation in multiple human cancers, also stimulates tumorigenesis. These findings indicate that Neudesin plays roles in neural functions, energy metabolism, and tumorigenesis and is expected to be a novel target for obesity and anti-cancer treatments.

  7. Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport

    Directory of Open Access Journals (Sweden)

    Tjeldhorn Lena

    2010-09-01

    Full Text Available Abstract Background Activated protein C (PC is a serine protease that regulates blood coagulation by inactivating coagulation factors Va and VIIIa. PC deficiency is an autosomally inherited disorder associated with a high risk of recurrent venous thrombosis. The aim of the study was to explore the mechanisms responsible for severe PC deficiency in a patient with the protein C A267T mutation by in-vitro expression studies. Results Huh7 and CHO-K1 cells were transiently transfected with expression vectors containing wild-type (WT PC and mutated PC (A267T PC cDNAs. PC mRNA levels were assessed by qRT-PCR and the PC protein levels were measured by ELISA. The mRNA levels of WT PC and A267T PC were similar, while the intracellular protein level of A267T PC was moderately decreased compared to WT PC. The secretion of A267T PC into the medium was severely impaired. No differences in molecular weights were observed between WT and A267T PC before and after treatment with endo-β-N-acetylglucosaminidase. Proteasomal and lysosomal degradations were examined using lactacystin and bafilomycin, respectively, and revealed that A267T PC was slightly more susceptible for proteasomal degradation than WT PC. Intracellular co-localization analysis indicated that A267T PC was mainly located in the endoplasmic reticulum (ER, whereas WT PC was observed in both ER and Golgi. Conclusions In contrast to what has been reported for other PC mutants, intracellular degradation of A267T PC was not the main/dominant mechanism underlying the reduced intracellular and secretion levels of PC. Our results indicate that the A267T mutation most likely caused misfolding of PC, which might lead to increased retention of the mutated PC in ER.

  8. Identification of SopE2, a Salmonella Secreted Protein Which Is Highly Homologous to SopE and Involved in Bacterial Invasion of Epithelial Cells

    OpenAIRE

    2000-01-01

    Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying Salmonella pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the sopE2 gene which is present and conserved in pathogenic strains of Salmonella. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present i...

  9. Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

    Science.gov (United States)

    Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

    2011-04-01

    Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

  10. Lipopolysaccharide binding protein and serum amyloid A secretion by human intestinal epithelial cells during the acute phase response.

    Science.gov (United States)

    Vreugdenhil, A C; Dentener, M A; Snoek, A M; Greve, J W; Buurman, W A

    1999-09-01

    The acute phase proteins LPS binding protein (LBP) and serum amyloid A (SAA) are produced by the liver and are present in the circulation. Both proteins have been shown to participate in the immune response to endotoxins. The intestinal mucosa forms a large surface that is continuously exposed to these microbial products. By secretion of antimicrobial and immunomodulating agents, the intestinal epithelium contributes to the defense against bacteria and their products. The aim of this study was to explore the influence of the inflammatory mediators TNF-alpha, IL-6, and IL-1beta on the release of LBP and SAA by intestinal epithelial cells (IEC). In addition, the induction of LBP and SAA release by cell lines of intestinal epithelial cells and hepatic cells was compared. The data obtained show that in addition to liver cells, IEC also expressed LBP mRNA and released bioactive LBP and SAA upon stimulation. Regulation of LBP and SAA release by IEC and hepatocytes was typical for class 1 acute phase proteins, although differences in regulation between the cell types were observed. Endotoxin did not induce LBP and SAA release. Glucocorticoids were demonstrated to strongly enhance the cytokine-induced release of LBP and SAA by IEC, corresponding to hepatocytes. The data from this study, which imply that human IEC can produce LBP and SAA, suggest a role for these proteins in the local defense mechanism of the gut to endotoxin. Furthermore, the results demonstrate that tissues other than the liver are involved in the acute phase response.

  11. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG.

    Directory of Open Access Journals (Sweden)

    Ingmar J J Claes

    Full Text Available Lactobacillus rhamnosus GG (LGG produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75 and Msp2 (LGG_00031 or p40, which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

  12. Preparation of Monoclonal Antibodies Against SpiC Protein Secreted by T3SS-2 of Salmonella spp.

    Science.gov (United States)

    Geng, Shizhong; Qian, Shanshan; Pan, Zhiming; Sun, Lin; Chen, Xiang; Jiao, Xinan

    2015-12-01

    SpiC protein, a member of Salmonella spp. type III secretion system (T3SS)-2, is necessary for the survival of Salmonella within macrophages, and it plays a vital role in Salmonella pathogenesis. To develop and test monoclonal antibodies (MAbs) against SpiC protein, two recombinant proteins, rHis-SpiC and rGST-SpiC, were expressed in vitro in the prokaryotic expression vectors pET-30(a) and pGEX-6p-1, respectively, and rHis-SpiC protein used to immunize mice. Hybridomas were generated from the splenocytes of these mice and the monoclonal antibodies produced by these cells were assessed using indirect enzyme-linked immunosorbent assay (ELISA) with rGST-SpiC as the coating antigen. An immunoblotting analysis indicated that all seven of the MAbs developed in this study could specifically recognize the SpiC protein. These MAbs will be very useful in the study of SpiC function and for use in the immunodiagnosis of Salmonella infection.

  13. Conjugated linoleic acid reduces hepatic steatosis and restores liver triacylglycerol secretion and the fatty acid profile during protein repletion in rats.

    Science.gov (United States)

    Andreoli, María F; Illesca, Paola G; González, Marcela A; Bernal, Claudio A

    2010-11-01

    Protein depletion is associated with hepatic steatosis and decreased circulating triacylglycerol (TAG). Since conjugated linoleic acid (CLA) increases lean body mass, protects against muscle catabolism, and modulates lipid metabolism, the aim of this work was to investigate the effects of CLA with two different amounts of dietary fat on the regulation of plasma and hepatic TAG concentration, and its possible connections with changes in fatty acid (FA) profile in plasma, liver and adipose tissue and hepatic oxidative status during protein repletion. Rats were fed a low protein diet (14 days) and then a protein repletion diet (30 days), supplemented or not with CLA, containing 7% (w/w) or 20% (w/w) of fat. Hepatic TAG secretion and removal by muscle and adipose tissue lipoprotein lipase, FA profile and liver oxidative status were evaluated. Protein depletion affected hepatic TAG secretion and peripheral removal, decreasing plasma and increasing liver TAG concentration, whereas protein repletion with CLA improved these abnormalities independently of the amount of dietary fat by increasing hepatic TAG secretion. This prevention in the absence of CLA was not observed. CLA was incorporated in plasma and tissues (adipose > liver > plasma, and c9,t11-CLA > t10,c12-CLA), accompanied by alterations in FA composition, mainly in adipose tissue. The hepatic oxidative stress was overcome by protein repletion. CLA had a beneficial impact on TAG metabolism in protein repleted animals, preventing hepatic steatosis through higher hepatic TAG secretion.

  14. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    DEFF Research Database (Denmark)

    Li, Yiping; Kang, H.N.; Babiuk, L.A.

    2006-01-01

    AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models. METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without...

  15. Epidermal secretions of terrestrial flatworms and slugs: Lehmannia valentiana mucus contains matrilin-like proteins.

    Science.gov (United States)

    Li, Dongmei; Graham, Lloyd D

    2007-11-01

    We describe the epidermal mucus of two types of terrestrial invertebrates: free-living flatworms (Tricladida: Terricola), and the slug Lehmannia valentiana (Gastropoda: Pulmonata). Both exhibited similar dry shear strengths (1.4-1.7 MPa). In denaturing gel electrophoresis, the protein fraction of flatworm mucus migrated mainly as a broad band (200-300 kDa). Slug mucus had a higher protein content than flatworm mucus but it contained more carbohydrate than protein, mainly as large heparan sulfate-like glycosaminoglycans. Proteins and glycosaminoglycans were both essential for the mechanical integrity of the slug hydrogel. The protein fraction of slug mucus contained approximately 12 larger proteins (30-300 kDa) and approximately 6 smaller ones (10-28 kDa). Complete cDNA clones were obtained for the slug mucus 40 kDa protein (Sm40; Genbank accession EF634345) and 85 kDa protein (Sm85; Genbank accession EF634346). Both proteins contain EGF-like repeats and von Willebrand A-domains, and therefore resemble vertebrate matrilins. Many of the larger slug mucus proteins appear to contain A-domains, and these may play a role in the unusual rheological properties of gastropod mucus.

  16. The 2.5 Å structure of the enterococcus conjugation protein TraM resembles VirB8 type IV secretion proteins.

    Science.gov (United States)

    Goessweiner-Mohr, Nikolaus; Grumet, Lukas; Arends, Karsten; Pavkov-Keller, Tea; Gruber, Christian C; Gruber, Karl; Birner-Gruenberger, Ruth; Kropec-Huebner, Andrea; Huebner, Johannes; Grohmann, Elisabeth; Keller, Walter

    2013-01-18

    Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G-) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G-) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G- bacteria, we propose a new classification scheme of VirB8-like proteins.

  17. Real-time quantification of proteins secreted by artificial connective tissue made from uni- or multidirectional collagen I scaffolds and oral mucosa fibroblasts.

    Science.gov (United States)

    Bustos, Rosa Helena; Suesca, Edward; Millán, Diana; González, José Manuel; Fontanilla, Marta R

    2014-03-04

    Previously, we found that oral autologous artificial connective tissue (AACT) had a different protein secretion profile to that of clot-embedded AACT. Other oral mucosa substitutes, having different cell types and scaffolds, had dissimilar secretion profiles of proteins (including that for AACT) that influence healing outcome; thus, to ascertain the profiles of factors secreted by artificial tissue and whether they are influenced by their microstructure might help in understanding their bioactivity. An important component of tissue microstructure is the fiber orientation of the scaffold used for manufacturing it. This work developed a surface plasmon resonance (SPR) methodology to quantify factors secreted by oral artificial connective tissue (ACT) in culture medium, and a method to manufacture unidirectional laminar collagen I scaffolds. The SPR methodology was used for assessing differences in the protein secretion profile of ACT made with collagen scaffolds having different fiber orientation (unidirectional vs multidirectional). Oral fibroblasts seeded onto unidirectional scaffolds increased the secretion of six factors involved in modulating healing compared to those seeded onto multidirectional scaffolds. Histological analysis of uni- and multidirectional ACT showed that cells differ in their alignment and morphology. This SPR-methodology led to nanoscale detection of paracrine factors and might be useful to study biomarkers of three-dimensional cell growth, cell differentiation, and wound-healing progression.

  18. Perivascular adipose tissue-secreted angiopoietin-like protein 2 (Angptl2) accelerates neointimal hyperplasia after endovascular injury.

    Science.gov (United States)

    Tian, Zhe; Miyata, Keishi; Tazume, Hirokazu; Sakaguchi, Hisashi; Kadomatsu, Tsuyoshi; Horio, Eiji; Takahashi, Otowa; Komohara, Yoshihiro; Araki, Kimi; Hirata, Yoichiro; Tabata, Minoru; Takanashi, Shuichiro; Takeya, Motohiro; Hao, Hiroyuki; Shimabukuro, Michio; Sata, Masataka; Kawasuji, Michio; Oike, Yuichi

    2013-04-01

    Much attention is currently focused on the role of perivascular adipose tissue in development of cardiovascular disease (CVD). Some researchers view it as promoting CVD through secretion of cytokines and growth factors called adipokines, while recent reports reveal that perivascular adipose tissue can exert a protective effect on CVD development. Furthermore, adiponectin, an anti-inflammatory adipokine, reportedly suppresses neointimal hyperplasia after endovascular injury, whereas such vascular remodeling is enhanced by pro-inflammatory adipokines secreted by perivascular adipose, such as tumor necrosis factor-α (TNF-α). These findings suggest that extent of vascular remodeling, a pathological process associated with CVD development, depends on the balance between pro- and anti-inflammatory adipokines secreted from perivascular adipose tissue. We previously demonstrated that angiopoietin-like protein 2 (Angptl2), a pro-inflammatory factor secreted by adipose tissue, promotes adipose tissue inflammation and subsequent systemic insulin resistance in obesity. Here, we examined whether Angptl2 secreted by perivascular adipose tissue contributes to vascular remodeling after endovascular injury in studies of transgenic mice expressing Angptl2 in adipose tissue (aP2-Angptl2 transgenic mice) and Angptl2 knockout mice (Angptl2(-/-) mice). To assess the role of Angptl2 secreted by perivascular adipose tissue on vascular remodeling after endovascular injury, we performed adipose tissue transplantation experiments using these mice. Wild-type mice with perivascular adipose tissue derived from aP2-Angptl2 mice exhibited accelerated neointimal hyperplasia after endovascular injury compared to wild-type mice transplanted with wild-type tissue. Conversely, vascular inflammation and neointimal hyperplasia after endovascular injury were significantly attenuated in wild-type mice transplanted with Angptl2(-/-) mouse-derived perivascular adipose tissue compared to wild-type mice

  19. Production, characterization, and immunogenicity of a secreted form of Plasmodium falciparum merozoite surface protein 4 produced in Bacillus subtilis.

    Science.gov (United States)

    Chittibabu, G; Ma, Charles; Netter, Hans J; Noronha, Santosh B; Coppel, Ross L

    2014-04-01

    Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.

  20. A regulator of G Protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH-stimulated luteinizing hormone (LH secretion

    Directory of Open Access Journals (Sweden)

    Musgrove Lois C

    2001-11-01

    Full Text Available Abstract Background Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs. These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells. Results A truncated version of RGS3 (RGS3T = RGS3 314–519 inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqα than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqα protein. Conclusions RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqα protein function. A version of RGS3 that is amino

  1. Identification of the essential EPE1 gene involved in retention of secreted proteins on the cell surface of Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Alexieva, K I; Klis, F; Wedler, H; Wambutt, R; Venkov, P

    1999-09-01

    Saccharomyces cerevisiae yeast cells secrete extracellularly low amounts of a few proteins. The reasons for retardation of secreted proteins on the cell surface remain obscure. We describe here a mutant able to export enhanced amount of proteins. Classical genetic methods, nucleic acids manipulations and cloning procedures were used to isolate and characterize the mutant and to clone and sequence the corresponding wild type gene. The isolated Saccharomyces cerevisiae mutant MW11, is temperature sensitive and exports on average twenty-fold more proteins at 37 degrees C than parental wild type strain (80 micrograms of proteins/1 x 10(8) mutant cells, SEM +/- 5, n22; versus 3 micrograms of proteins/1 x 10(8) parental cells, SEM +/- 1, n22). Protein overexport in the mutant requires a functional SEC1 pathway and is independent of cell lysis. Cloning and sequencing of the corresponding wild type gene identified an open reading frame of 786 bp coding for a hydrophilic protein with predicted molecular mass of 30 kDa and cytosolic localization. The newly identified gene, designated EPE1, is an essential gene. Its DNA and amino acids sequence showed no homology with other yeast genes and proteins. It is concluded that the function of unknown yet genes, such as EPE1 is needed for retention of secreted proteins on the surface of Saccharomyces cerevisiae cells.

  2. Identification of Antithrombin-Modulating Genes. Role of LARGE, a Gene Encoding a Bifunctional Glycosyltransferase, in the Secretion of Proteins?

    Science.gov (United States)

    de la Morena-Barrio, María Eugenia; Buil, Alfonso; Antón, Ana Isabel; Martínez-Martínez, Irene; Miñano, Antonia; Gutiérrez-Gallego, Ricardo; Navarro-Fernández, José; Aguila, Sonia; Souto, Juan Carlos; Vicente, Vicente; Soria, José Manuel; Corral, Javier

    2013-01-01

    The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families). Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02). Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteins. PMID:23705025

  3. Inhibition effect of tea tree oil on Listeria monocytogenes growth and exotoxin proteins listeriolysin O and p60 secretion.

    Science.gov (United States)

    Liu, Z; Meng, R; Zhao, X; Shi, C; Zhang, X; Zhang, Y; Guo, N

    2016-12-01

    Listeria monocytogenes (L. monocytogenes) is a Gram-positive bacterium that causes infections in humans. In this study, the effects of tea tree oil (TTO) at subinhibitory concentrations on L. monocytogenes growth and two important exotoxin proteins secreted by L. monocytogenes were researched. Treatment with half of minimal inhibitory concentration of TTO demonstrated very little or no reduction in numbers of viable ATCC 19115 cells. Listeriolysin O (LLO) and p60, were investigated. A listeriolysin assay was used to investigate the hemolytic activities of L. monocytogenes exposed to TTO, and the secretion of LLO and p60 was detected by immunoblot analysis. Additionally, real-time RT-PCR was used to analyse the influence of TTO on the transcription of LLO and p60 encoded genes hly and iap respectively. According to our experimental results, we propose that TTO could be used as a promising natural compound against L. monocytogenes and its virulence factors.

  4. Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

    Directory of Open Access Journals (Sweden)

    Boutin Jean A

    2010-10-01

    Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

  5. Hepatitis B Virus X Protein Up-regulates TNF-α and IL-1β Secretion of Macrophages

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus (HBV) X protein (HBx) on hepatocellular carcinoma. Methods: Reconstructed plasmid pcDNA3.1(+)-HBx was transfected into THP-1 macrophages. Expression of HBx was assayed in macrophages lysate by Western-blotting, and TNF-α and IL-1β contents were detected respectively by ELISA. All the data were analyzed by SPSS13.0. Results: In THP-1 macrophages, the pcDNA3.1(+)-HBx plasmid expressed HBx with a molecular weight of about 17 KDa demonstrated by Western-blotting. The secreted TNF-α and IL-1β from macrophages were determined by ELISA, the results from analysis of all groups showed as following: control group was different from LPS group and pcDNA3.1(+) group (P<0.01), and so was pcDNA3.1(+)-HBx group; but there was no obvious difference between pcDNA3.1(+) group and LPS group (P>0.05), all of which indicated that transient overexpression of HBx enhanced LPS-induced production of TNF-α and IL-1β by macrophages.Conclusion: Transient overexpression of HBx up-regulates LPS-induced TNF-α and IL-1β secretion of macrophages.

  6. Identification of antithrombin-modulating genes. Role of LARGE, a gene encoding a bifunctional glycosyltransferase, in the secretion of proteins?

    Directory of Open Access Journals (Sweden)

    María Eugenia de la Morena-Barrio

    Full Text Available The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families. Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02. Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteins.

  7. Stimulation of a Gs-like G protein in the osteoclast inhibits bone resorption but enhances tartrate-resistant acid phosphatase secretion.

    Science.gov (United States)

    Moonga, B S; Pazianas, M; Alam, A S; Shankar, V S; Huang, C L; Zaidi, M

    1993-01-29

    Previous studies have demonstrated that G-protein agonists induce quiescence (Q effect) or retraction (R effect) in isolated osteoclasts. We now report the functional effects of such agonists on osteoclastic bone resorption and enzyme release. Exposure of osteoclasts to tetrafluoro-aluminate anions (AlF4-), a universal G protein stimulator, resulted in a marked concentration-dependent inhibition of bone resorption. This was associated with a dramatic increase in the secretion of the osteoclast-specific enzyme, tartrate-resistant acid phosphatase (TRAP). Cholera toxin, a Gs stimulator and a selective Q effect agonist, similarly abolished bone resorption and enhanced TRAP secretion. In contrast, pertussis toxin, a Gi inhibitor and a selective R effect agonist, inhibited bone resorption significantly, but slightly reduced enzyme release. The results suggest an involvement of a Gs-like G protein in TRAP secretion from the osteoclast, possibly through a cyclic AMP-dependent mechanism.

  8. Proteomic screening of glucose-responsive and glucose non-reponsive MIN-6 beta cells reveals differential expression of protein involved in protein folding, secretion and oxidative stress

    DEFF Research Database (Denmark)

    Dowling, P.; O´Driscoll, L.; O´Sullivan, F.;

    2006-01-01

    The glucose-sensitive insulin-secretion (GSIS) phenotype is relatively unstable in long-term culture of beta cells. The purpose of this study was to investigate relative changes in the proteome between glucose-responsive (low passage) and glucose non-responsive (high passage) murine MIN-6...... pancreatic beta cells. The 2D-DIGE and subsequent DeCyder analysis detected 3351 protein spots in the pH range of 4-7. Comparing MIN-6(H) to MIN-6(L) and using a threshold of 1.2-fold, the number of proteins with a decrease in expression level was 152 (4.5%), similar was 3140 (93.7%) and increased 59 (1.......8%). From the differentially expressed proteins identified in this study, groups of proteins associated with the endoplasmic reticulum (ER) and proteins involved in oxidative stress were found to be significantly decreased in the high-passage (H passage) cells. These proteins included endoplasmic reticulum...

  9. The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology.

    Science.gov (United States)

    Guerriero, Christopher J; Brodsky, Jeffrey L

    2012-04-01

    Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding "problem," as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates.

  10. Effect of dietary protein quality and feeding level on milk secretion and mammary protein synthesis in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Sampson, D.A.; Jansen, G.R.

    1985-04-01

    Protein synthesis was studied in mammary tissue of rats fed diets deficient in protein quality and/or restricted in food intake throughout gestation and lactation. Diets containing 25% wheat gluten (WG), wheat gluten plus lysine and threonine (WGLT), or casein (C) were pair-fed from conception until day 15 of lactation at 100% or 85% of WG ad libitum consumption (PF100 and PF85, respectively). A seventh group was fed C ad libitum. Rates of protein synthesis were measured in vivo at day 15 of lactation from incorporation of (3-/sup 3/H)phenylalanine. At both PF100 and PF85, fractional and absolute rates of mammary gland protein synthesis were two- to three-fold higher in rats fed C than in those fed WG. Pup weights showed similar treatment effects. Both mammary protein synthesis rates and pup weights were significantly higher in rats fed C at PF85 than rats fed WG ad libitum. Food restriction from PF100 to PF85 depressed pup weights and mammary protein synthesis rates in rats fed WGLT, but had no effect in rats fed WG. These results demonstrate that when food intake is restricted, improvement of protein quality of the maternal diet increases milk output in the rat in association with increased rates of mammary protein synthesis.

  11. Contributions of the pre- and pro-regions of a Staphylococcus hyicus lipase to secretion of a heterologous protein by Bacillus subtilis.

    Science.gov (United States)

    Kouwen, Thijs R H M; Nielsen, Allan K; Denham, Emma L; Dubois, Jean-Yves F; Dorenbos, Ronald; Rasmussen, Michael D; Quax, Wim J; Freudl, Roland; van Dijl, Jan Maarten

    2010-02-01

    Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre(lip)) and pro (pro(lip)) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the pro(lip)-peptide, in combination with the lipase signal peptide (pre(lip)), contributes significantly to the efficient secretion of PhoA by B. subtilis and that pre(lip) directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.

  12. Comparative genomics of Fusarium oxysporum f. sp. melonis reveals the secreted protein recognized by the Fom-2 resistance gene in melon.

    Science.gov (United States)

    Schmidt, Sarah Maria; Lukasiewicz, Joanna; Farrer, Rhys; van Dam, Peter; Bertoldo, Chiara; Rep, Martijn

    2016-01-01

    Development of resistant crops is the most effective way to control plant diseases to safeguard food and feed production. Disease resistance is commonly based on resistance genes, which generally mediate the recognition of small proteins secreted by invading pathogens. These proteins secreted by pathogens are called 'avirulence' proteins. Their identification is important for being able to assess the usefulness and durability of resistance genes in agricultural settings. We have used genome sequencing of a set of strains of the melon wilt fungus Fusarium oxysporum f. sp. melonis (Fom), bioinformatics-based genome comparison and genetic transformation of the fungus to identify AVRFOM2, the gene that encodes the avirulence protein recognized by the melon Fom-2 gene. Both an unbiased and a candidate gene approach identified a single candidate for the AVRFOM2 gene. Genetic complementation of AVRFOM2 in three different race 2 isolates resulted in resistance of Fom-2-harbouring melon cultivars. AvrFom2 is a small, secreted protein with two cysteine residues and weak similarity to secreted proteins of other fungi. The identification of AVRFOM2 will not only be helpful to select melon cultivars to avoid melon Fusarium wilt, but also to monitor how quickly a Fom population can adapt to deployment of Fom-2-containing cultivars in the field.

  13. The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

    Directory of Open Access Journals (Sweden)

    Ofir Bahar

    2014-01-01

    Full Text Available Pattern recognition receptors (PRRs play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo. In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

  14. Disruption of Lipid Raft Function Increases Expression and Secretion of Monocyte Chemoattractant Protein-1 in 3T3-L1 Adipocytes.

    Science.gov (United States)

    Lu, Juu-Chin; Chiang, Yu-Ting; Lin, Yu-Chun; Chang, Yu-Tzu; Lu, Chia-Yun; Chen, Tzu-Yu; Yeh, Chia-Shan

    2016-01-01

    The adipocyte is unique in its capacity to store lipids. In addition to triglycerides, the adipocyte stores a significant amount of cholesterol. Moreover, obese adipocytes are characterized by a redistribution of cholesterol with depleted cholesterol in the plasma membrane, suggesting that cholesterol perturbation may play a role in adipocyte dysfunction. We used methyl-β-cyclodextrin (MβCD), a molecule with high affinity for cholesterol, to rapidly deplete cholesterol level in differentiated 3T3-L1 adipocytes. We tested whether this perturbation altered adipocyte secretion of monocyte chemoattractant protein-1 (MCP-1), a chemokine that is elevated in obesity and is linked to obesity-associated chronic diseases. Depletion of cholesterol by MβCD increased MCP-1 secretion as well as the mRNA and protein levels, suggesting perturbation at biosynthesis and secretion. Pharmacological inhibition revealed that NF-κB, but not MEK, p38 and JNK, was involved in MβCD-stimulated MCP-1 biosynthesis and secretion in adipocytes. Finally, another cholesterol-binding drug, filipin, also induced MCP-1 secretion without altering membrane cholesterol level. Interestingly, both MβCD and filipin disturbed the integrity of lipid rafts, the membrane microdomains enriched in cholesterol. Thus, the depletion of membrane cholesterol in obese adipocytes may result in dysfunction of lipid rafts, leading to the elevation of proinflammatory signaling and MCP-1 secretion in adipocytes.

  15. Clinical evaluation of MPT-64 and MPT-59, two proteins secreted from Mycobacterium tuberculosis, for skin test reagents

    DEFF Research Database (Denmark)

    Wilcke, J T; Jensen, B N; Ravn, P;

    1996-01-01

    SETTING: Department of Pulmonary Medicine P, Bispebjerg Hospital, Copenhagen, Denmark. OBJECTIVE: To study the ability of two proteins secreted from Mycobacterium tuberculosis, MPT-64 and MPT-59 to induce delayed type hypersensitivity (DTH) reactions following intradermal administration. DESIGN......: In a small scale clinical investigation, skin reactions to these antigens were compared to reactions to tuberculin PPD RT23 in 1) patients with active tuberculosis, 2) BCG vaccinated healthy subjects with close contact with tuberculous patients, and 3) BCG vaccinated healthy subjects without contact...... with tuberculous patients. Tests for in vitro reactivity to these antigens were carried out in similar groups. RESULTS: All subjects gave positive reaction to tuberculin PPD RT23, whereas approximately half of the subjects in each of the three groups reacted to MPT-59. Two subjects (one patient with tuberculosis...

  16. Secreted Protein Acidic and Rich in Cysteine Modulates Molecular Arterial Homeostasis of Human Arterial Smooth Muscle Cells In Vitro.

    Science.gov (United States)

    Ye, Geng-Fan; Zhu, Shao-Wei; Zhu, Shu-Gan; Li, Feng; Wang, Yun-Yan

    2016-12-01

    Secreted protein acidic and rich in cysteine (SPARC) is widely expressed in the vascular smooth muscle cells (VSMCs) of human intracranial aneurysms (IAs), but the effect and underlying mechanism of SPARC on VSMCs during the formation and progression of IAs needs to be probed. Human umbilical arterial smooth muscle cells (HUASMCs) were treated with a gradient concentrations of SPARC in vitro for different time. Cell counting kit-8 (CCK-8) assay, cell cycle, and cell apoptosis were used to investigate the effect of SPARC on HUASMCs. After exposure to 2 and 4 μg/ml SPARC, cell viability were 89.3 ± 2.00 %, and 87.57 ± 2.17 % (P IAs.

  17. The function of breast cancer resistance protein in epithelial barriers, stem cells and milk secretion of drugs and xenotoxins.

    Science.gov (United States)

    van Herwaarden, Antonius E; Schinkel, Alfred H

    2006-01-01

    The breast cancer resistance protein [BCRP (also known as ABCG2)] belongs to the ATP binding cassette (ABC) family of transmembrane drug transporters. BCRP has a broad substrate specificity and actively extrudes a wide variety of drugs, carcinogens and dietary toxins from cells. Situated in the apical plasma membrane of epithelial cells of the small and large intestine and renal proximal tubules and in the bile canalicular membrane of hepatocytes, BCRP decreases the oral availability and systemic exposure of its substrates. In several blood-tissue barriers BCRP reduces tissue penetration of its substrates and it protects haematopoietic stem cells from cytotoxic substrates. Moreover, BCRP is expressed in mammary gland alveolar epithelial cells during pregnancy and lactation, where it actively secretes a variety of drugs, toxins and carcinogens into milk. In apparent contradiction with the detoxifying role of BCRP in mothers, this contamination of milk exposes suckling infants and dairy consumers to xenotoxins. BCRP thus affects many important aspects of pharmacology and toxicology.

  18. Proteins involved in uptake, intracellular transport and basolateral secretion of fat-soluble vitamins and carotenoids by mammalian enterocytes.

    Science.gov (United States)

    Reboul, Emmanuelle; Borel, Patrick

    2011-10-01

    Our understanding of the molecular mechanisms responsible for fat-soluble vitamin uptake and transport at the intestinal level has advanced considerably over the past decade. On one hand, it has long been considered that vitamin D and E as well as β-carotene (the main provitamin A carotenoid in human diet) were absorbed by a passive diffusion process, although this could not explain the broad inter-individual variability in the absorption efficiency of these molecules. On the other hand, it was assumed that preformed vitamin A (retinol) and vitamin K1 (phylloquinone) absorption occurred via energy-dependent processes, but the transporters involved have not yet been identified. The recent discovery of intestinal proteins able to facilitate vitamin E and carotenoid uptake and secretion by the enterocyte has spurred renewed interest in studying the fundamental mechanisms involved in the absorption of these micronutrients. The proteins identified so far are cholesterol transporters such as SR-BI (scavenger receptor class B type I), CD36 (cluster determinant 36), NPC1L1 (Niemann-Pick C1-like 1) or ABCA1 (ATP-Binding Cassette A1) displaying a broad substrate specificity, but it is likely that other membrane proteins are also involved. After overviewing the metabolism of fat-soluble vitamins and carotenoids in the human upper gastrointestinal lumen, we will focus on the putative or identified proteins participating in the intestinal uptake, intracellular transport and basolateral secretion of these fat-soluble vitamins and carotenoids, and outline the uncertainties that need to be explored in the future. Identifying the proteins involved in intestinal uptake and transport of fat-soluble vitamins and carotenoids across the enterocyte is of great importance, especially as some of them are already targets for the development of drugs able to slow cholesterol absorption. Indeed, these drugs may also interfere with lipid vitamin uptake. A better understanding of the molecular

  19. Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding

    Science.gov (United States)

    Pinto, Antônio F. M.; Moresco, James; Yates, John R.; da Silva Vaz, Itabajara; Mulenga, Albert

    2016-01-01

    Ixodes scapularis is the most medically important tick species and transmits five of the 14 reportable human tick borne disease (TBD) agents in the USA. This study describes LC-MS/MS identification of 582 tick- and 83 rabbit proteins in saliva of I. scapularis ticks that fed for 24, 48, 72, 96, and 120 h, as well as engorged but not detached (BD), and spontaneously detached (SD). The 582 tick proteins include proteases (5.7%), protease inhibitors (7.4%), unknown function proteins (22%), immunity/antimicrobial (2.6%), lipocalin (3.1%), heme/iron binding (2.6%), extracellular matrix/ cell adhesion (2.2%), oxidant metabolism/ detoxification (6%), transporter/ receptor related (3.2%), cytoskeletal (5.5%), and housekeeping-like (39.7%). Notable observations include: (i) tick saliva proteins of unknown function accounting for >33% of total protein content, (ii) 79% of proteases are metalloproteases, (iii) 13% (76/582) of proteins in this study were found in saliva of other tick species and, (iv) ticks apparently selectively inject functionally similar but unique proteins every 24 h, which we speculate is the tick's antigenic variation equivalent strategy to protect important tick feeding functions from host immune system. The host immune responses to proteins present in 24 h I. scapularis saliva will not be effective at later feeding stages. Rabbit proteins identified in our study suggest the tick's strategic use of host proteins to modulate the feeding site. Notably fibrinogen, which is central to blood clotting and wound healing, was detected in high abundance in BD and SD saliva, when the tick is preparing to terminate feeding and detach from the host. A remarkable tick adaptation is that the feeding lesion is completely healed when the tick detaches from the host. Does the tick concentrate fibrinogen at the feeding site to aide in promoting healing of the feeding lesion? Overall, these data provide broad insight into molecular mechanisms regulating different tick

  20. Ixodes scapularis Tick Saliva Proteins Sequentially Secreted Every 24 h during Blood Feeding.

    Directory of Open Access Journals (Sweden)

    Tae Kwon Kim

    2016-01-01

    Full Text Available Ixodes scapularis is the most medically important tick species and transmits five of the 14 reportable human tick borne disease (TBD agents in the USA. This study describes LC-MS/MS identification of 582 tick- and 83 rabbit proteins in saliva of I. scapularis ticks that fed for 24, 48, 72, 96, and 120 h, as well as engorged but not detached (BD, and spontaneously detached (SD. The 582 tick proteins include proteases (5.7%, protease inhibitors (7.4%, unknown function proteins (22%, immunity/antimicrobial (2.6%, lipocalin (3.1%, heme/iron binding (2.6%, extracellular matrix/ cell adhesion (2.2%, oxidant metabolism/ detoxification (6%, transporter/ receptor related (3.2%, cytoskeletal (5.5%, and housekeeping-like (39.7%. Notable observations include: (i tick saliva proteins of unknown function accounting for >33% of total protein content, (ii 79% of proteases are metalloproteases, (iii 13% (76/582 of proteins in this study were found in saliva of other tick species and, (iv ticks apparently selectively inject functionally similar but unique proteins every 24 h, which we speculate is the tick's antigenic variation equivalent strategy to protect important tick feeding functions from host immune system. The host immune responses to proteins present in 24 h I. scapularis saliva will not be effective at later feeding stages. Rabbit proteins identified in our study suggest the tick's strategic use of host proteins to modulate the feeding site. Notably fibrinogen, which is central to blood clotting and wound healing, was detected in high abundance in BD and SD saliva, when the tick is preparing to terminate feeding and detach from the host. A remarkable tick adaptation is that the feeding lesion is completely healed when the tick detaches from the host. Does the tick concentrate fibrinogen at the feeding site to aide in promoting healing of the feeding lesion? Overall, these data provide broad insight into molecular mechanisms regulating different tick

  1. Nuclear localization and secretion competence is conserved amongst henipavirus matrix proteins.

    Science.gov (United States)

    McLinton, Elisabeth C; Wagstaff, Kylie M; Lee, Alexander; Moseley, Gregory W; Marsh, Glenn A; Wang, Lin-Fa; Jans, David A; Lieu, Kim G; Netter, Hans

    2017-01-05

    Viruses of the genus Henipavirus of the family Paramyxoviridae are zoonotic pathogens, which have emerged in South East Asia, Australia and Africa. Nipah virus (NiV) and Hendra virus (HeV) are highly virulent pathogens transmitted from bats to animals and humans, whilst the henipavirus Cedar virus (CedV) seems to be non-pathogenic in infection studies. The full replication cycle of the Paramyxoviridae occurs in the host cell's cytoplasm where viral assembly is orchestrated by the matrix (M) protein. Unexpectedly, the NiV-M protein traffics through the nucleus as an essential step to engage the plasma membrane in preparation for viral budding/release. Comparative studies were performed to assess whether M protein nuclear localization is a common feature of the henipaviruses including the recently sequenced (although not yet isolated) Ghanaian bat henipavirus (Kumasi virus, GH-M74a virus, KV) and Mojiang virus (MojV). Live-cell confocal microscopy revealed that nuclear translocation of GFP-fused M protein is conserved between henipaviruses in both human and bat-derived cell lines. However, the efficiency of M protein nuclear localization and virus-like particle budding competency varied. Additionally, CedV-, KV- and MojV-M proteins were mutated in a bipartite nuclear localization signal indicating that a key lysine residue is essential for nuclear import, export and for the induction of budding events as previously reported for NiV-M. The results of this study suggest that the M proteins of henipaviruses may utilize a similar nucleocytoplasmic trafficking pathway as an essential step during viral replication in both humans and bats.

  2. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins.

    Science.gov (United States)

    Lone, Abdul G; Atci, Erhan; Renslow, Ryan; Beyenal, Haluk; Noh, Susan; Fransson, Boel; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R; Call, Douglas R

    2015-08-01

    A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MS(E)). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2- to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

  3. Isolation and molecular cloning of a secreted immunosuppressant protein from Dermacentor andersoni salivary gland.

    Science.gov (United States)

    Bergman, D K; Palmer, M J; Caimano, M J; Radolf, J D; Wikel, S K

    2000-06-01

    A 36-kDa immunosuppressant protein (Da-p36) was isolated from salivary glands of feeding female ixodid ticks Dermacentor andersoni, using its affinity for UltraLink Biosupport Medium (Pierce, Rockford, Illinois)/protein complexes. Using a nested set of forward degenerate oligonucleotide primers corresponding to Da-p36 N-terminal amino acids, a cDNA encoding the immunosuppressant protein was isolated by 3' rapid amplification of cDNA ends. The resulting 772-base pair cDNA encodes a novel protein with predicted molecular weight of 24.9 kDa. Sequence analysis revealed the presence of 5 potential glycosylation sites and 1 myristylation site. Immunoblot analyses showed native Da-p36 is present in salivary glands and saliva from both male and female D. andersoni but not in salivary glands or saliva from Amblyomma americanum or Ixodes scapularis. Reverse transcription polymerase chain reaction and immunoblot analyses showed that Da-p36 expression is temporally regulated in salivary glands with maximum mRNA levels preceding maximum Da-p36 accumulation that occurred at day 6 of feeding. The levels of Da-p36 mRNA and protein were greatly reduced in salivary glands from near-replete females removed from sheep after 8 days of feeding. These data are consistent with a role of Da-p36 in immunosuppression during feeding.

  4. Pathogenic Acanthamoeba spp secrete a mannose-induced cytolytic protein that correlates with the ability to cause disease.

    Science.gov (United States)

    Hurt, Michael; Neelam, Sudha; Niederkorn, Jerry; Alizadeh, Hassan

    2003-11-01

    The pathogenesis of Acanthamoeba keratitis begins when Acanthamoeba trophozoites bind specifically to mannosylated glycoproteins upregulated on the surfaces of traumatized corneal epithelial cells. When Acanthamoeba castellanii trophozoites are grown in methyl-alpha-D-mannopyranoside, they are induced to secrete a novel 133-kDa protein that is cytolytic to corneal epithelial cells. Clinical isolates of Acanthamoeba spp., and not the soil isolates, were proficient at producing a mannose-induced protein (MIP-133) and generating disease in Chinese hamsters. The purified protein was efficient at killing corneal epithelial cells, the first mechanistic barrier, by inducing apoptosis in a caspase 3-dependent pathway. Subsequent steps in pathogenesis require the amoebae to penetrate and degrade collagen. Only the clinical isolates tested were efficient at migrating through a collagenous matrix in vitro, presumably by MIP-133 degradation of both human type I and human type IV collagen. A chicken anti-MIP-133 antiserum effectively bound to the protein and blocked collagenolytic activity, migration, and cytopathic effects (CPE) against corneal cells in vitro. Chinese hamsters orally immunized with MIP-133 displayed a >30% reduction in disease. Immunoglobulin A isolated from immunized animals bound MIP-133 and blocked CPE on corneal cells in vitro. Animals induced to generate severe chronic infections displayed significant reductions in disease symptoms upon oral immunization postinfection. These data suggest that MIP-133 production might be necessary to initiate corneal disease and that it may play an important role in the subsequent steps of the pathogenic cascade of Acanthamoeba keratitis. Furthermore, as antibodies produced both prior to and after infection reduced clinical symptoms of disease, the protein may represent an important immunotherapeutic target for Acanthamoeba keratitis.

  5. Succination of thiol groups in adipose tissue proteins in diabetes: succination inhibits polymerization and secretion of adiponectin.

    Science.gov (United States)

    Frizzell, Norma; Rajesh, Mathur; Jepson, Matthew J; Nagai, Ryoji; Carson, James A; Thorpe, Suzanne R; Baynes, John W

    2009-09-18

    S-(2-Succinyl)cysteine (2SC) is formed by reaction of the Krebs cycle intermediate fumarate with cysteine residues in protein, a process termed succination of protein. Both fumarate and succination of proteins are increased in adipocytes cultured in high glucose medium (Nagai, R., Brock, J. W., Blatnik, M., Baatz, J. E., Bethard, J., Walla, M. D., Thorpe, S. R., Baynes, J. W., and Frizzell, N. (2007) J. Biol. Chem. 282, 34219-34228). We show here that succination of protein is also increased in epididymal, mesenteric, and subcutaneous adipose tissue of diabetic (db/db) mice and that adiponectin is a major target for succination in both adipocytes and adipose tissue. Cys-39, which is involved in cross-linking of adiponectin monomers to form trimers, was identified as a key site of succination of adiponectin in adipocytes. 2SC was detected on two of seven monomeric forms of adiponectin immunoprecipitated from adipocytes and epididymal adipose tissue. Based on densitometry, 2SC-adiponectin accounted for approximately 7 and 8% of total intracellular adiponectin in cells and tissue, respectively. 2SC was found only in the intracellular, monomeric forms of adiponectin and was not detectable in polymeric forms of adiponectin in cell culture medium or plasma. We conclude that succination of adiponectin blocks its incorporation into trimeric and higher molecular weight, secreted forms of adiponectin. We propose that succination of proteins is a biomarker of mitochondrial stress and accumulation of Krebs cycle intermediates in adipose tissue in diabetes and that succination of adiponectin may contribute to the decrease in plasma adiponectin in diabetes.

  6. Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion

    DEFF Research Database (Denmark)

    Duguez, S.; Duddy, W.; Johnston, H.

    2013-01-01

    Duchenne muscular dystrophy results from loss of the protein dystrophin, which links the intracellular cytoskeletal network with the extracellular matrix, but deficiency in this function does not fully explain the onset or progression of the disease. While some intracellular events involved...... of new potential therapeutic targets....

  7. Expression, secretion and antigenic variation of bacterial S-layer proteins

    NARCIS (Netherlands)

    Boot, H.J.; Pouwels, P.H.

    1996-01-01

    The function of the S-layer, a regularly arranged structure on the outside of numerous bacteria, appears to be different for bacteria living in different environments. Almost no similarity exists between the primary sequences of S-proteins, although their amino acid composition is comparable. S-prot

  8. Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal multidrug resistance proteins

    NARCIS (Netherlands)

    Gajic, O; Buist, G; Kojic, M; Topisirovic, L; Kuipers, OP; Kok, J

    2003-01-01

    A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB. The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein. LsbA is a

  9. RNA-seq based expression analysis of the CHO cell protein secretion pathway

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Kildegaard, Helene Faustrup;

    The Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant therapeutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies...

  10. Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.

    Science.gov (United States)

    Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V; Sampey, Gavin C; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

    2014-08-08

    Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

  11. Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein*

    Science.gov (United States)

    Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V.; Sampey, Gavin C.; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

    2014-01-01

    Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. PMID:24939845

  12. Expression of a secreted protein in mammalian cells using baculovirus particles.

    Science.gov (United States)

    Jardin, Barbara Ann; Elias, Cynthia B; Prakash, Satya

    2012-01-01

    There are many methods presently available to produce recombinant proteins in mammalian systems. The BacMam system is a simple straightforward method which overlaps two well-established technologies, namely the BEVS insect cell system and the transduction of mammalian cells in vitro. This chapter describes a method for the study of gene expression in mammalian cells in a series of simple steps. Protocols outlined include the design and construction of the recombinant baculovirus, cell culture techniques required to maintain both insect and mammalian cells, generation of baculovirus stocks, and methods to obtain maximal and reproducible gene expression in mammalian cells. Currently available statistical techniques using factorial design of experiment to optimize conditions for recombinant protein in vitro are outlined. Then details with respect to process scale-up in disposable bioreactors are included.

  13. Inhibition of cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal.

    Directory of Open Access Journals (Sweden)

    Tyler M Sharp

    Full Text Available Protein trafficking between the endoplasmic reticulum (ER and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development.

  14. Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis.

    Science.gov (United States)

    Villeneuve, Julien; Duran, Juan; Scarpa, Margherita; Bassaganyas, Laia; Van Galen, Josse; Malhotra, Vivek

    2017-01-01

    Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi-cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor-mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain-containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions.

  15. Allozyme-specific modification of a maize seed chitinase by a protein secreted by the fungal pathogen Stenocarpella maydis.

    Science.gov (United States)

    Naumann, Todd A; Wicklow, Donald T

    2010-07-01

    Stenocarpella maydis causes both dry-ear rot and stalk rot of maize. Maize inbred lines have varying levels of resistance to ear rot caused by S. maydis. The genetic basis of resistance appears to rely on multiple genetic factors, none of which are known. The commonly used stiff-stalk inbred line B73 has been shown to be strongly susceptible to ear rot caused by S. maydis. Here, we report that the ChitA protein alloform from B73, ChitA-F, encoded by a known allele of the chiA gene, is susceptible to modification by a protein (Stm-cmp) secreted by S. maydis. We also identify a new allele of chiA (from inbred line LH82) which encodes ChitA-S, an alloform of ChitA that is resistant to Stm-cmp modification. Chitinase zymogram analysis of seed from a commercial field showed the presence of both ChitA alloforms in healthy ears, and showed that ChitA-F but not ChitA-S was modified in ears rotted by S. maydis. The ChitA-F protein was purified from inbred line B73 and ChitA-S from LH82. ChitA-F was modified more efficiently than ChitA-S by S. maydis protein extracts in vitro. The chiA gene from LH82 was cloned and sequenced. It is a novel allele that encodes six polymorphisms relative to the known allele from B73. This is the first demonstration that the susceptibility to modification of a fungal targeted plant chitinase differs among inbred lines. These findings suggest that the LH82 chiA allele may be a specific genetic determinant that contributes to resistance to ear rot caused by S. maydis whereas the B73 allele may contribute to susceptibility.

  16. Crystal structure and self-interaction of the type VI secretion tail-tube protein from enteroaggregative Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Badreddine Douzi

    Full Text Available The type VI secretion system (T6SS is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp and terminated by a trimeric valine-glycine repeat protein G (VgrG component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae.

  17. Secreted protein acidic and rich in cysteine (SPARC is associated with nasopharyngeal carcinoma metastasis and poor prognosis

    Directory of Open Access Journals (Sweden)

    Wang Hai-Yun

    2012-02-01

    Full Text Available Abstract Background The aim of the present study was to analyse the expression of Secreted protein acidic and rich in cysteine (SPARC in nasopharyngeal carcinoma (NPC specimens, and to evaluate its correlation with clinicopathologic features, including survival of patients with NPC Methods NPC tissue microarrays (TMAs were constructed from Sun Yat-sen University Cancer Center (SYSUCC, another three centers on mainland China, Singapore and Hong Kong. Using quantitative RT-PCR and Western-blotting techniques, we detected mRNA and protein expression of SPARC in NPC cell lines and immortalized nasopharyngeal epithelial cells (NPECs induced by Bmi-1 (NPEC2 Bmi-1. The difference of SPARC expression in the cell lines was tested using a t-test method. The relationship between the SPARC expression and clinicopathological data was assessed by chi-square. Survival analysis was estimated using the Kaplan-Meier approach with log-rank test. Univariate and multivariate analyses of clinical variables were performed using Cox proportional hazards regression models. Results The expression levels of SPARC mRNA and protein were markedly higher in NPC cell lines than in NPEC2 Bmi-1. Especially, the expression levels of SPARC mRNA and protein were much lower in the 6-10B than in the 5-8 F (P = 0.002, P = 0.001. SPARC immunostaining revealed cytoplasmic localization in NPC cells and no staining in the stroma and epithelium. In addition, high level of SPARC positively correlated with the status of distant metastasis (P = 0.001 and WHO histological classification (P = 0.023. NPC patients with high SPARC expression also had a significantly poorer prognosis than patients with low SPARC expression (log-rank test, P P P Conclusions SPARC expression is common in NPC patients. Our data shows that elevated SPARC expression is a potential unfavorable prognostic factor for patients with NPC.

  18. Acute-phase protein α1-antitrypsin--a novel regulator of angiopoietin-like protein 4 transcription and secretion.

    Science.gov (United States)

    Frenzel, Eileen; Wrenger, Sabine; Immenschuh, Stephan; Koczulla, Rembert; Mahadeva, Ravi; Deeg, H Joachim; Dinarello, Charles A; Welte, Tobias; Marcondes, A Mario Q; Janciauskiene, Sabina

    2014-06-01

    The angiopoietin-like protein 4 (angptl4, also known as peroxisome proliferator-activated receptor [PPAR]γ-induced angiopoietin-related protein) is a multifunctional protein associated with acute-phase response. The mechanisms accounting for the increase in angptl4 expression are largely unknown. This study shows that human α1-antitrypsin (A1AT) upregulates expression and release of angplt4 in human blood adherent mononuclear cells and in primary human lung microvascular endothelial cells in a concentration- and time-dependent manner. Mononuclear cells treated for 1 h with A1AT (from 0.1 to 4 mg/ml) increased mRNA of angptl4 from 2- to 174-fold, respectively, relative to controls. In endothelial cells, the maximal effect on angptl4 expression was achieved at 8 h with 2 mg/ml A1AT (11-fold induction versus controls). In 10 emphysema patients receiving A1AT therapy (Prolastin), plasma angptl4 levels were higher relative to patients without therapy (nanograms per milliliter, mean [95% confidence interval] 127.1 [99.5-154.6] versus 76.8 [54.8-98.8], respectively, p = 0.045) and correlated with A1AT levels. The effect of A1AT on angptl4 expression was significantly diminished in cells pretreated with a specific inhibitor of ERK1/2 activation (UO126), irreversible and selective PPARγ antagonist (GW9662), or genistein, a ligand for PPARγ. GW9662 did not alter the ability of A1AT to induce ERK1/2 phosphorylation, suggesting that PPARγ is a critical mediator in the A1AT-driven angptl4 expression. In contrast, the forced accumulation of HIF-1α, an upregulator of angptl4 expression, enhanced the effect of A1AT. Thus, acute-phase protein A1AT is a physiological regulator of angptl4, another acute-phase protein.

  19. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

    Directory of Open Access Journals (Sweden)

    Lijuan Han

    2016-05-01

    Full Text Available Abstract Background Somatic calreticulin (CALR, Janus kinase 2 (JAK2, and thrombopoietin receptor (MPL mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN, suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These

  20. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lone , Abdul G.; Atci, Erhan; Renslow, Ryan S.; Beyenal, Haluk; Noh, S.; Fransson, B.; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R.; Call, Douglas R.

    2015-08-31

    A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using MSE mass spectrometry. We found that S. aureus biofilm grows predominantly in sebum-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2-3 fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after four days of culture. The colonized explants released significantly (P< 0.01) more anti-oxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentration found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in media from infected explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

  1. Function of FlhB, a Membrane Protein Implicated in the Bacterial Flagellar Type III Secretion System

    Science.gov (United States)

    Meshcheryakov, Vladimir A.; Barker, Clive S.; Kostyukova, Alla S.; Samatey, Fadel A.

    2013-01-01

    The membrane protein FlhB is a highly conserved component of the flagellar secretion system, and it plays an active role in the regulation of protein export. In this study conserved properties of FlhB that are important for its function were investigated. Replacing the flhB gene (or part of the gene) in Salmonella typhimurium with the flhB gene of the distantly related bacterium Aquifex aeolicus greatly reduces motility. However, motility can be restored to some extent by spontaneous mutations in the part of flhB gene coding for the cytoplasmic domain of Aquifex FlhB. Structural analysis suggests that these mutations destabilize the structure. The secondary structure and stability of the mutated cytoplasmic fragments of FlhB have been studied by circular dichroism spectroscopy. The results suggest that conformational flexibility could be important for FlhB function. An extragenic suppressor mutation in the fliS gene, which decreases the affinity of FliS to FliC, partially restores motility of the FlhB substitution mutants. PMID:23874605

  2. Function of FlhB, a membrane protein implicated in the bacterial flagellar type III secretion system.

    Directory of Open Access Journals (Sweden)

    Vladimir A Meshcheryakov

    Full Text Available The membrane protein FlhB is a highly conserved component of the flagellar secretion system, and it plays an active role in the regulation of protein export. In this study conserved properties of FlhB that are important for its function were investigated. Replacing the flhB gene (or part of the gene in Salmonella typhimurium with the flhB gene of the distantly related bacterium Aquifex aeolicus greatly reduces motility. However, motility can be restored to some extent by spontaneous mutations in the part of flhB gene coding for the cytoplasmic domain of Aquifex FlhB. Structural analysis suggests that these mutations destabilize the structure. The secondary structure and stability of the mutated cytoplasmic fragments of FlhB have been studied by circular dichroism spectroscopy. The results suggest that conformational flexibility could be important for FlhB function. An extragenic suppressor mutation in the fliS gene, which decreases the affinity of FliS to FliC, partially restores motility of the FlhB substitution mutants.

  3. Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells.

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    Gunther Doehlemann

    2009-02-01

    Full Text Available The basidiomycete Ustilago maydis causes smut disease in maize. Colonization of the host plant is initiated by direct penetration of cuticle and cell wall of maize epidermis cells. The invading hyphae are surrounded by the plant plasma membrane and proliferate within the plant tissue. We identified a novel secreted protein, termed Pep1, that is essential for penetration. Disruption mutants of pep1 are not affected in saprophytic growth and develop normal infection structures. However, Deltapep1 mutants arrest during penetration of the epidermal cell and elicit a strong plant defense response. Using Affymetrix maize arrays, we identified 116 plant genes which are differentially regulated in Deltapep1 compared to wild type infections. Most of these genes are related to plant defense. By in vivo immunolocalization, live-cell imaging and plasmolysis approaches, we detected Pep1 in the apoplastic space as well as its accumulation at sites of cell-to-cell passages. Site-directed mutagenesis identified two of the four cysteine residues in Pep1 as essential for function, suggesting that the formation of disulfide bridges is crucial for proper protein folding. The barley covered smut fungus Ustilago hordei contains an ortholog of pep1 which is needed for penetration of barley and which is able to complement the U. maydis Deltapep1 mutant. Based on these results, we conclude that Pep1 has a conserved function essential for establishing compatibility that is not restricted to the U. maydis / maize interaction.

  4. NMR characterization of the interaction of the Salmonella type III secretion system protein SipD and bile salts.

    Science.gov (United States)

    Wang, Yu; Nordhues, Bryce A; Zhong, Dalian; De Guzman, Roberto N

    2010-05-18

    Salmonella and Shigella bacteria require the type III secretion system (T3SS) to inject virulence proteins into their hosts and initiate infections. The tip proteins SipD and IpaD are critical components of the Salmonella and Shigella T3SS, respectively. Recently, SipD and IpaD have been shown to interact with bile salts, which are enriched in the intestines, and are hypothesized to act as environmental sensors for these enteric pathogens. Bile salts activate the Shigella T3SS but repress the Salmonella T3SS, and the mechanism of this differing response to bile salts is poorly understood. Further, how SipD binds to bile salts is currently unknown. Computer modeling predicted that IpaD binds the bile salt deoxycholate in a cleft formed by the N-terminal domain and the long central coiled coil of IpaD. Here, we used NMR methods to determine which SipD residues are affected by the interaction with the bile salts deoxycholate, chenodeoxycholate, and taurodeoxcholate. The bile salts perturbed nearly the same set of SipD residues; however, the largest chemical shift perturbations occurred away from what was predicted for the bile salt binding site in IpaD. Our NMR results indicate that that bile salt interaction of SipD will be different from what was predicted for IpaD, suggesting a possible mechanism for the differing response of Salmonella and Shigella to bile salts.

  5. Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Gasser Brigitte

    2006-12-01

    Full Text Available Abstract Background Secretion of heterologous proteins depends both on biomass concentration and on the specific product secretion rate, which in turn is not constant at varying specific growth rates. As fed batch processes usually do not maintain a steady state throughout the feed phase, it is not trivial to model and optimize such a process by mathematical means. Results We have developed a model for product accumulation in fed batch based on iterative calculation in Microsoft Excel spreadsheets, and used the Solver software to optimize the time course of the media feed in order to maximize the volumetric productivity. The optimum feed phase consisted of an exponential feed at maximum specific growth rate, followed by a phase with linearly increasing feed rate and consequently steadily decreasing specific growth rate. The latter phase could be modeled also by exact mathematical treatment by the calculus of variations, yielding the explicit shape of the growth function, however, with certain indeterminate parameters. To evaluate the latter, one needs a numerical optimum search algorithm. The explicit shape of the growth function provides additional evidence that the Excel model results in correct data. Experimental evaluation in two independent fed batch cultures resulted in a good correlation to the optimized model data, and a 2.2 fold improvement of the volumetric productivity. Conclusion The advantages of the procedure we describe here are the ease of use and the flexibility, applying software familiar to every scientist and engineer, and rapid calculation which makes predictions extremely easy, so that many options can be tested in silico quickly. Additional options like further biological and technological constraints or different functions for specific productivity and biomass yield can easily be integrated.

  6. Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes.

    Science.gov (United States)

    Mazzeo, C; Calvo, V; Alonso, R; Mérida, I; Izquierdo, M

    2016-01-01

    Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.

  7. Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

    Science.gov (United States)

    Senderskiy, Igor V; Timofeev, Sergey A; Seliverstova, Elena V; Pavlova, Olga A; Dolgikh, Viacheslav V

    2014-01-01

    Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.

  8. Secretion of Antonospora (Paranosema locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

    Directory of Open Access Journals (Sweden)

    Igor V Senderskiy

    Full Text Available Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species, strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.

  9. Bone morphogenetic protein 4 inhibits insulin secretion from rodent beta cells through regulation of calbindin1 expression and reduced voltage-dependent calcium currents

    DEFF Research Database (Denmark)

    Christensen, Gitte L.; Jacobsen, Maria L. B.; Wendt, Anna

    2015-01-01

    AIMS/HYPOTHESIS: Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression...

  10. Gene delivery into primary brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Lichota, Jacek;

    model was established by co-culturing primary BCECs together with primary astrocytes, both of which were isolated from rats. This was done in order to study the possibility of using gene transfection in an environment closer to the in-vivo BBB situation. The in-vitro BBB barrier model showed trans...... for recombinant protein production. We have previously shown that it is possible to transfect human microvascular endothelial cells (HBMEC) and rat brain endothelial (RBE4) cells with genetic material encoding the human growth hormone (GH1) (Thomsen et al 2011). In the present study, however, an in-vitro BBB...

  11. Gene delivery of therapeutic polypeptides into brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Moos, Torben;

    has been to investigate the usage of BCEC as factories for recombinant protein production. A non-viral gene carrier was prepared from pullulan-spermine conjugated with plasmid DNA (Thomsen et al., 2011). In vitro transfection of Rat Brain Endothelial Cells (RBE4) and Human Brain Microvascular...... Endothelial cells (HBMECs) were conducted with three plasmids bearing cDNA encoding human BDNF, EPO or the FGL peptide. Results revealed a high expression of BDNF, EPO and FGL transcripts in transfected cells compared to the non-transfected cells, which strongly suggest that transfection were successful...

  12. Protein phosphatase 1 (PP-1)-dependent inhibition of insulin secretion by leptin in INS-1 pancreatic β-cells and human pancreatic islets.

    Science.gov (United States)

    Kuehnen, Peter; Laubner, Katharina; Raile, Klemens; Schöfl, Christof; Jakob, Franz; Pilz, Ingo; Päth, Günter; Seufert, Jochen

    2011-05-01

    Leptin inhibits insulin secretion from pancreatic β-cells, and in turn, insulin stimulates leptin biosynthesis and secretion from adipose tissue. Dysfunction of this adipoinsular feedback loop has been proposed to be involved in the development of hyperinsulinemia and type 2 diabetes mellitus. At the molecular level, leptin acts through various pathways, which in combination confer inhibitory effects on insulin biosynthesis and secretion. The aim of this study was to identify molecular mechanisms of leptin action on insulin secretion in pancreatic β-cells. To identify novel leptin-regulated genes, we performed subtraction PCR in INS-1 β-cells. Regulated expression of identified genes was confirmed by RT-PCR and Northern and Western blotting. Furthermore, functional impact on β-cell function was characterized by insulin-secretion assays, intracellular Ca²(+) concentration measurements, and enzyme activity assays. PP-1α, the catalytic subunit of protein phosphatase 1 (PP-1), was identified as a novel gene down-regulated by leptin in INS-1 pancreatic β-cells. Expression of PP-1α was verified in human pancreatic sections. PP-1α mRNA and protein expression is down-regulated by leptin, which culminates in reduction of PP-1 enzyme activity in β-cells. In addition, glucose-induced insulin secretion was inhibited by nuclear inhibitor of PP-1 and calyculin A, which was in part mediated by a reduction of PP-1-dependent calcium influx into INS-1 β-cells. These results identify a novel molecular pathway by which leptin confers inhibitory action on insulin secretion, and impaired PP-1 inhibition by leptin may be involved in dysfunction of the adipoinsular axis during the development of hyperinsulinemia and type 2 diabetes mellitus.

  13. Expression by Streptomyces lividans of the Rat α Integrin CD11b A-Domain as a Secreted and Soluble Recombinant Protein

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    Dorra Zouari Ayadi

    2007-01-01

    Full Text Available We already reported the use of a long synthetic signal peptide (LSSP to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte β2 integrin CD11/CD18 alpha M subunit (CD11b. This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the “LSSP” synthetic signal peptide.

  14. Purification and characterization of a novel antifungal protein secreted by Penicillium chrysogenum from an Arctic sediment.

    Science.gov (United States)

    Chen, Zhiteng; Ao, Jingqun; Yang, Wenchuan; Jiao, Liping; Zheng, Tianling; Chen, Xinhua

    2013-12-01

    A fungal strain, Penicillium chrysogenum A096, was isolated from an Arctic sediment sample. Its culture supernatant inhibited mycelial growth of some plant pathogenic fungi. After saturation of P. chrysogenum A096 culture supernatant with ammonium sulfate and ion exchange chromatography, a novel antifungal protein (Pc-Arctin) was purified and identified by matrix assisted laser desorption ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS). The gene encoding for Pc-Arctin consisting of 195 nucleotides was cloned from P. chrysogenum A096 to confirm the mass spectrometry result. Pc-Arctin displays antifungal activity against Paecilomyces variotii, Alternaria longipes, and Trichoderma viride at minimum inhibitory concentrations (MIC) of 24, 48, and 192 ng/disc, respectively. Pc-Arctin was most sensitive to proteinase K and then to trypsin but insensitive to papain. Pc-Arctin possesses high thermostability and cannot be antagonized by common surfactants, except for sodium dodecyl sulfate (SDS). Divalent ions, such as Mn(2+), Mg(2+), and Zn(2+), inhibited the antifungal activity of Pc-Arctin. Hemagglutination assays showed that Pc-Arctin had no hemagglutinating or hemolytic activity against red blood cells (RBC) from rabbits, rats, and guinea pigs. Therefore, Pc-Arctin from Arctic P. chrysogenum may represent a novel antifungal protein with potential for application in controlling plant pathogenic fungal infection.

  15. Structural and functional similarity between the bacterial type III secretion system needle protein PrgI and the eukaryotic apoptosis Bcl-2 proteins.

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    Matthew D Shortridge

    Full Text Available BACKGROUND: Functional similarity is challenging to identify when global sequence and structure similarity is low. Active-sites or functionally relevant regions are evolutionarily more stable relative to the remainder of a protein structure and provide an alternative means to identify potential functional similarity between proteins. We recently developed the FAST-NMR methodology to discover biochemical functions or functional hypotheses of proteins of unknown function by experimentally identifying ligand binding sites. FAST-NMR utilizes our CPASS software and database to assign a function based on a similarity in the structure and sequence of ligand binding sites between proteins of known and unknown function. METHODOLOGY/PRINCIPAL FINDINGS: The PrgI protein from Salmonella typhimurium forms the needle complex in the type III secretion system (T3SS. A FAST-NMR screen identified a similarity between the ligand binding sites of PrgI and the Bcl-2 apoptosis protein Bcl-xL. These ligand binding sites correlate with known protein-protein binding interfaces required for oligomerization. Both proteins form membrane pores through this oligomerization to release effector proteins to stimulate cell death. Structural analysis indicates an overlap between the PrgI structure and the pore forming motif of Bcl-xL. A sequence alignment indicates conservation between the PrgI and Bcl-xL ligand binding sites and pore formation regions. This active-site similarity was then used to verify that chelerythrine, a known Bcl-xL inhibitor, also binds PrgI. CONCLUSIONS/SIGNIFICANCE: A structural and functional relationship between the bacterial T3SS and eukaryotic apoptosis was identified using our FAST-NMR ligand affinity screen in combination with a bioinformatic analysis based on our CPASS program. A similarity between PrgI and Bcl-xL is not readily apparent using traditional global sequence and structure analysis, but was only identified because of conservation in

  16. Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1, a Hybrid Proline-Rich Protein from Arabidopsis

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    Andrea Pitzschke

    2016-01-01

    Full Text Available Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs consist of a putative N-terminal secretion signal, a proline-rich domain (PRD, and a characteristic eight-cysteine-motif (8-CM. They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

  17. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    Institute of Scientific and Technical Information of China (English)

    Yi-Ping Li; Hye Na Kang; Lorne A Babiuk; Qiang Liu

    2006-01-01

    AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models.METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation,ELISPOT for the number of interferon-γ secreting cells,and cytotoxic T lymphocyte assays.RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-like ones in piglets.CONCLUSION: A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations.

  18. TtsI, a key regulator of Rhizobium species NGR234 is required for type III-dependent protein secretion and synthesis of rhamnose-rich polysaccharides.

    Science.gov (United States)

    Marie, Corinne; Deakin, William J; Ojanen-Reuhs, Tuula; Diallo, Ericka; Reuhs, Brad; Broughton, William J; Perret, Xavier

    2004-09-01

    Formation of nitrogen-fixing nodules on legume roots by Rhizobium sp. NGR234 requires an array of bacterial factors, including nodulation outer proteins (Nops) secreted through a type III secretion system (TTSS). Secretion of Nops is abolished upon inactivation of ttsI (formerly y4xI), a protein with characteristics of two-component response regulators that was predicted to activate transcription of TTSS-related genes. During the symbiotic interaction, the phenotype of NGR omega ttsI differs from that of a mutant with a nonfunctional secretion machine, however. This indicated that TtsI regulates the synthesis of other symbiotic factors as well. Conserved sequences, called tts boxes, proposed to act as binding sites for TtsI, were identified not only within the TTSS cluster but also in the promoter regions of i) genes predicted to encode homologs of virulence factors secreted by pathogenic bacteria, ii) loci involved in the synthesis of a rhamnose-rich component (rhamnan) of the lipopolysaccharides (LPS), and iii) open reading frames that play roles in plasmid partitioning. Transcription studies showed that TtsI and tts boxes are required for the activation of TTSS-related genes and those involved in rhamnose synthesis. Furthermore, extraction of polysaccharides revealed that inactivation of ttsI abolishes the synthesis of the rhamnan component of the LPS. The phenotypes of mutants impaired in TTSS-dependent protein secretion, rhamnan synthesis, or in both functions were compared to assess the roles of some of the TtsI-controlled factors during symbiosis.

  19. Analysis of expression of secreted phospholipases A2 in mouse tissues at protein and mRNA levels.

    Science.gov (United States)

    Eerola, Leena I; Surrel, Fanny; Nevalainen, Timo J; Gelb, Michael H; Lambeau, Gérard; Laine, V Jukka O

    2006-07-01

    Secreted phospholipases A(2) (sPLA(2)) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA(2)s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA(2)s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA(2)s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA(2) proteins corresponded to the expression of sPLA(2)s at the mRNA level. With a few exceptions, the sPLA(2) proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA(2) is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA(2) in the intestine of GIIA sPLA(2)-deficient C57BL/6J mice is not compensated by other sPLA(2)s under normal conditions. Outside the gastrointestinal tract, sPLA(2)s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA(2) proteins in mouse serum and tissues in various experimental settings.

  20. Role of alpha-crystallin, early-secreted antigenic target 6-kDa protein and culture filtrate protein 10 as novel diagnostic markers in osteoarticular tuberculosis

    Directory of Open Access Journals (Sweden)

    Nazia Rizvi

    2016-07-01

    Full Text Available Osteoarticular tuberculosis constitutes about 3% of all tuberculosis cases. Early and accurate diagnosis of tuberculosis is a challenging problem especially in the case of osteoarticular tuberculosis owing to the lower number of bacilli. However, an accurate and timely diagnosis of the disease results in an improved efficacy of the given treatment. Besides the limitations of conventional methods, nowadays molecular diagnostic techniques have emerged as a major breakthrough for the early diagnosis of tuberculosis with high sensitivity and specificity. Alpha-crystallin is a dominantly expressed protein responsible for the long viability of the pathogen during the latent phase under certain stress conditions such as hypoxia and nitric oxide stress. Two other proteins—early secreted antigenic target-6 and culture filtrate protein-10—show high expression in the active infective phase of Mycobacterium tuberculosis. In this article, we focus on the different proteins expressed dominantly in latent/active tuberculosis, and which may be further used as prognostic biomarkers for diagnosing tuberculosis, both in latent and active phases.

  1. Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animal-pathogenic bacteria.

    Science.gov (United States)

    Büttner, Daniela

    2012-06-01

    Flagellar and translocation-associated type III secretion (T3S) systems are present in most gram-negative plant- and animal-pathogenic bacteria and are often essential for bacterial motility or pathogenicity. The architectures of the complex membrane-spanning secretion apparatuses of both systems are similar, but they are associated with different extracellular appendages, including the flagellar hook and filament or the needle/pilus structures of translocation-associated T3S systems. The needle/pilus is connected to a bacterial translocon that is inserted into the host plasma membrane and mediates the transkingdom transport of bacterial effector proteins into eukaryotic cells. During the last 3 to 5 years, significant progress has been made in the characterization of membrane-associated core components and extracellular structures of T3S systems. Furthermore, transcriptional and posttranscriptional regulators that control T3S gene expression and substrate specificity have been described. Given the architecture of the T3S system, it is assumed that extracellular components of the secretion apparatus are secreted prior to effector proteins, suggesting that there is a hierarchy in T3S. The aim of this review is to summarize our current knowledge of T3S system components and associated control proteins from both plant- and animal-pathogenic bacteria.

  2. Vaccination with a single CD4 T cell peptide epitope from a Salmonella type III-secreted effector protein provides protection against lethal infection.

    Science.gov (United States)

    Kurtz, Jonathan R; Petersen, Hailey E; Frederick, Daniel R; Morici, Lisa A; McLachlan, James B

    2014-06-01

    Salmonella infections affect millions worldwide and remain a significant cause of morbidity and mortality. It is known from mouse studies that CD4 T cells are essential mediators of immunity against Salmonella infection, yet it is not clear whether targeting CD4 T cell responses directly with peptide vaccines against Salmonella can be effective in combating infection. Additionally, it is not known whether T cell responses elicited against Salmonella secreted effector proteins can provide protective immunity against infection. In this study, we investigated both of these possibilities using prime-boost immunization of susceptible mice with a single CD4 T cell peptide epitope from Salmonella secreted effector protein I (SseI), a component of the Salmonella type III secretion system. This immunization conferred significant protection against lethal oral infection, equivalent to that conferred by whole heat-killed Salmonella bacteria. Surprisingly, a well-characterized T cell epitope from the flagellar protein FliC afforded no protection compared to immunization with an irrelevant control peptide. The protective response appeared to be most associated with polyfunctional CD4 T cells raised against the SseI peptide, since no antibodies were produced against any of the peptides and very little CD8 T cell response was observed. Overall, this study demonstrates that eliciting CD4 T cell responses against components of the Salmonella type III secretion system can contribute to protection against infection and should be considered in the design of future Salmonella subunit vaccines.

  3. Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Seo, MiRan [Department of Biochemistry and Molecular Biology and Cancer Research Institute, Seoul National University College of Medicine, Seoul (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology and Cancer Research Institute, Seoul National University College of Medicine, Seoul (Korea, Republic of)

    2010-03-05

    Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the {alpha} subunit of Gq protein (G{alpha}q) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active G{alpha}q (G{alpha}qQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of G{alpha}q with shRNA in HaCaT human keratinocytes. G{alpha}q was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipase C (PLC), protein kinase C{delta} (PKC{delta}), and matrix metaloprotease-2 (MMP-2). Moreover, G{alpha}qQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that G{alpha}q mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKC{delta} and MMP-2 in HaCaT cells.

  4. Protein hydrolysate-induced cholecystokinin secretion from enteroendocrine cells is indirectly mediated by the intestinal oligopeptide transporter PepT1.

    Science.gov (United States)

    Liou, Alice P; Chavez, Diana I; Espero, Elvis; Hao, Shuzhen; Wank, Stephen A; Raybould, Helen E

    2011-05-01

    Dietary protein is a major stimulant for cholecystokinin (CCK) secretion by the intestinal I cell, however, the mechanism by which protein is detected is unknown. Indirect functional evidence suggests that PepT1 may play a role in CCK-mediated changes in gastric motor function. However, it is unclear whether this oligopeptide transporter directly or indirectly activates the I cell. Using both the CCK-expressing enteroendocrine STC-1 cell and acutely isolated native I cells from CCK-enhanced green fluorescent protein (eGFP) mice, we aimed to determine whether PepT1 directly activates the enteroendocrine cell to elicit CCK secretion in response to oligopeptides. Both STC-1 cells and isolated CCK-eGFP cells expressed PepT1 transcripts. STC-1 cells were activated, as measured by ERK(1/2) phosphorylation, by both peptone and the PepT1 substrate Cefaclor; however, the PepT1 inhibitor 4-aminomethyl benzoic acid (AMBA) had no effect on STC-1 cell activity. The PepT1-transportable substrate glycyl-sarcosine dose-dependently decreased gastric motility in anesthetized rats but had no affect on activation of STC-1 cells or on CCK secretion by CCK-eGFP cells. CCK secretion was significantly increased in response to peptone but not to Cefaclor, cephalexin, or Phe-Ala in CCK-eGFP cells. Taken together, the data suggest that PepT1 does not directly mediate CCK secretion in response to PepT1 specific substrates. PepT1, instead, may have an indirect role in protein sensing in the intestine.

  5. Secretion of pro- and mature Rhizopus arrhizus lipases by Pichia pastoris and properties of the proteins.

    Science.gov (United States)

    Niu, Wei-ning; Li, Zhao-peng; Tan, Tianwei

    2006-01-01

    The lipases of Rhizopus spp. share a high 1,3-regiospecificity toward triacylglycerols, which makes them important enzymes in lipid modification. In the present study, the extracellularly active production of recombinant Rhizopus arrhizuslipase was carried out with genes encoding the mature region (mRAL) and the mRAL having the prosequence (ProRAL) in Pichia pastoris. Two transformed P. pastoris clones containing the multicopy of mRAL and ProRAL genes were separately selected for the production of recombinant enzymes. In a fed-batch cultivation, where methanol feeding was controlled by an on-line methanol analyzer, the supernatant contained 91 mg/L recombinant pro-form lipase (rProRAL) and 80 mg/L recombinant mature lipase (rRAL) after 92 h of cultivation. rProRAL and rRAL were purified by ultrafiltration, SP-Sepharose Fast Flow chromatography, and Butyl-Sepharose Fast Flow chromatography. Molecular weights of rProRAL and rRAL are 32 kDa and 29 kDa, respectively. The amino-terminal analysis showed that the 32-kDa protein was mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL). The specific lipase activities of mRAL attached with 28 amino acids of the carboxy-terminal part of the prosequence (rPro28RAL) and rRAL were 1543 U/mg and 2437 U/mg. The rPro28RAL was more stable than rRAL at pH 4.0-7.0, whereas rRAL was more stable at pH 7.0-10.0. The rPro28RAL had the highest lipase activity toward tributyrin (C4), whereas rRAL had the highest lipase activity toward tricaprylin (C8).

  6. Peripheral biomarkers in Autism: secreted amyloid precursor protein-alpha as a probable key player in early diagnosis.

    Science.gov (United States)

    Bailey, Antoinette R; Giunta, Brian N; Obregon, Demian; Nikolic, William V; Tian, Jun; Sanberg, Cyndy D; Sutton, Danielle T; Tan, Jun

    2008-01-01

    Autism is a pervasive developmental disorder characterized by impairments in socialization and communication. There is currently no single molecular marker or laboratory tool capable of diagnosing autism at an early age. The purpose of this study is to explore the plausible use of peripheral biomarkers in the early diagnosis of autism via a sensitive ELISA. Here, we measured plasma secreted amyloid precursor protein alpha (sAPP-alpha) levels in autistic and aged-matched control blood samples and found a significantly increased level of sAPP-alpha in 60% of the known autistic children. We then tested 150 human umbilical cord blood (HUCB) samples and found significantly elevated levels of plasma sAPP-alpha in 10 of 150 samples. As an additional confirmatory measure, we performed Western blot analysis on these samples which consistently showed increased sAPP-alpha levels in autistic children and 10 of 150 HUCB samples; suggesting a group of autistic patients which could be identified in early childhood by levels of sAPP-alpha. While there is need for further studies of this concept, the measurement of sAPP-alpha levels in serum and human umbilical cord blood by ELISA is a potential tool for early diagnosis of autism.

  7. Brain Endothelial Cells Produce Amyloid β from Amyloid Precursor Protein 770 and Preferentially Secrete the O-Glycosylated Form*

    Science.gov (United States)

    Kitazume, Shinobu; Tachida, Yuriko; Kato, Masaki; Yamaguchi, Yoshiki; Honda, Takashi; Hashimoto, Yasuhiro; Wada, Yoshinao; Saito, Takashi; Iwata, Nobuhisa; Saido, Takaomi; Taniguchi, Naoyuki

    2010-01-01

    Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia. PMID:20952385

  8. Supplementation with a fish protein hydrolysate (Micromesistius poutassou: effects on body weight, body composition, and CCK/GLP-1 secretion

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    Vincenzo Nobile

    2016-01-01

    Full Text Available Background: Fish protein hydrolysates (FPHs have been reported as a suitable source of proteins for human nutrition because of their balanced amino acid composition and positive effect on gastrointestinal absorption. Objective: Here, we investigated the effect of a FPH, Slimpro®, obtained from blue whiting (Micromesistius poutassou muscle by enzymatic hydrolysis, on body composition and on stimulating cholecystokinin (CCK and glucagon-like peptide-1 (GLP-1 secretion. Design: A randomized clinical study was carried out on 120, slightly overweight (25 kg/m2 ≤ BMI<30 kg/m2, male (25% and female (75% subjects. FPH was tested in a food supplement at two doses (1.4 and 2.8 g to establish if a dose–effect relationship exists. Product use was associated with a mild hypocaloric diet (−300 kcal/day. Body composition (body weight; fat mass; extracellular water; and circumference of waist, thighs, and hips and CCK/GLP-1 blood levels were measured at the beginning of the study and after 45 and 90 days of product use. CCK/GLP-1 levels were measured since they are involved in controlling food intake. Results: Treated subjects reported an improvement of body weight composition and an increased blood concentration of both CCK and GLP-1. No differences were found between the 1.4 and 2.8 g FPH doses, indicating a plateau effect starting from 1.4 g FPH. Conclusions: Both 1.4 and 2.8 g of FPH were effective in improving body composition and in increasing CCK and GLP-1 blood levels.

  9. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

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    Gülin Güler-Gane

    Full Text Available The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine, and a detrimental effect of others (cysteine, proline, tyrosine and glutamine. When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that

  10. Changes of inducible protein-10 and regulated upon activation, normal T cell expressed and secreted protein in acute rejection of pancreas transplantation in rats

    Institute of Scientific and Technical Information of China (English)

    Jun Zhu; Ze-Kuan Xu; Yi Miao; Xun-Liang Liu; Hong Zhang

    2006-01-01

    AIM: To investigate the role of IFN-γ inducible protein -10 (Ip-10) and regulated upon activation, normal T cell expressed and secreted (RANTES) protein in acute pancreatic allograft rejection in rats.METHODS: An experimental pancreas transplantation model was established using diabetic SD rats as the recipient, induced by applying streptozocin (STZ).Pancreas transplantation was performed with a physiologic method of portal venous and enteric drainage. Rats were divided into two groups, isograft group (group A, n = 24) and allograft group (group B, n = 24) in which either healthy SD rats or Wistar rats served as donors, respectively. Twelve diabetic or healthy SD rats were used as controls. At d 1, 4, 7, and 10 post transplantation, serum IP-10 and RANTES were assessed by ELISA and their expression in the allografts was determined by immunohistochemistry.RESULTS: In group B (allograft group), the development of acute rejection was significantly correlated with increased serum concentration and tissue expression of IP-10 and RANTES, with a peak level at d 7 post transplantation. In contrast, there was no obvious change before and after transplantation in group A (isograft group).CONCLUSION: Our study suggests a possible role of IP-10 and RANTES in acute rejection and early monitoring of chemokines may be helpful in predicting the outcome of pancreas transplantation.

  11. Characterization of virulence profile, protein secretion and immunogenicity of different Sporothrix schenckii sensu stricto isolates compared with S. globosa and S. brasiliensis species.

    Science.gov (United States)

    Fernandes, Geisa Ferreira; dos Santos, Priscila Oliveira; Rodrigues, Anderson Messias; Sasaki, Alexandre Augusto; Burger, Eva; de Camargo, Zoilo Pires

    2013-04-01

    A comparative study about protein secretion, immunogenicity and virulence was performed in order to characterize and to compare eight Sporothrix schenckii sensu stricto isolates. For virulence characterization, a murine model, based on survival assay and CFU counting was used. S. brasiliensis and S. globosa, a highly virulent and a non-virulent isolates, respectively were used as external controls. Exoantigen profiles showed different secreted molecules; the 46- and 60-kDa molecules were commonly secreted by all three species. The S. schenckii s. str. isolates could be classified as non-virulent or presenting low, medium or high virulence, based on survival times after infection and recovery of viable fungi. The humoral response profiles of mice infected with S. schenckii s. str., S. globosa and S. brasiliensis were heterogeneous; five virulent isolates (S. schenckii s. str., n = 4 and S. brasiliensis, n = 1) had in common the recognition of the 60-kDa molecule by their respective antisera, suggesting that this antigen may be involved in virulence. Furthermore, the 110-kDa molecule was secreted and recognized by antisera from four virulent isolates (S. schenckii s. str., n = 3 and S. brasiliensis, n = 1), so there is a possibility that this molecule is also related to virulence. Our findings reveal different degrees of virulence in S. schenckii s. str. isolates and suggest the correlation of protein secretion and immunogenicity with virulence of S. schenckii complex. These findings provide new insights into the pathogenesis of S. schenckii s. str. and improve the knowledge about immunogenicity and protein profiles in S. schenckii complex.

  12. Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger

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    Dai, Ziyu; Aryal, Uma K.; Shukla, Anil; Qian, Wei-Jun; Smith, Richard D.; Magnuson, Jon K.; Adney, William S.; Beckham, Gregg T.; Brunecky, Roman; Himmel, Michael E.; Decker, Stephen R.; Ju, Xiaohui; Zhang, Xiao; Baker, Scott E.

    2013-12-01

    ALG3 is a Family 58 glycosyltransferase enzyme involved in early N-linked glycan synthesis. Here, we investigated the effect of the alg3 gene disruption on growth, development, metabolism, and protein secretion in Aspergillus niger. The alg3 gene deletion resulted in a significant reduction of growth on complete (CM) and potato dextrose agar (PDA) media and a substantial reduction of spore production on CM. It also delayed spore germination in the liquid cultures of both CM and PDA media, but led to a significant accumulation of red pigment on both CM and liquid modified minimal medium (MM) supplemented with yeast extract. The relative abundance of 55 proteins of the total 190 proteins identified in the secretome was significantly different as a result of alg3 gene deletion. Comparison of a Trichoderma reesei cellobiohydrolase (Cel7A) heterologously expressed in A. niger parental and Δalg3 strains showed that the recombinant Cel7A expressed in the mutant background was smaller in size than that from the parental strains. This study suggests that ALG3 is critical for growth and development, pigment production, and protein secretion in A. niger. Functional analysis of recombinant Cel7A with aberrant glycosylation demonstrates the feasibility of this alternative approach to evaluate the role of N-linked glycosylation in glycoprotein secretion and function.

  13. Collagen and Stretch Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells

    Science.gov (United States)

    Perrone, Carmen E.; Fenwick-Smith, Daniela; Vandenburgh, Herman H.

    1995-01-01

    Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

  14. Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate.

    Science.gov (United States)

    Klaus, Tomasz; Kulesza, Małgorzata; Bzowska, Monika; Wyroba, Barbara; Kilarski, Witold W; Bereta, Joanna

    2015-06-01

    The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.

  15. Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva.

    Science.gov (United States)

    Nakayama, Yohei; Kobayashi, Ryoki; Matsui, Sari; Matsumura, Hiroyoshi; Iwai, Yasunobu; Noda, Keisuke; Yamazaki, Mizuho; Kurita-Ochiai, Tomoko; Yoshimura, Atsutoshi; Shinomura, Tamayuki; Ganss, Bernhard; Ogata, Yorimasa

    2016-11-02

    The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.

  16. Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins.

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    Pei-Gang Wang

    Full Text Available BACKGROUND: Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E. METHODOLOGY/PRINCIPAL FINDINGS: We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs for all four dengue virus (DV serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant. CONCLUSIONS/SIGNIFICANCE: Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.

  17. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.

    Science.gov (United States)

    Hensel, M; Shea, J E; Waterman, S R; Mundy, R; Nikolaus, T; Banks, G; Vazquez-Torres, A; Gleeson, C; Fang, F C; Holden, D W

    1998-10-01

    The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

  18. Evidence of a Novel Mechanism for Partial γ-Secretase Inhibition Induced Paradoxical Increase in Secreted Amyloid β Protein

    Science.gov (United States)

    Baranello, Robert; Pacheco-Quinto, Javier; Crosson, Craig; Ablonczy, Zsolt; Eckman, Elizabeth; Eckman, Christopher B.; Ramakrishnan, Viswanathan; Greig, Nigel H.; Pappolla, Miguel A.; Sambamurti, Kumar

    2014-01-01

    BACE1 (β-secretase) and α-secretase cleave the Alzheimer's amyloid β protein (Aβ) precursor (APP) to C-terminal fragments of 99 aa (CTFβ) and 83 aa (CTFα), respectively, which are further cleaved by γ-secretase to eventually secrete Aβ and Aα (a.k.a. P3) that terminate predominantly at residues 40 and 42. A number of γ-secretase inhibitors (GSIs), such as N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), have been developed with the goal of reducing Aβ to treat Alzheimer's disease (AD). Although most studies show that DAPT inhibits Aβ in a dose-dependent manner several studies have also detected a biphasic effect with an unexpected increase at low doses of DAPT in cell cultures, animal models and clinical trials. In this article, we confirm the increase in Aβ40 and Aβ42 in SH-SY5Y human neuroblastoma cells treated with low doses of DAPT and identify one of the mechanisms for this paradox. We studied the pathway by first demonstrating that stimulation of Aβ, a product of γ-secretase, was accompanied by a parallel increase of its substrate CTFβ, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an Aβ degrading activity, endothelin converting enzyme (ECE), yielded more Aβ, but abolished the DAPT-induced stimulation. Finally, we have demonstrated that Aα, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of γ-secretase can paradoxically increase Aβ by transiently skirting Aβ degradation in the endosome. This study adds to the growing body of literature suggesting that preserving γ-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration. PMID:24658363

  19. Evidence of a novel mechanism for partial γ-secretase inhibition induced paradoxical increase in secreted amyloid β protein.

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    Eliza Barnwell

    Full Text Available BACE1 (β-secretase and α-secretase cleave the Alzheimer's amyloid β protein (Aβ precursor (APP to C-terminal fragments of 99 aa (CTFβ and 83 aa (CTFα, respectively, which are further cleaved by γ-secretase to eventually secrete Aβ and Aα (a.k.a. P3 that terminate predominantly at residues 40 and 42. A number of γ-secretase inhibitors (GSIs, such as N-[N-(3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, have been developed with the goal of reducing Aβ to treat Alzheimer's disease (AD. Although most studies show that DAPT inhibits Aβ in a dose-dependent manner several studies have also detected a biphasic effect with an unexpected increase at low doses of DAPT in cell cultures, animal models and clinical trials. In this article, we confirm the increase in Aβ40 and Aβ42 in SH-SY5Y human neuroblastoma cells treated with low doses of DAPT and identify one of the mechanisms for this paradox. We studied the pathway by first demonstrating that stimulation of Aβ, a product of γ-secretase, was accompanied by a parallel increase of its substrate CTFβ, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an Aβ degrading activity, endothelin converting enzyme (ECE, yielded more Aβ, but abolished the DAPT-induced stimulation. Finally, we have demonstrated that Aα, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of γ-secretase can paradoxically increase Aβ by transiently skirting Aβ degradation in the endosome. This study adds to the growing body of literature suggesting that preserving γ-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration.

  20. Secreted recombinant human IL-24 protein inhibits the proliferation of esophageal squamous cell carcinoma Eca-109 cells in vitro and in vivo.

    Science.gov (United States)

    Ma, Qunfeng; Jin, Bangming; Zhang, Yao; Shi, Yinan; Zhang, Chi; Luo, Dan; Wang, Pengkun; Duan, Cuimi; Song, Heyu; Li, Xue; Deng, Xuefeng; Chen, Zhinan; Wang, Ziling; Jiang, Hong; Liu, Yan

    2016-05-01

    Interleukin-24 (IL-24) displays cancer-specific apoptosis-inducing properties in a broad spectrum of human tumors without harmful effects on normal cells. The human IL-24 protein is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and a potent antiangiogenic molecule. However, the function of secreted recombinant human IL-24 (srhIL-24) protein in esophageal squamous cell carcinoma (ESCC) cells has not been studied. In the present study, we prepared a stable site-specific-integrated cell line, Flp-InTMCHO/IL-24 (FCHO/IL-24), which secreted rhIL-24 at a higher level than three random-integrated cell lines. In vitro, we identified that the purified srhIL-24 inhibited proliferation and induced the apoptosis of ESCC Eca-109 cells and activated STAT3, which was related with the IL-20 receptors. In vivo, the tumorigenicity of Eca-109 cells was significantly inhibited by s.c. injection of FCHO/IL-24 cells. Decreased tumor microvessel density and an increased number of TUNEL-positive tumor cells were associated with tumor growth inhibition, indicating the presence of antiangiogenic activity and induction of apoptotic activity. In summary, the present study demonstrated that srhIL-24 induced growth inhibition and apoptosis in ESCC Eca-109 cells in vitro and in vivo, which may be mediated by the receptor pathway.

  1. Cyclic AMP-dependent protein kinase (cAPK) regulatory subunits are packaged and secreted by many exocrine and endocrine cells

    Energy Technology Data Exchange (ETDEWEB)

    Mednieks, M.I.; Hand, A.R.

    1986-05-01

    Regulatory (R) subunits of cAPK were identified by us as components of rat and human saliva by photoaffinity labeling with (/sup 32/P)-8-azido cyclic AMP. Photoaffinity labeling of purified rat parotid granule contents and immunogold labeling of thin sections with monoclonal antibodies showed the presence of R subunits in granules. The authors now report that cAPK R subunits are present in secretory granules and are apparently secreted by many exocrine and endocrine cell types. Labeling of thin sections of rat tissues with antibody to R subunits and protein A-gold shows gold particles over secretory granules of endocrine cells of the pituitary, pancreas and intestine. Zymogen granules of exocrine pancreatic acinar cells, the dense cores of secretory granules of seminal vesicle epithelial cells and secretory product in the seminal vesicle lumina were prominently labeled with gold. Photoaffinity labeling shows that pancreatic secretions and seminal vesicle contents have cAPK components. Phosphorylative modification of cellular proteins by cAMP controls hormonally stimulated protein secretion by many cell types. Although no catalytic activity was detected, identification of R subunits in granules and as secretory products indicates that they may have multiple roles in cellular mechanisms of action of cyclic AMP-mediated events in secretory cells.

  2. The CELF1 RNA-Binding Protein Regulates Decay of Signal Recognition Particle mRNAs and Limits Secretion in Mouse Myoblasts

    Science.gov (United States)

    Russo, Joseph; Lee, Jerome E.; López, Carolina M.; Anderson, John; Nguyen, Thuy-mi P.; Heck, Adam M.; Wilusz, Jeffrey

    2017-01-01

    We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and in vitro binding assays using recombinant CELF1, we now provide evidence that CELF1 directly binds the mRNAs encoding each of the subunits of the SRP. Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts. Our results indicate CELF1 is a destabilizer of at least five of the six Srp transcripts and that the relative abundance of the SRP proteins is out of balance when CELF1 is depleted. CELF1 knockdown myoblasts exhibit altered secretion of a luciferase reporter protein and are impaired in their ability to migrate and close a wound, consistent with a defect in the secreted extracellular matrix. Importantly, similar defects in wound healing are observed when SRP subunit imbalance is induced by over-expression of SRP68. Our studies support the existence of an RNA regulon containing Srp mRNAs that is controlled by CELF1. One implication is that altered function of CELF1 in myotonic dystrophy may contribute to changes in the extracellular matrix of affected muscle through defects in secretion. PMID:28129347

  3. L-4F Inhibits Oxidized Low-density Lipoprotein-induced Inflammatory Adipokine Secretion via Cyclic AMP/Protein Kinase A-CCAAT/Enhancer Binding Protein β Signaling Pathway in 3T3-L1 Adipocytes

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    Xiang-Zhu Xie

    2016-01-01

    Conclusions: OxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

  4. Immune responses and protective efficacy induced by 85B antigen and early secreted antigenic target-6 kDa antigen fusion protein secreted by recombinant bacille Calmette-Guérin.

    Science.gov (United States)

    Shi, Changhong; Wang, Xiaowu; Zhang, Hai; Xu, Zhikai; Li, Yuan; Yuan, Lintian

    2007-04-01

    In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin (rBCG) strains expressing an 85B antigen (Ag85B) and early secreted antigenic target-6 kDa antigen (ESAT6) of Mycobacterium tuberculosis (MTB) fusion protein. Both rBCG strains have the same protein insertion but in a different order (Ag85B-ESAT6 and ESAT6-Ag85B). The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The gamma-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group (p0.05).

  5. Unconventional Pathways of Secretion Contribute to Inflammation

    Science.gov (United States)

    Daniels, Michael J. D.; Brough, David

    2017-01-01

    In the conventional pathway of protein secretion, leader sequence-containing proteins leave the cell following processing through the endoplasmic reticulum (ER) and Golgi body. However, leaderless proteins also enter the extracellular space through mechanisms collectively known as unconventional secretion. Unconventionally secreted proteins often have vital roles in cell and organism function such as inflammation. Amongst the best-studied inflammatory unconventionally secreted proteins are interleukin (IL)-1β, IL-1α, IL-33 and high-mobility group box 1 (HMGB1). In this review we discuss the current understanding of the unconventional secretion of these proteins and highlight future areas of research such as the role of nuclear localisation. PMID:28067797

  6. Modulation of adrenal catecholamine secretion by in vivo gene transfer and manipulation of G protein-coupled receptor kinase-2 activity.

    Science.gov (United States)

    Lymperopoulos, Anastasios; Rengo, Giuseppe; Zincarelli, Carmela; Soltys, Stephen; Koch, Walter J

    2008-02-01

    We recently reported that the upregulation of adrenal G protein-coupled receptor kinase-2 (GRK2) causes enhanced catecholamine (CA) secretion by desensitizing sympatho-inhibitory alpha (2)-adrenergic receptors (alpha (2)ARs) of chromaffin cells, and thereby aggravating heart failure (HF). In this study, we sought to develop an efficient and reproducible in vivo adrenal gene transfer method to determine whether manipulation of adrenal GRK2 levels/activity regulates physiological CA secretion in rats. We specifically investigated two different in vivo gene delivery methods: direct injection into the suprarenal glands, and retrograde delivery through the suprarenal veins. We delivered adenoviral (Ad) vectors containing either GRK2 or an inhibitor of GRK2 activity, the beta ARKct. We found both delivery approaches equally effective at supporting robust (>80% of the whole organ) and adrenal-restricted transgene expression, in the cortical region as well as in the medullar region. Additionally, rats with AdGRK2-infected adrenals exhibit enhanced plasma CA levels when compared with control rats (AdGFP-injected adrenals), whereas plasma CA levels after Ad beta ARKct infection were significantly lower. Finally, in isolated chromaffin cells, alpha (2)ARs of AdGRK2-infected cells failed to inhibit CA secretion whereas Ad beta ARKct-infected cells showed normal alpha (2)AR responsiveness. These results not only indicate that in vivo adrenal gene transfer is an effective way of manipulating adrenal gland signalling, but also identify GRK2 as a critically important molecule involved in CA secretion.

  7. Interleukin-22 restored mitochondrial damage and impaired glucose-stimulated insulin secretion through down-regulation of uncoupling protein-2 in INS-1 cells.

    Science.gov (United States)

    Hu, Minling; Lin, Hanxiao; Yang, Li; Cheng, Yanzhen; Zhang, Hua

    2017-01-07

    Defective glucose-stimulated insulin secretion (GSIS) induced by chronic exposure to fatty acids is a hallmark of type 2 diabetes (T2D). Interleukin-22 (IL-22) has been shown to exert beneficial effects on insulin secretion and to protect pancreatic β-cells from stress. Moreover, uncoupling protein-2 (UCP-2) plays a central role in the regulation of GSIS and β-cell dysfunction, whereas the role of UCP-2 in IL-22-enhanced glycemic control under conditions of lipotoxicity remains unclear. In this present study, we investigated the effects of IL-22 on rat insulin-secreting cells (INS-1 cells) and the mechanisms that underlie IL-22 and lipotoxicity-impaired GSIS in vitro. Chronic palmitate (PA) treatment impaired insulin secretion and activated UCP-2 expression in INS-1 cells. Furthermore, in INS-1 cells, both reduced mitochondrial membrane potential (ΔΨm) and impaired GSIS induced by PA treatment were effectively reversed by an inhibitor of UCP-2 (genipin). Additionally, compared with the PA-treated group, INS-1 cells treated with IL-22 down-regulated UCP-2 expression, increased mitochondrial membrane potential, and restored GSIS. Together, our findings indicate that chronic exposure to PA could activate UCP-2, resulting in mitochondrial damage and impaired GSIS in INS-1 cells. We also suggest that IL-22 plays a protective role in this process via the down-regulation of UCP-2.

  8. Secretion of multi-protein migratory complex induced by Toxoplasma gondii infection in macrophages involves the uPA/uPAR activation system.

    Science.gov (United States)

    Schuindt, Sara Hellen Santos; Oliveira, Bruno Cabral de Lima; Pimentel, Pollyana Maria de Oliveira; Resende, Thatiane Lacerda; Retamal, Cláudio A; DaMatta, Renato A; Seipel, Daniele; Arnholdt, Andrea Cristina Vetö

    2012-05-25

    Toxoplasmosis is a world wide spread zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells for dispersal throughout the body. However, the molecular mechanisms or outcomes of the subversion of the host cell are largely unknown. Recently our group established that metalloproteinases are involved in migration of infected macrophages. Herein, we evaluated the recruitment of host invasive machinery components in T. gondii infected murine macrophages. We showed by immunoprecipitation assays that MMP-9, CD44 TIMP-1 and uPAR were secreted as a multi-protein complex by infected macrophages. Zymographic analysis revealed that MMP-9 was present in its pro- and active form. Moreover, inhibition of uPA/uPAR pathway by PAI-1 decreased secretion of MMP-9 active forms, as well those associated to uPAR and TIMP-1, but not to CD44. Data presented here suggest that MMP-9 is secreted as a multiprotein complex by T. gondii infected macrophages, similar to that observed in metastatic cells. We further speculate that uPA/uPAR system is involved in the expression/secretion of complexes containing active MMP-9 forms.

  9. Whey proteins have beneficial effects on intestinal enteroendocrine cells stimulating cell growth and increasing the production and secretion of incretin hormones.

    Science.gov (United States)

    Gillespie, Anna L; Calderwood, Danielle; Hobson, Laura; Green, Brian D

    2015-12-15

    Whey protein has been indicated to curb diet-induced obesity, glucose intolerance and delay the onset of type 2 diabetes mellitus. Here the effects of intact crude whey, intact individual whey proteins and beta-lactoglobulin hydrolysates on an enteroendocrine (EE) cell model were examined. STC-1 pGIP/neo cells were incubated with several concentrations of yogurt whey (YW), cheese whey (CW), beta-lactoglobulin (BLG), alpha-lactalbumin (ALA) and bovine serum albumin (BSA). The findings demonstrate that BLG stimulates EE cell proliferation, and also GLP-1 secretion (an effect which is lost following hydrolysis with chymotrypsin or trypsin). ALA is a highly potent GLP-1 secretagogue which also increases the intracellular levels of GLP-1. Conversely, whey proteins and hydrolysates had little impact on GIP secretion. This appears to be the first investigation of the effects of the three major proteins of YW and CW on EE cells. The anti-diabetic potential of whey proteins should be further investigated.

  10. Secrets Law

    Directory of Open Access Journals (Sweden)

    Luz Helena Guamanzara Torres

    2013-01-01

    Full Text Available This paper provides a review of the book The Law of Secrets, of the author Juan Carlos Martínez-Villalba Riofrío studying the secrets and how law does protect. To this end, the author has analyzed the general theory of secrecy, secrets and methodology, its overall rating, essential elements and their different legal dimensions, the secret as a subjective right. It also establishes that professional secrecy is protected by constitutional principles such as the right to privacy.

  11. The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles

    Science.gov (United States)

    Santiago, André da Silva; Mendes, Juliano S.; dos Santos, Clelton A.; de Toledo, Marcelo A. S.; Beloti, Lilian L.; Crucello, Aline; Horta, Maria A. C.; Favaro, Marianna T. de Pinho; Munar, Duber M. M.; de Souza, Alessandra A.; Cotta, Mônica A.; de Souza, Anete P.

    2016-01-01

    The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible

  12. Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.

    Science.gov (United States)

    Gangalum, Rajendra K; Bhat, Ankur M; Kohan, Sirus A; Bhat, Suraj P

    2016-06-17

    Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.

  13. Erwinia carotovora DsbA mutants: evidence for a periplasmic-stress signal transduction system affecting transcription of genes encoding secreted proteins.

    Science.gov (United States)

    Vincent-Sealy, L V; Thomas, J D; Commander, P; Salmond, G P

    1999-08-01

    The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approximately two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene

  14. Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein

    Science.gov (United States)

    Sanclemente, Manuel; Iturralde, María; Naval, Javier; Alava, María Angeles; Martínez-Lostao, Luis; Thierse, Hermann-Josef; Anel, Alberto

    2016-01-01

    We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion. PMID:27086912

  15. The Role of Protein Kinase C and Its Effect on GHRH in the Regulation of Hormone Secretion by Somatotrophinomas

    Institute of Scientific and Technical Information of China (English)

    LIU Kui; BAI Xiangjun; LEI Ting; XUE Delin; LIU Qin

    2000-01-01

    Phorbol ester-induced release of growth hormone (GH) and prolactin (PRL) from human somatotrophic tumors was examined in vitro. 12-O-tetradecanoyl-phorbol-13- acetate (TPA)strongly stimulated GH and PRL secretion and showed an additive effect on GH secretion if used in combination with GH releasing hormone (GHRH). In contrast, staurosporine exerted a variable inhibitory effect on GH release. There was no correlation between such effects and gsp mutations.The findings suggested that TPA doesn't act directly through cAMP signal transduction system.

  16. Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27’s Phosphorylation Status, and Is Mediated by Exosome Liberation

    Directory of Open Access Journals (Sweden)

    Matthias B. Stope

    2017-01-01

    Full Text Available The heat shock protein HSP27 has been correlated in ovarian cancer (OC patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker.

  17. A Catalog of Proteins Expressed in the AG Secreted Fluid during the Mature Phase of the Chinese Mitten Crabs (Eriocheir sinensis).

    Science.gov (United States)

    He, Lin; Li, Qing; Liu, Lihua; Wang, Yuanli; Xie, Jing; Yang, Hongdan; Wang, Qun

    2015-01-01

    The accessory gland (AG) is an important component of the male reproductive system of arthropods, its secretions enhance fertility, some AG proteins bind to the spermatozoa and affect its function and properties. Here we report the first comprehensive catalog of the AG secreted fluid during the mature phase of the Chinese mitten crab (Eriocheir sinensis). AG proteins were separated by one-dimensional gel electrophoresis and analyzed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Altogether, the mass spectra of 1173 peptides were detected (1067 without decoy and contaminants) which allowed for the identification of 486 different proteins annotated upon the NCBI database (http://www.ncbi.nlm.nih.gov/) and our transcritptome dataset. The mass spectrometry proteomics data have been deposited at the ProteomeXchange with identifier PXD000700. An extensive description of the AG proteome will help provide the basis for a better understanding of a number of reproductive mechanisms, including potentially spermatophore breakdown, dynamic functional and morphological changes in sperm cells and sperm acrosin enzyme vitality. Thus, the comprehensive catalog of proteins presented here can serve as a valuable reference for future studies of sperm maturation and regulatory mechanisms involved in crustacean reproduction.

  18. SOLiD-SAGE of endophyte-infected red fescue reveals numerous effects on host transcriptome and an abundance of highly expressed fungal secreted proteins.

    Directory of Open Access Journals (Sweden)

    Karen V Ambrose

    Full Text Available One of the most important plant-fungal symbiotic relationships is that of cool season grasses with endophytic fungi of the genera Epichloë and Neotyphodium. These associations often confer benefits, such as resistance to herbivores and improved drought tolerance, to the hosts. One benefit that appears to be unique to fine fescue grasses is disease resistance. As a first step towards understanding the basis of the endophyte-mediated disease resistance in Festuca rubra we carried out a SOLiD-SAGE quantitative transcriptome comparison of endophyte-free and Epichloë festucae-infected F. rubra. Over 200 plant genes involved in a wide variety of physiological processes were statistically significantly differentially expressed between the two samples. Many of the endophyte expressed genes were surprisingly abundant, with the most abundant fungal tag representing over 10% of the fungal mapped tags. Many of the abundant fungal tags were for secreted proteins. The second most abundantly expressed fungal gene was for a secreted antifungal protein and is of particular interest regarding the endophyte-mediated disease resistance. Similar genes in Penicillium and Aspergillus spp. have been demonstrated to have antifungal activity. Of the 10 epichloae whole genome sequences available, only one isolate of E. festucae and Neotyphodium gansuense var inebrians have an antifungal protein gene. The uniqueness of this gene in E. festucae from F. rubra, its transcript abundance, and the secreted nature of the protein, all suggest it may be involved in the disease resistance conferred to the host, which is a unique feature of the fine fescue-endophyte symbiosis.

  19. SOLiD-SAGE of endophyte-infected red fescue reveals numerous effects on host transcriptome and an abundance of highly expressed fungal secreted proteins.

    Science.gov (United States)

    Ambrose, Karen V; Belanger, Faith C

    2012-01-01

    One of the most important plant-fungal symbiotic relationships is that of cool season grasses with endophytic fungi of the genera Epichloë and Neotyphodium. These associations often confer benefits, such as resistance to herbivores and improved drought tolerance, to the hosts. One benefit that appears to be unique to fine fescue grasses is disease resistance. As a first step towards understanding the basis of the endophyte-mediated disease resistance in Festuca rubra we carried out a SOLiD-SAGE quantitative transcriptome comparison of endophyte-free and Epichloë festucae-infected F. rubra. Over 200 plant genes involved in a wide variety of physiological processes were statistically significantly differentially expressed between the two samples. Many of the endophyte expressed genes were surprisingly abundant, with the most abundant fungal tag representing over 10% of the fungal mapped tags. Many of the abundant fungal tags were for secreted proteins. The second most abundantly expressed fungal gene was for a secreted antifungal protein and is of particular interest regarding the endophyte-mediated disease resistance. Similar genes in Penicillium and Aspergillus spp. have been demonstrated to have antifungal activity. Of the 10 epichloae whole genome sequences available, only one isolate of E. festucae and Neotyphodium gansuense var inebrians have an antifungal protein gene. The uniqueness of this gene in E. festucae from F. rubra, its transcript abundance, and the secreted nature of the protein, all suggest it may be involved in the disease resistance conferred to the host, which is a unique feature of the fine fescue-endophyte symbiosis.

  20. Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

    DEFF Research Database (Denmark)

    Sørensen, Mette G; Henriksen, Kim; Sørensen, Mette Guldmann;

    2010-01-01

    Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we...

  1. Involvement of breast cancer resistance protein (BCRP/ABCG2) in the secretion of danofloxacin into milk: interaction with ivermectin.

    Science.gov (United States)

    Real, R; Egido, E; Pérez, M; González-Lobato, L; Barrera, B; Prieto, J G; Alvarez, A I; Merino, G

    2011-08-01

    Danofloxacin, a veterinary fluoroquinolone antimicrobial drug, is actively secreted into milk by an as yet unknown mechanism. One of the main determinants of active drug secretion into milk is the transporter (BCRP/ABCG2). The main purpose was to determine whether danofloxacin is an in vitro substrate for Bcrp1/BCRP and to assess its involvement in danofloxacin secretion into milk. In addition, the role of potential drug-drug interactions in this process was assessed using ivermectin. Danofloxacin was transported in vitro by Bcrp1/BCRP, and ivermectin efficiently blocked this transport. Experiments with Bcrp1(-/-) mice showed no evidence of the involvement of Bcrp1 in plasma pharmacokinetics of danofloxacin. However, the milk concentration and milk-to-plasma ratio of danofloxacin were almost twofold higher in wild-type compared with Bcrp1(-/-) mice. The in vivo interaction with ivermectin was studied in sheep after co-administration of danofloxacin (1.25 mg/kg, i.m.) and ivermectin (0.2 mg/kg, s.c.). Ivermectin had no significant effect on the plasma levels of danofloxacin but significantly decreased danofloxacin concentrations in milk by almost 40%. Concomitant administration of multiple drugs, often used in veterinary therapy, may not only affect their pharmacological activity but also their secretion into milk, because of potential drug-drug interactions mediated by BCRP.

  2. Immune Responses and Protective Efficacy Induced by 85B Antigen and Early Secreted Antigenic Target-6 kDa Antigen Fusion Protein Secreted by Recombinant Bacille Calmette-Guérin

    Institute of Scientific and Technical Information of China (English)

    Changhong SHI; Xiaowu WANG; Hai ZHANG; Zhikai XU; Yuan LI; Lintian YUAN

    2007-01-01

    In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin (rBCG) strains expressing an 85B antigen (Ag85B) and early secreted antigenic target-6 kDa antigen (ESAT6) of Mycobacterium tuberculosis (MTB) fusion protein. Both rBCG strains have the same protein insertion but in a different order (Ag85B-ESAT6 and ESAT6-Ag85B). The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The γ-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group (P<0.05). Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups.Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups (P>0.05).

  3. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

  4. Crystal structure of the C-terminal domain of the Salmonella type III secretion system export apparatus protein InvA.

    Science.gov (United States)

    Worrall, Liam J; Vuckovic, Marija; Strynadka, Natalie C J

    2010-05-01

    InvA is a prominent inner-membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N-terminal integral membrane domain and a C-terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C-terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V-type ATPase and a ring building motif found in other T3SS proteins respectively.

  5. Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

    Science.gov (United States)

    Saitoh, Hiromasa; Fujisawa, Shizuko; Mitsuoka, Chikako; Ito, Akiko; Hirabuchi, Akiko; Ikeda, Kyoko; Irieda, Hiroki; Yoshino, Kae; Yoshida, Kentaro; Matsumura, Hideo; Tosa, Yukio; Win, Joe; Kamoun, Sophien; Takano, Yoshitaka; Terauchi, Ryohei

    2012-01-01

    To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively. PMID:22589729

  6. Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens.

    Directory of Open Access Journals (Sweden)

    Hiromasa Saitoh

    Full Text Available To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46 in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

  7. N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD.

    Science.gov (United States)

    Dickenson, Nicholas E; Arizmendi, Olivia; Patil, Mrinalini K; Toth, Ronald T; Middaugh, C Russell; Picking, William D; Picking, Wendy L

    2013-12-10

    The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri , providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is composed of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g., deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. Although the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study because of the hydrophobic nature of the IpaB and IpaC translocator proteins. Here, we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11-27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together, these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation.

  8. The Effect of Human Recombinant Interferon Gamma (hrIFN-γ) on hCG Secretion of Trophoblast and Protein Synthesis of Decidual Tissue in Vitro

    Institute of Scientific and Technical Information of China (English)

    曹咏清; 陈幼珍

    1996-01-01

    In this study the effect of human recombinant interferon gamma (hrIFN-γ) on hCG secretion of human first trimester trophoblast and protein synthesis of decidual tissue was investigated in vitro. The results indicated that hrIFN-γ at the doses of 250 U/ml medium and 2500 U/ml medium decreased hCG secretion of trophoblast obviously (P < 0. 0,5, P<0. 01 ) in a dose dependent manner. The effect of hrIFN-γ on protein synthesis at the doses of 10 U to 1,000 U/ml medium inhibited the3 H leucine incorporation obviously. The cpm values between control and experimental groups were significantly different (P <0. 05 ) in a dosedependent manner. Furthermore its inhibitory effect is in a dose-dependent manner and was neutralized by IFN-γ antiserum. The IFN-α at the doses used did not find any effect on protein synthesis in decidual tissue.

  9. Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation.

    Science.gov (United States)

    Bottai, Daria; Di Luca, Mariagrazia; Majlessi, Laleh; Frigui, Wafa; Simeone, Roxane; Sayes, Fadel; Bitter, Wilbert; Brennan, Michael J; Leclerc, Claude; Batoni, Giovanna; Campa, Mario; Brosch, Roland; Esin, Semih

    2012-03-01

    The chromosome of Mycobacterium tuberculosis encodes five type VII secretion systems (ESX-1-ESX-5). While the role of the ESX-1 and ESX-3 systems in M. tuberculosis has been elucidated, predictions for the function of the ESX-5 system came from data obtained in Mycobacterium marinum, where it transports PPE and PE_PGRS proteins and modulates innate immune responses. To define the role of the ESX-5 system in M. tuberculosis, in this study, we have constructed five M. tuberculosis H37Rv ESX-5 knockout/deletion mutants, inactivating eccA(5), eccD(5), rv1794 and esxM genes or the ppe25-pe19 region. Whereas the Mtbrv1794ko displayed no obvious phenotype, the other four mutants showed defects in secretion of the ESX-5-encoded EsxN and PPE41, a representative member of the large PPE protein family. Strikingly, the MtbeccD(5) ko mutant also showed enhanced sensitivity to detergents and hydrophilic antibiotics. When the virulence of the five mutants was evaluated, the MtbeccD(5) ko and MtbΔppe25-pe19 mutants were found attenuated both in macrophages and in the severe combined immune-deficient mouse infection model. Altogether these findings indicate an essential role of ESX-5 for transport of PPE proteins, cell wall integrity and full virulence of M. tuberculosis, thereby opening interesting new perspectives for the study of this human pathogen.

  10. Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

    Science.gov (United States)

    Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

    2016-03-01

    The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria.

  11. Estrogen secreting adrenal adenocarcinoma in an 18-month-old boy: aromatase activity, protein expression, mRNA and utilization of gonadal type promoter.

    Science.gov (United States)

    Watanabe, T; Yasuda, T; Noda, H; Wada, K; Kazukawa, I; Someya, T; Minamitani, K; Minagawa, M; Wataki, K; Matsunaga, T; Ohnuma, N; Kohno, Y; Harada, N

    2000-12-01

    We examined clinical, endocrinological and molecular biological aspects of an estrogen-secreting adrenal carcinoma in an 18-month-old male to clarify the pathogenesis of this condition. An 18-month-old boy was referred for evaluation of progressive bilateral gynecomastia and appearance of pubic hair. The patient had elevated plasma estradiol (349 pg/ml) and testosterone (260 ng/dl) levels that completely suppressed FSH and LH levels, and was subsequently diagnosed with an adrenal tumor on the right side. After removal of a 300-g adenocarcinoma, gynecomastia regressed and essentially normal hormone levels were restored. Aromatase activity in the tumor tissue determined by the 3H-water method was 71.0-104.4 pmol/min/mg protein. High levels of aromatase protein and mRNA in the tumor tissue were also demonstrated, while neither aromatase activity nor protein was detected in normal adrenal glands. To investigate the regulation of aromatase expression in the adrenal carcinoma, we examined the usage of alternate promoters responsible for aromatase gene transcription. In the present case, the amounts of aromatase mRNA utilizing gonadal types of exon 1c (1.3) and 1d (II) were significantly higher than those that using other exon 1s. This result suggested that the utilization of a gonadal-type exon 1 might be involved in the over-production of aromatase in estrogen-secreting adrenal carcinoma.

  12. SecDF as part of the Sec-translocase facilitates efficient secretion of Bacillus cereus toxins and cell wall-associated proteins.

    Science.gov (United States)

    Vörös, Aniko; Simm, Roger; Slamti, Leyla; McKay, Matthew J; Hegna, Ida K; Nielsen-LeRoux, Christina; Hassan, Karl A; Paulsen, Ian T; Lereclus, Didier; Økstad, Ole Andreas; Molloy, Mark P; Kolstø, Anne-Brit

    2014-01-01

    The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system.

  13. Immunoglobulin superantigen protein L induces IL-4 and IL-13 secretion from human Fc epsilon RI+ cells through interaction with the kappa light chains of IgE.

    Science.gov (United States)

    Genovese, Arturo; Borgia, Guglielmo; Björck, Lars; Petraroli, Angelica; de Paulis, Amato; Piazza, Marcello; Marone, Gianni

    2003-02-15

    Peptostreptococcus magnus protein L is a multidomain bacterial surface protein that correlates with virulence. It consists of up to five homologous Ig-binding domains (B1-B5) that interact with the variable domain of Ig kappa L chains. Intact protein L stimulates the synthesis and the release of IL-4 and IL-13 from human basophils in vitro. A protein L fragment covering the Ig-binding domains B1-B4 also induced IL-4 and IL-13 release from basophils. There was an excellent correlation (r(s) = 0.82; p ADZ (kappa chains) blocked both anti-IgE- and protein L-induced secretion. Cyclosporin A, but not cyclosporin H, inhibited protein L-induced release of IL-4 and IL-13 from basophils. Thus, protein L acts as a bacterial Ig superantigen to induce the synthesis and release of IL-4 and IL-13 from basophils by interacting with kappa L chains of the IgE isotype.

  14. Hepatocarcinoma-Intestine-Pancreas/Pancreatic Associated Protein (HIP/PAP) Is Expressed and Secreted by Proliferating Ductules as well as by Hepatocarcinoma and Cholangiocarcinoma Cells

    Science.gov (United States)

    Christa, Laurence; Simon, Marie-Thérèse; Brezault-Bonnet, Catherine; Bonte, Eric; Carnot, Françoise; Zylberberg, Hervé; Franco, Dominique; Capron, Frédérique; Roskams, Tania; Bréchot, Christian

    1999-01-01

    Hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP) gene was identified because of its increased expression in 25% of human hepatocellular carcinoma. HIP/PAP protein, a C-type lectin, binds laminin, acts as an adhesion molecule for hepatocytes, and has also been described as an acute phase secretory protein during acute pancreatitis in humans and rats. We investigated HIP/PAP protein expression in patients with various liver diseases associated with ductular reaction. At the same time, we analyzed patients with hepatocellular carcinoma and cholangiocarcinoma, and tested HIP/PAP protein levels in sera to establish the pattern of secretion. Our data show that HIP/PAP expression was not restricted to hepatocellular carcinoma, but was also detected in cholangiocarcinoma cells as well as in reactive non-malignant bile ductules. In contrast, HIP/PAP protein expression was undetectable in normal mature hepatocytes, but some ductular cells localized at the interface of portal tracts with parenchyma were HIP/PAP immunoreactive in normal liver. Finally, we present evidence that HIP/PAP serum levels were increased in 21/28 (75%) patients with hepatocellular carcinoma, and in 25/51 (49%) patients with nonmalignant cirrhosis. Altogether, these results suggest that HIP/PAP protein may be implicated in hepatocytic and cholangiolar differentiation and proliferation. PMID:10550309

  15. Pancreatic stone protein/regenerating protein (PSP/reg): a novel secreted protein up-regulated in type 2 diabetes mellitus.

    Science.gov (United States)

    Yang, Jiayue; Li, Ling; Raptis, Dimitri; Li, Xiaoshan; Li, Fengfei; Chen, Bijun; He, Jiajia; Graf, Rolf; Sun, Zilin

    2015-04-01

    Type 2 diabetes mellitus (T2DM) has insulin resistance (IR) or reduced β-cell mass, partially due to an increased β-cell apoptosis rate. Pancreatic stone protein/regenerating protein (PSP/reg) is a secretory protein produced in the pancreas and up-regulated dramatically during pancreatic disease. Recent studies revealed that β-cells undergoing apoptosis induce PSP/reg expression in surviving neighboring cells. Further experiments demonstrated that PSP/reg was elevated during disease progression in type 1 diabetes mellitus (T1DM). However, the association between PSP/reg and T2DM patients is unknown. The aim of this pilot study was to investigate PSP/reg in different clinical stages of T2DM and evaluate its correlation with chronic complications of diabetes. A total of 1,121 participants (479 males, 642 females; age range 23-80 years) were enrolled in this study. PSP/reg serum values were measured by a newly developed enzyme-linked immunosorbent assay (ELISA). We analyzed its correlation with clinical and biochemical parameters in subjects with T2DM at different clinical phases. Statistical analyses were conducted using SPSS 17.0 software. Correlations of PSP/reg and clinical parameters were performed using Spearman's rank correlation coefficient. Differences between groups were determined by Nemenyi test. PSP/reg was elevated in high-risk and impaired glucose regulation (IGR) patients (pPSP/reg was significantly up-regulated in newly diagnosed T2DM patients and long-term diabetes patients with complications (pPSP/reg levels correlated with the duration of diabetes (pPSP/reg is significantly up-regulated in T2DM patients, and PSP/reg levels are related to the duration of diabetes. Therefore, PSP/reg might be useful as a predictor of T2DM and disease progression.

  16. Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger.

    Science.gov (United States)

    Dai, Ziyu; Aryal, Uma K; Shukla, Anil; Qian, Wei-Jun; Smith, Richard D; Magnuson, Jon K; Adney, William S; Beckham, Gregg T; Brunecky, Roman; Himmel, Michael E; Decker, Stephen R; Ju, Xiaohui; Zhang, Xiao; Baker, Scott E

    2013-12-01

    Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl α-1,3-mannosyltransferase (also known as "asparagine-linked glycosylation 3", or ALG3) is involved in early N-linked glycan synthesis and thus is essential for formation of N-linked protein glycosylation. In this study, we examined the effects of alg3 gene deletion (alg3Δ) on growth, development, pigment production, protein secretion and recombinant Trichoderma reesei cellobiohydrolase (rCel7A) expressed in Aspergillus niger. The alg3Δ delayed spore germination in liquid cultures of complete medium (CM), potato dextrose (PD), minimal medium (MM) and CM with addition of cAMP (CM+cAMP), and resulted in significant reduction of hyphal growth on CM, potato dextrose agar (PDA), and CM+cAMP and spore production on CM. The alg3Δ also led to a significant accumulation of red pigment on both liquid and solid CM cultures. The relative abundances of 54 of the total 215 proteins identified in the secretome were significantly altered as a result of alg3Δ, 63% of which were secreted at higher levels in alg3Δ strain than the parent. The rCel7A expressed in the alg3Δ mutant was smaller in size than that expressed in both wild-type and parental strains, but still larger than T. reesei Cel7A. The circular dichroism (CD)-melt scans indicated that change in glycosylation of rCel7A does not appear to impact the secondary structure or folding. Enzyme assays of Cel7A and rCel7A on nanocrystalline cellulose and bleached kraft pulp demonstrated that the rCel7As have improved activities on hydrolyzing the nanocrystalline cellulose. Overall, the results suggest that alg3 is critical for growth, sporulation, pigment production, and protein secretion in A. niger, and demonstrate the feasibility of this alternative approach to evaluate the roles of N-linked glycosylation in glycoprotein secretion and function.

  17. Decreased basal chloride secretion and altered cystic fibrosis transmembrane conductance regulatory protein, Villin, GLUT5 protein expression in jejunum from leptin-deficient mice

    Directory of Open Access Journals (Sweden)

    Leung L

    2014-07-01

    Full Text Available Lana Leung, Jonathan Kang, Esa Rayyan, Ashesh Bhakta, Brennan Barrett, David Larsen, Ryan Jelinek, Justin Willey, Scott Cochran, Tom L Broderick, Layla Al-NakkashDepartment of Physiology, Arizona College of Osteopathic Medicine, Midwestern University, Glendale, AZ, USAAbstract: Patients with diabetes and obesity are at increased risk of developing disturbances in intestinal function. In this study, we characterized jejunal function in the clinically relevant leptin-deficient ob/ob mouse, a model of diabetes and obesity. We measured transepithelial short circuit current (Isc, across freshly isolated segments of jejunum from 12-week-old ob/ob and lean C57BL/6J (female and male mice. The basal Isc was significantly decreased (~30% in the ob/ob mice (66.5±5.7 µA/cm2 [n=20] (P< 0.05 compared with their lean counterparts (95.1±9.1 µA/cm2 [n=19]. Inhibition with clotrimazole (100 µM, applied bilaterally was significantly reduced in the ob/ob mice (−7.92%±3.67% [n=15] (P<0.05 compared with the lean mice (10.44%±7.92% [n=15], indicating a decreased contribution of Ca2+-activated K+ (KCa channels in the ob/ob mice. Inhibition with ouabain (100 µM, applied serosally was significantly reduced in the ob/ob mice (1.40%±3.61%, n=13 (P< 0.05 versus the lean mice (18.93%±3.76% [n=18], suggesting a potential defect in the Na+/K+-adenosine triphosphate (ATPase pump with leptin-deficiency. Expression of cystic fibrosis transmembrane conductance regulatory protein (CFTR (normalized to glyceraldehyde-3-phosphate dehydrogenase [GAPDH] was significantly decreased ~twofold (P<0.05 in the ob/ob mice compared with the leans, whilst crypt depth was unchanged. Villi length was significantly increased by ~25% (P<0.05 in the ob/ob mice compared with the leans and was associated with an increase in Villin and GLUT5 expression. GLUT2 and SGLT-1 expression were both unchanged. Our data suggests that reduced basal jejunal Isc in ob/ob mice is likely a consequence of

  18. Modification of the secretion pattern of proteases, inflammatory mediators, and extracellular matrix proteins by human aortic valve is key in severe aortic stenosis.

    Science.gov (United States)

    Alvarez-Llamas, Gloria; Martín-Rojas, Tatiana; de la Cuesta, Fernando; Calvo, Enrique; Gil-Dones, Felix; Dardé, Veronica M; Lopez-Almodovar, Luis F; Padial, Luis R; Lopez, Juan-Antonio; Vivanco, Fernando; Barderas, Maria G

    2013-09-01

    One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention.

  19. Identification and quantification of peptides and proteins secreted from prostate epithelial cells by unbiased liquid chromatography tandem mass spectrometry using goodness of fit and analysis of variance.

    Science.gov (United States)

    Florentinus, Angelica K; Bowden, Peter; Sardana, Girish; Diamandis, Eleftherios P; Marshall, John G

    2012-02-01

    The proteins secreted by prostate cancer cells (PC3(AR)6) were separated by strong anion exchange chromatography, digested with trypsin and analyzed by unbiased liquid chromatography tandem mass spectrometry with an ion trap. The spectra were matched to peptides within proteins using a goodness of fit algorithm that showed a low false positive rate. The parent ions for MS/MS were randomly and independently sampled from a log-normal population and therefore could be analyzed by ANOVA. Normal distribution analysis confirmed that the parent and fragment ion intensity distributions were sampled over 99.9% of their range that was above the background noise. Arranging the ion intensity data with the identified peptide and protein sequences in structured query language (SQL) permitted the quantification of ion intensity across treatments, proteins and peptides. The intensity of 101,905 fragment ions from 1421 peptide precursors of 583 peptides from 233 proteins separated over 11 sample treatments were computed together in one ANOVA model using the statistical analysis system (SAS) prior to Tukey-Kramer honestly significant difference (HSD) testing. Thus complex mixtures of proteins were identified and quantified with a high degree of confidence using an ion trap without isotopic labels, multivariate analysis or comparing chromatographic retention times.

  20. An efficient heat-inducible Bacillus subtilis bacteriophage 105 expression and secretion system for the production of the Streptomyces clavuligerus beta-lactamase inhibitory protein (BLIP).

    Science.gov (United States)

    Liu, Hong-Bing; Chui, Ka-Shun; Chan, Chi-Leong; Tsang, Chun-Wai; Leung, Yun-Chung

    2004-03-18

    The Streptomyces clavuligerus beta-lactamase inhibitory protein (BLIP) has been shown to be a potent inhibitor of class A beta-lactamases including the Escherichia coli TEM-1 beta-lactamase (Ki = 0.6 nM). A heat-inducible BLIP expression system was constructed based on a derivative of Bacillus subtilis phage phi105. The recombinant BLIP produced by this system was secreted to the culture medium, purified to homogeneity, and fully active. We have shown that the signal peptide of BLIP functions well in B. subtilis to secrete BLIP out of the cells, which facilitates purification. The absence of a His-tag also avoids the activity and structure of BLIP being altered. An unprecedented high yield of recoverable protein in culture supernatant (3.6mg of >95% pure BLIP/l culture) was achieved by a simple purification protocol. We have developed an efficient production process in which the culture time before heat-induction was 3-4h and the culture supernatant could be collected 5h after induction. This total time of 8-9h is considered to be very short compared to that of the native S. clavuligerus culturing (60-70h). We achieved a very efficient BLIP production rate of 0.8-0.9mg/l/h. Heterologous gene expression was tightly controlled and no production of BLIP was observed before heat-induction, suggesting that cell density can be further increased to improve enzyme yield.

  1. Hepatitis E virus ORF1 encoded macro domain protein interacts with light chain subunit of human ferritin and inhibits its secretion.

    Science.gov (United States)

    Ojha, Nishant Kumar; Lole, Kavita S

    2016-06-01

    Hepatitis E Virus (HEV) is the major causative agent of acute hepatitis in developing countries. Its genome has three open reading frames (ORFs)-called as ORF1, ORF2, and ORF3. ORF1 encodes nonstructural polyprotein having multiple domains, namely: Methyltransferase, Y domain, Protease, Macro domain, Helicase, and RNA-dependent RNA polymerase. In the present study, we show that HEV-macro domain specifically interacts with light chain subunit of human ferritin (FTL). In cultured hepatoma cells, HEV-macro domain reduces secretion of ferritin without causing any change in the expression levels of FTL. This inhibitory effect was further enhanced upon Brefeldin-A treatment. The levels of transferrin Receptor 1 or ferroportin, two important proteins in iron metabolism, remained unchanged in HEV-macro domain expressing cells. Similarly, there were no alterations in the levels of cellular labile iron pool and reactive oxygen species, indicating that HEV-macro domain does not influence cellular iron homeostasis/metabolism. As ferritin is an acute-phase protein, secreted in higher level in infected persons and HEV-macro domain has the property of reducing synthesis of inflammatory cytokines, we propose that by directly binding to FTL, macro domain prevents ferritin from entering into circulation and helps in further attenuation of the host immune response.

  2. The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence.

    Science.gov (United States)

    Scarafoni, Alessio; Ronchi, Alessandro; Prinsi, Bhakti; Espen, Luca; Assante, Gemma; Venturini, Giovanni; Duranti, Marcello

    2013-03-01

    The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre-formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post-extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual-step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

  3. Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots.

    Science.gov (United States)

    Park, Sang-Wook; Lawrence, Christopher B; Linden, James C; Vivanco, Jorge M

    2002-09-01

    Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.

  4. A disordered region in the EvpP protein from the type VI secretion system of Edwardsiella tarda is essential for EvpC binding.

    Directory of Open Access Journals (Sweden)

    Wentao Hu

    Full Text Available The type VI secretion system (T6SS of pathogenic bacteria plays important roles in both virulence and inter-bacterial competitions. The effectors of T6SS are presumed to be transported either by attaching to the tip protein or by interacting with HcpI (haemolysin corregulated protein 1. In Edwardsiella tarda PPD130/91, the T6SS secreted protein EvpP (E. tarda virulent protein P is found to be essential for virulence and directly interacts with EvpC (Hcp-like, suggesting that it could be a potential effector. Using limited protease digestion, nuclear magnetic resonance heteronuclear Nuclear Overhauser Effects, and hydrogen-deuterium exchange mass spectrometry, we confirmed that the dimeric EvpP (40 kDa contains a substantial proportion (40% of disordered regions but still maintains an ordered and folded core domain. We show that an N-terminal, 10-kDa, protease-resistant fragment in EvpP connects to a shorter, 4-kDa protease-resistant fragment through a highly flexible region, which is followed by another disordered region at the C-terminus. Within this C-terminal disordered region, residues Pro143 to Ile168 are essential for its interaction with EvpC. Unlike the highly unfolded T3SS effector, which has a lower molecular weight and is maintained in an unfolded conformation with a dedicated chaperone, the T6SS effector seems to be relatively larger, folded but partially disordered and uses HcpI as a chaperone.

  5. Recombinant proteins secreted from tissue-engineered bioartificial muscle improve cardiac dysfunction and suppress cardiomyocyte apoptosis in rats with heart failure

    Institute of Scientific and Technical Information of China (English)

    RONG Shu-ling; WANG Yong-jin; WANG Xiao-lin; LU Yong-xin; WU Yin; LIU Qi-yun; MI Shao-hua; XU Yu-lan

    2010-01-01

    Background Tissue-engineered bioartificial muscle-based gene therapy represents a promising approach for the treatment of heart diseases. Experimental and clinical studies suggest that systemic administration of insulin-like growth factor-1 (IGF-1) protein or overexpression of IGF-1 in the heart exerts a favorable effect on cardiovascular function. This study aimed to investigate a chronic stage after myocardial infarction (MI) and the potential therapeutic effects of delivering a human IGF-1 gene by tissue-engineered bioartificial muscles (BAMs) following coronary artery ligation in Sprague-Dawley rats.Methods Ligation of the left coronary artery or sham operation was performed. Primary skeletal myoblasts were retrovirally transduced to synthesize and secrete recombinant human insulin-like growth factor-1 (rhIGF-1), and green fluorescent protein (GFP), and tissue-engineered into implantable BAMs. The rats that underwent ligation were randomly assigned to 2 groups: MI-IGF group (n=6) and MI-GFP group (n=6). The MI-IGF group received rhIGF-secreting BAM (IGF-BAMs) transplantation, and the MI-GFP group received GFP-secreting BAM (GFP-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: S-IGF group (n=6)and S-GFP group (n=6). The S-IGF group underwent IGF-1-BAM transplantation, and S-GFP group underwent GFP-BAM transplantation. IGF-1-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats after two weeks of operation was performed. Four weeks after the treatment, hemodynamics was performed. IGF-1 was measured by radioimmunoassay, and then the rats were sacrificed and ventricular samples were subjected to immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of bax and Bcl-2. TNF-α and caspase 3 expression in myocardium was examined by Western blotting.Results Primary rat myoblasts were retrovirally transduced to

  6. Gastric emptying, gastric secretion and enterogastrone response after administration of milk proteins or their peptide hydrolysates in humans

    DEFF Research Database (Denmark)

    Calbet, Jose A L; Holst, Jens Juul

    2004-01-01

    absorption and enterogastrone response, after the intragastric administration of complete cow milk proteins or their respective peptide hydrolysates in man. METHODS: Six healthy males were randomized to receive one of the following four solutions: whey whole protein (W), casein whole protein (C), whey......-like peptide-1 (GLP-1) and peptide YY (PYY) plasma responses were elicited by the four solutions. CONCLUSIONS: The rate of gastric emptying and the plasma GLP-1 and PYY responses to feeding with cow milk protein solutions in humans are independent of the degree of protein fractionation and are not altered...... by small differences in the amino acid composition or protein solubility. In contrast, the GIP response is accentuated when milk proteins are delivered as peptide hydrolysates....

  7. Excess of methyl donor in the perinatal period reduces postnatal leptin secretion in rat and interacts with the effect of protein content in diet.

    Directory of Open Access Journals (Sweden)

    Fanny Giudicelli

    Full Text Available Methionine, folic acid, betaine and choline interact in the one-carbon metabolism which provides methyl groups for methylation reactions. An optimal intake of these nutrients during pregnancy is required for successful completion of fetal development and evidence is growing that they could be involved in metabolic long-term programming. However, the biological pathways involved in the action of these nutrients are still poorly known. This study investigated the interaction between methyl donors and protein content in maternal diet during the preconceptual, pregnancy and lactation periods and the consequences on the rat offspring in the short and long term. Methyl donor supplementation reduced leptin secretion in offspring, whereas insulin levels were mostly affected by protein restriction. The joint effect of protein restriction and methyl donor excess strongly impaired postnatal growth in both gender and long term weight gain in male offspring only, without affecting food intake. In addition, rats born from protein restricted and methyl donor supplemented dams gained less weight when fed a hypercaloric diet. Methylation of the leptin gene promoter in adipose tissue was increased in methyl donor supplemented groups but not affected by protein restriction only. These results suggest that maternal methyl donor supplementation may influence energy homeostasis in a gender-dependent manner, without affecting food intake. Moreover, we showed that macronutrients and micronutrients in maternal diet interact to influence the programming of the offspring.

  8. Proteomic Analysis of Exosomes and Exosome-Free Conditioned Media From Human Osteosarcoma Cell Lines Reveals Secretion of Proteins Related to Tumor Progression.

    Science.gov (United States)

    Jerez, Sofía; Araya, Héctor; Thaler, Roman; Charlesworth, M Cristine; López-Solís, Remigio; Kalergis, Alexis M; Céspedes, Pablo F; Dudakovic, Amel; Stein, Gary S; van Wijnen, Andre J; Galindo, Mario

    2017-02-01

    Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro-environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno-blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC-MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome-free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential. J. Cell. Biochem. 118: 351-360, 2017. © 2016 Wiley Periodicals, Inc.

  9. Comparative genomic analysis of evolutionarily conserved but functionally uncharacterized membrane proteins in archaea: Prediction of novel components of secretion, membrane remodeling and glycosylation systems.

    Science.gov (United States)

    Makarova, Kira S; Galperin, Michael Y; Koonin, Eugene V

    2015-11-01

    A systematic comparative genomic analysis of all archaeal membrane proteins that have been projected to the last archaeal common ancestor gene set led to the identification of several novel components of predicted secretion, membrane remodeling, and protein glycosylation systems. Among other findings, most crenarchaea have been shown to encode highly diverged orthologs of the membrane insertase YidC, which is nearly universal in bacteria, eukaryotes, and euryarchaea. We also identified a vast family of archaeal proteins, including the C-terminal domain of N-glycosylation protein AglD, as membrane flippases homologous to the flippase domain of bacterial multipeptide resistance factor MprF, a bifunctional lysylphosphatidylglycerol synthase and flippase. Additionally, several proteins were predicted to function as membrane transporters. The results of this work, combined with our previous analyses, reveal an unexpected diversity of putative archaeal membrane-associated functional systems that remain to be functionally characterized. A more general conclusion from this work is that the currently available collection of archaeal (and bacterial) genomes could be sufficient to identify (almost) all widespread functional modules and develop experimentally testable predictions of their functions.

  10. Analysis of skin and secretions of Dybowski's frogs (Rana dybowskii) exposed to Staphylococcus aureus or Escherichia coli identifies immune response proteins.

    Science.gov (United States)

    Xiao, Xiang-Hong; Miao, Hui-Min; Xu, Yi-Gang; Zhang, Jing-Yu; Chai, Long-Hui; Xu, Jia-Jia

    2014-04-01

    The aim of the present study was to investigate responses in Dybowski's frogs (Rana dybowskii) exposed to bacteria, using proteomic and transcriptomic approaches. Staphylococcus aureus and Escherichia coli were used as representative Gram-positive and Gram-negative bacteria, respectively, in an infectious challenge model. Frog skin and skin secretions were collected and protein expression in infected frogs compared to control frogs by two-dimensional gel electrophoresis, silver staining, and image analysis. Proteins that demonstrated differential expression were analysed by mass spectrometry and identified by searching protein databases. More than 180 protein spots demonstrated differential expression in E. coli- or S. aureus-challenged groups and, of these, more than 55 spots were up- or down-regulated at least sixfold, post-infection. Proteins with a potential function in the immune response were identified, such as stathmin 1a, annexin A1, superoxide dismutase A, C-type lectin, lysozyme, antimicrobial peptides, cofilin-1-B, mannose receptor, histone H4, prohormone convertase 1, carbonyl reductase 1 and some components of the Toll-like receptor (TLR) signalling pathway. These molecules are potential candidates for further investigation of immune mechanisms in R. dybowskii; in particular, TLR-mediated responses, which might be activated in frogs exposed to pathogenic bacteria as part of innate immune defence, but which might also impact on adaptive immunity to infection.

  11. Differential effects of low-fat and high-fat diets on fed-state hepatic triacylglycerol secretion, hepatic fatty acid profiles, and DGAT-1 protein expression in obese-prone Sprague-Dawley rats.

    Science.gov (United States)

    Heden, Timothy D; Morris, E Matthew; Kearney, Monica L; Liu, Tzu-Wen; Park, Young-Min; Kanaley, Jill A; Thyfault, John P

    2014-04-01

    The purpose of this study was to compare the effects of short-term low-fat (LF) and high-fat (HF) diets on fed-state hepatic triacylglycerol (TAG) secretion, the content of proteins involved in TAG assembly and secretion, fatty acid oxidation (FAO), and the fatty acid profile of stored TAG. Using selectively bred obese-prone Sprague-Dawley rats, we directly measured fed-state hepatic TAG secretion, using Tyloxapol (a lipoprotein lipase inhibitor) and a standardized oral mixed meal (45% carbohydrate, 40% fat, 15% protein) bolus in animals fed a HF or LF diet for 2 weeks, after which the rats were maintained on their respective diet for 1 week (washout) prior to the liver being excised to measure protein content, FAO, and TAG fatty acid profiles. Hepatic DGAT-1 protein expression was ∼27% lower in HF- than in LF-fed animals (p hepatic TAG secretion rate was ∼39% lower (p Hepatic TAG content was ∼2-fold higher (p profile of liver TAG in HF-fed animals closely resembled the diet, whereas in LF-fed animals, the fatty acid profile consisted of mostly de novo synthesized fatty acids. FAO was not altered by diet. LF and HF diets differentially alter fed-state hepatic TAG secretion, hepatic fatty acid profiles, and DGAT-1 protein expression.

  12. Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation.

    Science.gov (United States)

    Udo, H; Munoz-Dorado, J; Inouye, M; Inouye, S

    1995-04-15

    A gene, pkn2, encoding a Myxococcus xanthus protein with significant similarities to eukaryotic protein serine/threonine kinases, was cloned using the polymerase chain reaction. The open reading frame for the protein, beginning with a GUG initiation codon, consists of 830 amino acids. The amino-terminal 279 residues show 37% identity to catalytic domain of Pkn1, another protein serine/threonine kinase expressed during the development at the onset of sporulation. The catalytic domain of Pkn2 contains 27% and 25% identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively. In the middle of the carboxy-terminal regulatory domain, there is a typical transmembrane domain consisting of 18 hydrophobic residues. The gene product, Pkn2, produced in Escherichia coli under a T7 promoter was phosphorylated at both serine and threonine residues. TEM-beta-lactamase produced in E. coli was found to serve as an effective substrate for Pkn2, phosphorylated only at threonine residues, shifting its apparent molecular mass from 29 to 44 kD. The phosphorylated beta-lactamase was unable to be secreted into the periplasmic space and localized in the cytoplasmic and membrane fractions. Analysis of phoA fusions with pkn2 demonstrated that Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the 207-residue carboxy-terminal domain outside the cytoplasmic membrane. Disruption of pkn2 showed no effect on vegetative growth but reduced the yield of myxospores by 30%-50%. On the basis of the present results, we propose that Pkn2 is a transmembrane protein serine/threonine kinase that regulates the activity of endogenous beta-lactamase or related enzymes in response to an external signal yet to be identified.

  13. The bone matrix protein secreted phosphoprotein 24 kD (Spp24): bone metabolism regulator and starting material for biotherapeutic materials.

    Science.gov (United States)

    Murray, Samuel S; Wang, Jeffrey C; Duarte, Maria Eugenia Leite; Zhao, Ke-Wei; Tian, Haijun; Francis, Timothy; Brochmann Murray, Elsa J

    2015-05-01

    Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that appears to be derived primarily from the liver and delivered to other tissues in a protective complex. A significant role in bone growth and turnover is suggested by genetic studies that associate the gene locus (SPP2) with bone mineral density and bone quality. The function of this protein in the normal bone environment is unknown but clues are given by the fact that Spp24, or proteolytic products of Spp24, bind cytokines of the TGF-β superfamily and also activate intracellular signaling pathways. Several potential biotherapeutics have been engineered from this protein including materials that enhance BMP-induced bone healing and, on the other hand, materials that inhibit BMPs in clinical situations where this is called for such as reducing BMP-induced inflammation and inhibiting tumors dependent on BMP autocrine systems. As understanding of the structure and function of this protein increases, more opportunities for rationally developed therapeutics will become apparent.

  14. Adrenergic Activation of Melatonin Secretion in Ovine Pineal Explants in Short-Term Superfusion Culture Occurs via Protein Synthesis Independent and Dependent Phenomena

    Directory of Open Access Journals (Sweden)

    Bogdan Lewczuk

    2014-01-01

    Full Text Available The ovine pineal is generally considered as an interesting model for the study on adrenergic regulation of melatonin secretion due to some functional similarities with this gland in the human. The present investigations, performed in the superfusion culture of pineal explants, demonstrated that the norepinephrine-induced elevation of melatonin secretion in ovine pinealocytes comprised of two subsequent periods: a rapid increase phase and a slow increase phase. The first one included the quick rise in release of N-acetylserotonin and melatonin, occurring parallel to elevation of NE concentration in the medium surrounding explants. This rapid increase phase was not affected by inhibition of translation. The second, slow increase phase began after NE level had reached the maximum concentration in the culture medium and lasted about two hours. It was completely abolished by the treatment with translation inhibitors. The obtained results showed for the first time that the regulation of N-acetylserotonin synthesis in pinealocytes of some species like the sheep involves the on/off mechanism, which is completely independent of protein synthesis and works very fast. They provided strong evidence pointing to the need of revision of the current opinion that arylalkylamines N-acetyltransferase activity in pinealocytes is controlled exclusively by changes in enzyme abundance.

  15. Effects of laminin and collagen type I on the morphology and secretion of proteins in human hepatoblastoma and hepatoma cell lines.

    Directory of Open Access Journals (Sweden)

    Tokiwa,Takayoshi

    1990-04-01

    Full Text Available The effects of laminin (LAM and collagen type I (C-I on human hepatoblastoma (HuH-6 and hepatoma (HuH-7 cell lines were investigated. C-I was superior to LAM in supporting the attachment of the cells, especially of HuH-6, to plastic surfaces. No effect of LAM and C-I on cellular morphology was recognizable by phase contrast microscopy. By scanning electron microscopy (SEM, much more microvilli were found on the cell surface of HuH-6 on LAM substrate than on C-I substrate. In HuH-7 cells, however, these microvilli were rarely found on either LAM substrate or C-I substrate. The gel profile of the proteins secreted by HuH-6 and HuH-7 cells was not affected by the culture substrate except for the major band, though the amount of alpha-fetoprotein (AFP secreted was larger when the cells were cultured on LAM substrate than on C-I substrate. These results indicate that the ability of LAM or C-I to enhance attachment is different from that to enhance AFP production or microvilli expression in HuH-6 cells and probably in HuH-7 cells.

  16. Effects of secretive bone morphogenetic protein 2 induced by gene transfection on the biological changes of NIH3T3 cells

    Institute of Scientific and Technical Information of China (English)

    SUN Wei-bin; WANG Juan; LU Chun; TANG Gui-xia

    2005-01-01

    Background Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta superfamily, are powerful regulators of cartilage and bone formation. This study investigated the biological changes of NIH3T3 cells incubated with secretive BMP2 that was induced by gene transfection through transwell. Methods Eukaryonic expression vector (pcDNA3.1-B2) was transfered into NIH3T3 cells with SofastTM,a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expressions of BMP2 were determined by immunohistochemical stain and enzyme-linked immunosorbent assay. NIH3T3 cells were co-cultured with hBMP2 gene transfecting cells through transwell, and the ultrastructure, alkaline phosphatase activity and the expression of osteocalcin (the marker of osteogenetic differentiation) changes were observed. Results There were cytoplasmic and extracellular expressions of BMP2 in transfecting NIH3T3 cells. The ultrastructural changes, the high activity of alkaline phosphatase and the positive stain of osteocalcin suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells. Conclusion Secretive BMP2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.

  17. Incretin hormone secretion over the day

    DEFF Research Database (Denmark)

    Ahren, B; Carr, RD; Deacon, Carolyn F.

    2010-01-01

    . Regulation of incretin hormone secretion is less well characterized. The main stimulus for incretin hormone secretion is presence of nutrients in the intestinal lumen, and carbohydrate, fat as well as protein all have the capacity to stimulate GIP and GLP-1 secretion. More recently, it has been established...

  18. The secreted immunoglobulin domain proteins ZIG-5 and ZIG-8 cooperate with L1CAM/SAX-7 to maintain nervous system integrity.

    Directory of Open Access Journals (Sweden)

    Claire Y Bénard

    Full Text Available During nervous system development, neuronal cell bodies and their axodendritic projections are precisely positioned through transiently expressed patterning cues. We show here that two neuronally expressed, secreted immunoglobulin (Ig domain-containing proteins, ZIG-5 and ZIG-8, have no detectable role during embryonic nervous system development of the nematode Caenorhabditis elegans but are jointly required for neuronal soma and ventral cord axons to maintain their correct position throughout postembryonic life of the animal. The maintenance defects observed upon removal of zig-5 and zig-8 are similar to those observed upon complete loss of the SAX-7 protein, the C. elegans ortholog of the L1CAM family of adhesion proteins, which have been implicated in several neurological diseases. SAX-7 exists in two isoforms: a canonical, long isoform (SAX-7L and a more adhesive shorter isoform lacking the first two Ig domains (SAX-7S. Unexpectedly, the normally essential function of ZIG-5 and ZIG-8 in maintaining neuronal soma and axon position is completely suppressed by genetic removal of the long SAX-7L isoform. Overexpression of the short isoform SAX-7S also abrogates the need for ZIG-5 and ZIG-8. Conversely, overexpression of the long isoform disrupts adhesion, irrespective of the presence of the ZIG proteins. These findings suggest an unexpected interdependency of distinct Ig domain proteins, with one isoform of SAX-7, SAX-7L, inhibiting the function of the most adhesive isoform, SAX-7S, and this inhibition being relieved by ZIG-5 and ZIG-8. Apart from extending our understanding of dedicated neuronal maintenance mechanisms, these findings provide novel insights into adhesive and anti-adhesive functions of IgCAM proteins.

  19. Genes encoding a group of related small secreted proteins from the gut of Hessian fly larvae [Mayetiola destructor (Say)

    Institute of Scientific and Technical Information of China (English)

    MING-SHUN CHEN; XIANG LIU; YU-CHENG ZHU; JOHN C. REESE; GERALD E. WILDE

    2006-01-01

    A group of related genes has been isolated and characterized from the gut of Hessian fly larvae [Mayetiola destructor (Say)]. Members in this group appear to encode proteins with secretary signal peptides at the N-terminals. The mature putative proteins are small, acidic proteins with calculated molecular masses of 14.5 to 15.3 kDa, and isoelectric points from 4.56 to 4.88. Northern blot analysis revealed that these genes are expressed predominantly in the gut of Hessian fly larvae and pupae. Two related genes, G10K1 and G10K2, were isolated as tandem repeats. Both genes contain three exons and two introns.The intron/exon boundaries were conserved in terms of amino acid encoding, suggesting that they arose by gene duplication. The fact that the frequency of this group of clones in a gut cDNA library higher than that of total cDNA clones encoding digestive enzymes suggested that this group of proteins may perform an important function in the gut physiology of this insect. However, the exact functions of these proteins are as yet known since no sequence similarity could be identified between these proteins and any known sequences in public databases using standard methods.

  20. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    DEFF Research Database (Denmark)

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kudryavtsev, Denis

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation......AChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding...

  1. HrpG and HrpV proteins from the Type III secretion system of Erwinia amylovora form a stable heterodimer.

    Science.gov (United States)

    Gazi, Anastasia D; Charova, Spyridoula; Aivaliotis, Michalis; Panopoulos, Nicholas J; Kokkinidis, Michael

    2015-01-01

    Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and pu