Science.gov

Sample records for seca2-dependent secretory protein

  1. RFP tags for labeling secretory pathway proteins

    SciTech Connect

    Han, Liyang; Zhao, Yanhua; Xu, Pingyong; Huan, Shuangyan

    2014-05-09

    Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.

  2. Secretory Granule Membrane Protein Recycles Through Multivesicular Bodies

    PubMed Central

    Bäck, Nils; Rajagopal, Chitra; Mains, Richard E.; Eipper, Betty A.

    2010-01-01

    The recycling of secretory granule membrane proteins that reach the plasma membrane following exocytosis is poorly understood. As a model, peptidylglycine α-amidating monooxygenase (PAM), a granule membrane protein that catalyzes a final step in peptide processing was examined. Ultrastructural analysis of antibody internalized by PAM and surface biotinylation demonstrated efficient return of plasma membrane PAM to secretory granules. Electron microscopy revealed the rapid movement of PAM from early endosomes to the limiting membranes of multivesicular bodies and then into intralumenal vesicles. Wheat germ agglutinin and PAM antibody internalized simultaneously were largely segregated when they reached multivesicular bodies. Mutation of basally phosphorylated residues (Thr946, Ser949) in the cytoplasmic domain of PAM to Asp (TS/DD) substantially slowed its entry into intralumenal vesicles. Mutation of the same sites to Ala (TS/AA) facilitated the entry of internalized PAM into intralumenal vesicles and its subsequent return to secretory granules. Entry of PAM into intralumenal vesicles is also associated with a juxtamembrane endoproteolytic cleavage that releases a 100 kDa soluble PAM fragment that can be returned to secretory granules. Controlled entry into the intralumenal vesicles of multivesicular bodies plays a key role in the recycling of secretory granule membrane proteins. PMID:20374556

  3. AP-1A controls secretory granule biogenesis and trafficking of membrane secretory granule proteins.

    PubMed

    Bonnemaison, Mathilde; Bäck, Nils; Lin, Yimo; Bonifacino, Juan S; Mains, Richard; Eipper, Betty

    2014-10-01

    The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-μ1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Coimmunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition.

  4. Progressive quality control of secretory proteins in the early secretory compartment by ERp44

    PubMed Central

    Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto

    2014-01-01

    ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval via interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here we show that also conserved histidines in the C-terminal tail regulate ERp44 in vivo. Mutants lacking these histidines are hyperactive in retaining substrates. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon over-expression of different partners. The ensuing gradients may help optimising folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes. PMID:25097228

  5. Progressive quality control of secretory proteins in the early secretory compartment by ERp44.

    PubMed

    Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto

    2014-10-01

    ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes.

  6. Secretory proteins as potential semiochemical carriers in the horse.

    PubMed

    D'Innocenzo, Barbara; Salzano, Anna Maria; D'Ambrosio, Chiara; Gazzano, Angelo; Niccolini, Alberto; Sorce, Carlo; Dani, Francesca Romana; Scaloni, Andrea; Pelosi, Paolo

    2006-11-14

    Two soluble proteins were isolated as major secretory products of horse sweat and of the parotid gland and characterized for structural and functional properties. The first protein, lipocalin allergen EquC1, was characterized for its glycosylation sites and bound glycosidic moieties. Only one (Asn53) of the two putative glycosylation sites within the sequence was post-translationally modified; a different glycosylation pattern was determined with respect to data previously reported. When purified from horse sweat, this protein contained oleamide and other organic molecules as natural ligands. Ligand binding experiments indicated good protein selectivity toward volatile compounds having a straight chain structure of 9-11 carbon atoms, suggesting a role of this lipocalin in chemical communication. The second protein, here reported for the first time in the horse, belongs to the group of parotid secretory proteins, part of a large superfamily of binding proteins whose function in most cases is still unclear. This protein was sequenced and characterized for its post-translational modifications. Of the three cysteine residues present, two were involved in a disulfide bridge (Cys155-Cys198). A model, built up on the basis of similar proteins, indicated a general fold characterized by the presence of a long hydrophobic barrel. Binding experiments performed with a number of different organic compounds failed to identify ligands for this protein with a well-defined physiological role.

  7. In vitro aggregation of the regulated secretory protein chromogranin A.

    PubMed Central

    Jain, Renu K; Chang, Wen Tzu; Geetha, Chitta; Joyce, Paul B M; Gorr, Sven-Ulrik

    2002-01-01

    Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation. PMID:12175332

  8. Neuroendocrine secretory protein 7B2: structure, expression and functions.

    PubMed Central

    Mbikay, M; Seidah, N G; Chrétien, M

    2001-01-01

    7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation. PMID:11439082

  9. Orchestration of secretory protein folding by ER chaperones

    PubMed Central

    Gidalevitz, Tali; Stevens, Fred; Argon, Yair

    2013-01-01

    The endoplasmic reticulum is a major compartment of protein biogenesis in the cell, dedicated to production of secretory, membrane and organelle proteins. The secretome has distinct structural and post-translational characteristics, since folding in the ER occurs in an environment that is distinct in terms of its ionic composition, dynamics and requirements for quality contol. The folding machinery in the ER therefore includes chaperones and folding enzymes that introduce, monitor and react to disulfide bonds, glycans, and fluctuations of luminal calcium. We describe the major chaperone networks in the lumen and discuss how they have distinct modes of operation that enable cells to accomplish highly efficient production of the secretome. PMID:23507200

  10. The secretory carrier membrane protein family: structure and membrane topology.

    PubMed

    Hubbard, C; Singleton, D; Rauch, M; Jayasinghe, S; Cafiso, D; Castle, D

    2000-09-01

    Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane

  11. Proteomic analysis of Toxocara canis excretory and secretory (TES) proteins.

    PubMed

    Sperotto, Rita Leal; Kremer, Frederico Schmitt; Aires Berne, Maria Elisabeth; Costa de Avila, Luciana F; da Silva Pinto, Luciano; Monteiro, Karina Mariante; Caumo, Karin Silva; Ferreira, Henrique Bunselmeyer; Berne, Natália; Borsuk, Sibele

    2017-01-01

    Toxocariasis is a neglected disease, and its main etiological agent is the nematode Toxocara canis. Serological diagnosis is performed by an enzyme-linked immunosorbent assay using T. canis excretory and secretory (TES) antigens produced by in vitro cultivation of larvae. Identification of TES proteins can be useful for the development of new diagnostic strategies since few TES components have been described so far. Herein, we report the results obtained by proteomic analysis of TES proteins using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. TES fractions were separated by one-dimensional SDS-PAGE and analyzed by LC-MS/MS. The MS/MS spectra were compared with a database of protein sequences deduced from the genome sequence of T. canis, and a total of 19 proteins were identified. Classification according to the signal peptide prediction using the SignalP server showed that seven of the identified proteins were extracellular, 10 had cytoplasmic or nuclear localization, while the subcellular localization of two proteins was unknown. Analysis of molecular functions by BLAST2GO showed that the majority of the gene ontology (GO) terms associated with the proteins present in the TES sample were associated with binding functions, including but not limited to protein binding (GO:0005515), inorganic ion binding (GO:0043167), and organic cyclic compound binding (GO:0097159). This study provides additional information about the exoproteome of T. canis, which can lead to the development of new strategies for diagnostics or vaccination.

  12. Brevibacillus expression system: host-vector system for efficient production of secretory proteins.

    PubMed

    Mizukami, Makoto; Hanagata, Hiroshi; Miyauchi, Akira

    2010-04-01

    Brevibacillus expression system is an effective bacterial expression system for secretory proteins. The host bacterium, Brevibacillus choshinensis, a gram-positive bacterium, has strong capacity to secrete a large amount of proteins (approximately 30 g/L), which mostly consist of cell wall protein. A host-vector system that utilizes such high expression capacity has been constructed for the production of secretory proteins and tested for various heterologous proteins, including cytokines, enzymes, antigens, and adjuvants.

  13. Cysteine rich secretory proteins in reproduction and venom.

    PubMed

    Gibbs, Gerard M; O'Bryan, Moira K

    2007-01-01

    The cysteine rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract and in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionarily diverse N-terminal domain and a characteristic cysteine rich C-terminal domain which we refer to as the Crisp domain. Since their identification 30 years ago Crisp research in mammals has focused on the characterisation of their expression localization to infer function. While no doubt important observations, these have not substantially led to an understanding of the biochemical activity of the Crisps and their role in sperm function or fertilisation. Recently, we demonstrated that the Crisp-2 Crisp domain has a structure similar to ion channel toxins ShK and BgK and was itself able to regulate Ca2+ flux through ryanodine receptors. These data build upon the previous characterizations of reptile venom Crisps as regulators of several types of ion channels and permits for the first time a dissection of the biochemical activity of mammalian Crisps.

  14. Modulation of Sertoli cell secretory function by rat round spermatid protein(s).

    PubMed

    Onoda, M; Djakiew, D

    1990-10-01

    The influence of rat round spermatid protein(s) (RSP) on protein synthesis and secretory function of Sertoli cells was used in the bicameral chamber system. Round spermatids (RS) were purified from 90-day-old rats by centrifugal elutriation. RS were incubated in a supplement-enriched culture medium that lacked exogenous proteins. The RS-conditioned media were dialysed and lyophilized to obtain RSP. Most de novo protein synthesized under basal conditions by Sertoli cells (18-day-old) was secreted into the apical chamber (apical/basal ratio: 3.42). Follicle-stimulating hormone (FSH, 100 ng/ml) stimulated total protein secretion from Sertoli cells by a factor of 1.54. The RSP (100 micrograms/ml) stimulated total protein secretion from Sertoli cells by a factor of 2.33. The enhancement of total Sertoli cell protein secretion by FSH and RSP additively increased by a factor of 2.82. The combined effect of FSH and RSP on total protein secretion from Sertoli cells was dose dependent and saturated at approximately 200 micrograms/ml of RSP. Polarity of total protein secretion from Sertoli cells (apical/basal ratio: 3.42) was stimulated by RSP predominantly in the apical direction (apical/basal ratio: 8.48). The modulation of radiolabeled Sertoli cell secretory proteins (ceruloplasmin, CP; sulfated glycoprotein-2, SGP-2; testins and transferrin, Tf) by cold (non-labeled) RSP was investigated by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The secretion of CP, SGP-2 and Tf was stimulated in a dose-dependent manner by the addition of RSP up to a saturating concentration of between 200 and 300 micrograms/ml, whereas the secretion of Sertoli cell testins did not reach saturation at 300 micrograms/ml RSP. These results indicate that FSH and RSP independently modulate Sertoli cell protein secretion, and that Sertoli cell secretory proteins may differentially respond to RSP stimulation.

  15. Lumenal protein within secretory granules affects fusion pore expansion.

    PubMed

    Weiss, Annita Ngatchou; Anantharam, Arun; Bittner, Mary A; Axelrod, Daniel; Holz, Ronald W

    2014-07-01

    It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ∼2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only

  16. Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

    PubMed

    Rindler, Michael J; Xu, Chong-Feng; Gumper, Iwona; Cen, Chuan; Sonderegger, Peter; Neubert, Thomas A

    2008-04-01

    Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.

  17. A hydrophobic patch in a charged alpha-helix is sufficient to target proteins to dense core secretory granules.

    PubMed

    Dikeakos, Jimmy D; Lacombe, Marie-Josée; Mercure, Chantal; Mireuta, Matei; Reudelhuber, Timothy L

    2007-01-12

    Many endocrine and neuroendocrine cells contain specialized secretory organelles called dense core secretory granules. These organelles are the repository of proteins and peptides that are secreted in a regulated manner when the cell receives a physiological stimulus. The targeting of proteins to these secretory granules is crucial for the generation of certain peptide hormones, including insulin and ACTH. Although previous work has demonstrated that proteins destined to a variety of cellular locations, including secretory granules, contain targeting sequences, no single consensus sequence for secretory granule-sorting signals has emerged. We have shown previously that alpha-helical domains in the C-terminal tail of the prohormone convertase PC1/3 play an important role in the ability of this region of the protein to direct secretory granule targeting (Jutras, I. Seidah, N. G., and Reudelhuber, T. L. (2000) J. Biol. Chem. 275, 40337-40343). In this study, we show that a variety of alpha-helical domains are capable of directing a heterologous secretory protein to granules. By testing a series of synthetic alpha-helices, we also demonstrate that the presence of charged (either positive or negative) amino acids spatially segregated from a hydrophobic patch in the alpha-helices of secretory proteins likely plays a critical role in the ability of these structures to direct secretory granule sorting.

  18. Multiplex Sequential Immunoprecipitation of Insulin Secretory Granule Proteins from Radiolabeled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Specific proteins can then be immunoprecipitated and analyzed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine, followed by multiplex sequential immunoprecipitation of insulin and three other secretory granule accessory proteins. This provided a means of distinguishing those pancreatic islet proteins with different biosynthetic rates in response to the media glucose concentrations.

  19. Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland

    SciTech Connect

    Marino, C.R.; Castle, J.D.; Gorelick, F.S. )

    1990-07-01

    An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with ({sup 32}P)orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

  20. Secretory COPII Protein SEC31B Is Required for Pollen Wall Development1[OPEN

    PubMed Central

    Zhao, Bingchun; Shi, Haidan; Wang, Wanlei; Liu, Xiaoyu; Gao, Hui; Wang, Xiaoxiao; Zhang, Yinghui; Yang, Meidi; Li, Rui

    2016-01-01

    The pollen wall protects pollen grains from abiotic and biotic stresses. During pollen wall development, tapetal cells play a vital role by secreting proteins, signals, and pollen wall material to ensure microspore development. But the regulatory mechanism underlying the secretory pathway of the tapetum is largely unknown. Here, we characterize the essential role of the Arabidopsis (Arabidopsis thaliana) COPII protein SECRETORY31B (SEC31B) in pollen wall development and the secretory activity of tapetal cells. The sporophyte-controlled atsec31b mutant exhibits severe pollen and seed abortion. Transmission electron microscopy observation indicates that pollen exine formation in the atsec31b mutant is disrupted significantly. AtSEC31B is a functional COPII protein revealed by endoplasmic reticulum (ER) exit site localization, interaction with AtSEC13A, and retarded ER-Golgi protein trafficking in the atsec31b mutant. A genetic tapetum-specific rescue assay indicates that AtSEC31B functions primarily in the tapetum. Moreover, deletion of AtSEC31B interrupted the formation of the ER-derived tapetosome and altered the location of the ATP-BINDING CASSETTE TRANSPORTER9 protein in the tapetum. Therefore, this work demonstrates that AtSEC31B plays a vital role in pollen wall development by regulating the secretory pathway of the tapetal cells. PMID:27634427

  1. Analysis of Protein Localization and Secretory Pathway Function Using the Yeast "Saccharomyces Cerevisiae"

    ERIC Educational Resources Information Center

    Vallen, Elizabeth

    2002-01-01

    The isolation and characterization of mutants has been crucial in understanding a number of processes in the field of cell biology. In this exercise, students examine the effects of mutations in the secretory pathway on protein localization. Yeast strains deficient for synthesis of histidinol dehydrogenase are transformed with a plasmid encoding a…

  2. Regulated and constitutive protein targeting can be distinguished by secretory polarity in thyroid epithelial cells

    PubMed Central

    1991-01-01

    We have studied concurrent apical/basolateral and regulated/constitutive secretory targeting in filter-grown thyroid epithelial monolayers in vitro, by following the exocytotic routes of two newly synthesized endogenous secretory proteins, thyroglobulin (Tg) and p500. Tg is a regulated secretory protein as indicated by its acute secretory response to secretagogues. Without stimulation, pulse-labeled Tg exhibits primarily two kinetically distinct routes: less than or equal to 80% is released in an apical secretory phase which is largely complete by 6-10 h, with most of the remaining Tg retained in intracellular storage from which delayed apical discharge is seen. The rapid export observed for most Tg is unlikely to be because of default secretion, since its apical polarity is preserved even during the period (less than or equal to 10 h) when p500 is released basolaterally by a constitutive pathway unresponsive to secretagogues. p500 also exhibits a second, kinetically distinct secretory route: at chase times greater than 10 h, a residual fraction (less than or equal to 8%) of p500 is secreted with an apical preponderance similar to that of Tg. It appears that this fraction of p500 has failed to be excluded from the regulated pathway, which has a predetermined apical polarity. From these data we hypothesize that a targeting hierarchy may exist in thyroid epithelial cells such that initial sorting to the regulated pathway may be a way of insuring apical surface delivery from one of two possible exocytotic routes originating in the immature storage compartment. PMID:1991788

  3. Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum.

    PubMed

    Yim, Sung Sun; Choi, Jae Woong; Lee, Roo Jin; Lee, Yong Jae; Lee, Se Hwa; Kim, So Young; Jeong, Ki Jun

    2016-01-01

    Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum.

  4. Cysteine-rich secretory proteins are not exclusively expressed in the male reproductive tract.

    PubMed

    Reddy, Thulasimala; Gibbs, Gerard M; Merriner, D Jo; Kerr, Jeffrey B; O'Bryan, Moira K

    2008-11-01

    The Cysteine-RIch Secretory Proteins (CRISPs) are abundantly produced in the male reproductive tract of mammals and within the venom of reptiles and have been shown to regulate ion channel activity. CRISPs, along with the Antigen-5 proteins and the Pathogenesis related-1 (Pr-1) proteins, form the CAP superfamily of proteins. Analyses of EST expression databases are increasingly suggesting that mammalian CRISPs are expressed more widely than in the reproductive tract. We, therefore, conducted a reverse transcription PCR expression profile and immunohistochemical analyses of 16 mouse tissues to define the sites of production of each of the four murine CRISPs. These data showed that each of the CRISPs have distinct and sometimes overlapping expression profiles, typically associated with the male and female reproductive tract, the secretory epithelia of exocrine glands, and immune tissues including the spleen and thymus. These investigations raise the potential for a role for CRISPs in general mammalian physiology.

  5. Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

  6. Intracellular and transcellular transport of secretory and membrane proteins in the rat hepatocyte

    SciTech Connect

    Sztul, E.S.

    1984-01-01

    The intra- and transcellular transport of hepatic secretory and membrane proteins was studied in rats in vivo using (/sup 3/H)fucose and (/sup 35/S)cyteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile and plasma were separated by SDS-PAGE and identified by fluorography. /sup 3/H-radioactivity in Golgi fractions peaked at 10 min post injection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from the Golgi complex occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 10 min later than the bulk of content proteins. A major 80K form of Secretory Component (SC) was identified in the bile by precipitation with an anti IgA antibody. A comparative study of kinetics of transport of /sup 35/S-labeled SC and /sup 35/S-labeled albumin showed that albumin peaked in bile at approx.45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins which are delivered to the bile canaliculus (BC).

  7. Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona.

    PubMed

    Hayashi, Yuya; Miclaus, Teodora; Engelmann, Péter; Autrup, Herman; Sutherland, Duncan S; Scott-Fordsmand, Janeck J

    2016-01-01

    Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect the corona composition, the extent to which nanoparticles influence the cells' protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time-dependent manner (2, 4, 8 and 24 h at a low-cytotoxic concentration), and examined the implication of the temporal changes in transcriptional profiles of secretory proteins with a particular reference to that of lysenin. NM-300K was accumulated in/at the cells and lysenin was, after transient induction, gradually suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll-like receptor (TLR) signaling. This offers an intriguing perspective of the nanosilver pathophysiology in earthworms, in which the conserved pattern recognition receptor TLRs may play an effector role.

  8. 2D Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically Labelled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids such is a common method for investigating the biosynthetic rates of proteins. In this way, the abundance of newly synthesized proteins can be determined by several proteomic techniques including 2D gel electrophoresis (2DE). This chapter describes a protocol for labelling pancreatic islets with (35)S-methionine in the presence of low and high concentrations of glucose, followed by subcellular fractionation enrichment of secretory granule proteins and analysis of the granule protein contents by 2DE. This demonstrated that the biosynthetic rates of most of the granule proteins are co-ordinately regulated in the presence of stimulatory glucose concentrations.

  9. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

    PubMed Central

    Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

    2013-01-01

    The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

  10. Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

    PubMed Central

    Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

    2012-01-01

    The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

  11. Cysteine-Rich Atrial Secretory Protein from the Snail Achatina achatina: Purification and Structural Characterization

    PubMed Central

    Shabelnikov, Sergey; Kiselev, Artem

    2015-01-01

    Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported. In this study, we describe an 18.1-kDa, cysteine-rich atrial secretory protein (CRASP) isolated from the terrestrial snail Achatina achatina that has no detectable sequence similarity to any known protein or nucleotide sequence. CRASP is an acidic, 158-residue, N-glycosylated protein composed of eight alpha-helical segments stabilized with five disulphide bonds. A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold. CRASP is most strongly expressed in the atrium in secretory atrial granular cells, and substantial amounts of CRASP are released from the heart upon nerve stimulation. CRASP is detected in the haemolymph of intact animals at nanomolar concentrations. CRASP is the first secretory protein expressed in molluscan atrium to be reported. We propose that CRASP is an example of a taxonomically restricted gene that might be responsible for adaptations specific for terrestrial pulmonates. PMID:26444993

  12. A Critical Role for Toxoplasma gondii Vacuolar Protein Sorting VPS9 in Secretory Organelle Biogenesis and Host Infection

    PubMed Central

    Sakura, Takaya; Sindikubwabo, Fabien; Oesterlin, Lena K.; Bousquet, Hugo; Slomianny, Christian; Hakimi, Mohamed-Ali; Langsley, Gordon; Tomavo, Stanislas

    2016-01-01

    Accurate sorting of proteins to the three types of parasite-specific secretory organelles namely rhoptry, microneme and dense granule in Toxoplasma gondii is crucial for successful host cell invasion by this obligate intracellular parasite. Despite its tiny body architecture and limited trafficking machinery, T. gondii relies heavily on transport of vesicles containing proteins, lipids and important virulence-like factors that are delivered to these secretory organelles. However, our understanding on how trafficking of vesicles operates in the parasite is still limited. Here, we show that the T. gondii vacuolar protein sorting 9 (TgVps9), has guanine nucleotide exchange factor (GEF) activity towards Rab5a and is crucial for sorting of proteins destined to secretory organelles. Our results illuminate features of TgVps9 protein as a key trafficking facilitator that regulates protein maturation, secretory organelle formation and secretion, thereby ensuring a primary role in host infection by T. gondii. PMID:27966671

  13. Machine Learning of Protein Interactions in Fungal Secretory Pathways.

    PubMed

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja; Rousu, Juho

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker's yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities.

  14. Machine Learning of Protein Interactions in Fungal Secretory Pathways

    PubMed Central

    Kludas, Jana; Arvas, Mikko; Castillo, Sandra; Pakula, Tiina; Oja, Merja; Brouard, Céline; Jäntti, Jussi; Penttilä, Merja

    2016-01-01

    In this paper we apply machine learning methods for predicting protein interactions in fungal secretion pathways. We assume an inter-species transfer setting, where training data is obtained from a single species and the objective is to predict protein interactions in other, related species. In our methodology, we combine several state of the art machine learning approaches, namely, multiple kernel learning (MKL), pairwise kernels and kernelized structured output prediction in the supervised graph inference framework. For MKL, we apply recently proposed centered kernel alignment and p-norm path following approaches to integrate several feature sets describing the proteins, demonstrating improved performance. For graph inference, we apply input-output kernel regression (IOKR) in supervised and semi-supervised modes as well as output kernel trees (OK3). In our experiments simulating increasing genetic distance, Input-Output Kernel Regression proved to be the most robust prediction approach. We also show that the MKL approaches improve the predictions compared to uniform combination of the kernels. We evaluate the methods on the task of predicting protein-protein-interactions in the secretion pathways in fungi, S.cerevisiae, baker’s yeast, being the source, T. reesei being the target of the inter-species transfer learning. We identify completely novel candidate secretion proteins conserved in filamentous fungi. These proteins could contribute to their unique secretion capabilities. PMID:27441920

  15. Secretory-granule dynamics visualized in vivo with a phogrin-green fluorescent protein chimaera.

    PubMed Central

    Pouli, A E; Emmanouilidou, E; Zhao, C; Wasmeier, C; Hutton, J C; Rutter, G A

    1998-01-01

    To image the behaviour in real time of single secretory granules in neuroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogrin (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 beta-cells and pheochromocytoma PC12 cells, the chimaera was localized efficiently (up to 95%) to dense-core secretory granules (diameter 200-1000 nm), identified by co-immunolocalization with anti-(pro-)insulin antibodies in INS-1 cells and dopamine beta-hydroxylase in PC12 cells. Using laser-scanning confocal microscopy and digital image analysis, we have used this chimaera to monitor the effects of secretagogues on the dynamics of secretory granules in single living cells. In unstimulated INS-1 beta-cells, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5-10 s and mean displacement <1 microm. Both elevated glucose concentrations (30 mM), and depolarization of the plasma membrane with K+, provoked large (5-10 microm) saltatory excursions of granules across the cell, which were never observed in cells maintained at low glucose concentration. By contrast, long excursions of granules occurred in PC12 cells without stimulation, and occurred predominantly from the cell body towards the cell periphery and neurite extensions. Purinergic-receptor activation with ATP provoked granule movement towards the membrane of PC12 cells, resulting in the transfer of fluorescence to the plasma membrane consistent with fusion of the granule and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin-EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule-cytoskeletal interactions and the trafficking of a granule-specific transmembrane protein during the cycle of exocytosis and endocytosis. PMID:9639579

  16. Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

    PubMed

    Schenk, Jörg A; Fettke, Joerg; Lenz, Christine; Albers, Katharina; Mallwitz, Frank; Gajovic-Eichelmann, Nenad; Ehrentreich-Förster, Eva; Kusch, Emely; Sellrie, Frank

    2012-03-31

    The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.

  17. Ranking Gene Ontology terms for predicting non-classical secretory proteins in eukaryotes and prokaryotes.

    PubMed

    Huang, Wen-Lin

    2012-11-07

    Protein secretion is an important biological process for both eukaryotes and prokaryotes. Several sequence-based methods mainly rely on utilizing various types of complementary features to design accurate classifiers for predicting non-classical secretory proteins. Gene Ontology (GO) terms are increasing informative in predicting protein functions. However, the number of used GO terms is often very large. For example, there are 60,020 GO terms used in the prediction method Euk-mPLoc 2.0 for subcellular localization. This study proposes a novel approach to identify a small set of m top-ranked GO terms served as the only type of input features to design a support vector machine (SVM) based method Sec-GO to predict non-classical secretory proteins in both eukaryotes and prokaryotes. To evaluate the Sec-GO method, two existing methods and their used datasets are adopted for performance comparisons. The Sec-GO method using m=436 GO terms yields an independent test accuracy of 96.7% on mammalian proteins, much better than the existing method SPRED (82.2%) which uses frequencies of tri-peptides and short peptides, secondary structure, and physicochemical properties as input features of a random forest classifier. Furthermore, when applying to Gram-positive bacterial proteins, the Sec-GO with m=158 GO terms has a test accuracy of 94.5%, superior to NClassG+ (90.0%) which uses SVM with several feature types, comprising amino acid composition, di-peptides, physicochemical properties and the position specific weighting matrix. Analysis of the distribution of secretory proteins in a GO database indicates the percentage of the non-classical secretory proteins annotated by GO is larger than that of classical secretory proteins in both eukaryotes and prokaryotes. Of the m top-ranked GO features, the top-four GO terms are all annotated by such subcellular locations as GO:0005576 (Extracellular region). Additionally, the method Sec-GO is easily implemented and its web tool of

  18. Structure and function of snake venom cysteine-rich secretory proteins.

    PubMed

    Yamazaki, Yasuo; Morita, Takashi

    2004-09-01

    Cysteine-rich secretory proteins (CRISPs) are primarily found in the epididymis of mammals and are expressed in diverse organisms. However, the functions of most CRISPs remain unknown. Recent studies reveal that CRISPs are widely distributed in snake venoms and that they inhibit smooth muscle contraction and cyclic nucleotide-gated ion channels. In this review, we discuss recent findings on several snake venom-derived CRISPs.

  19. Two Dimensional Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically-Labeled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2DE). This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine in the presence of basal and stimulatory concentrations of glucose, followed by subcellular fractionation to produce a secretory granule fraction and analysis of the granule protein contents by 2DE. This provides a means of determining whether or not the biosynthetic rates of the entire granule constituents are coordinately regulated.

  20. Sending proteins to dense core secretory granules: still a lot to sort out.

    PubMed

    Dikeakos, Jimmy D; Reudelhuber, Timothy L

    2007-04-23

    The intracellular sorting of peptide hormone precursors to the dense core secretory granules (DCSGs) is essential for their bioactivation. Despite the fundamental importance of this cellular process, the nature of the sorting signals for entry of proteins into DCSGs remains a source of vigorous debate. This review highlights recent discoveries that are consistent with a model in which several protein domains, acting in a cell-specific fashion and at different steps in the sorting process, act in concert to regulate the entry of proteins into DCSGs.

  1. Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland.

    PubMed

    Radha, Arumugam; Sree, Sreesha; Faisal, Kunnathodi; Kumar, G Pradeep; Oommen, Oommen V; Akbarsha, Mohammad A

    2014-10-01

    Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands.

  2. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs

    PubMed Central

    Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

    2016-01-01

    In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4+ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals’ endocrine system. PMID:27775083

  3. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs.

    PubMed

    Céspedes, María Virtudes; Fernández, Yolanda; Unzueta, Ugutz; Mendoza, Rosa; Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejando; Álamo, Patricia; Toledo-Rubio, Verónica; Ferrer-Miralles, Neus; Vázquez, Esther; Schwartz, Simó; Abasolo, Ibane; Corchero, José Luis; Mangues, Ramon; Villaverde, Antonio

    2016-10-24

    In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4(+) cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals' endocrine system.

  4. Subcellular location of secretory proteins retained in the liver during the ethanol-induced inhibition of hepatic protein secretion in the rat

    SciTech Connect

    Volentine, G.D.; Tuma, D.J.; Sorrell, M.F.

    1986-01-01

    Ethanol administration inhibits the secretion of proteins by the liver, resulting in their hepatocellular retention. Experiments were designed in this study to determine the subcellular location of the retained secretory proteins. Ethanol was administered acutely to nonfasted rats by gastric intubation, whereas control animals received an isocaloric dose of glucose. Two hours after intubation, when maximum blood ethanol levels (45 mM) were observed, (/sup 3/H)leucine and (/sup 14/C)fucose were injected simultaneously into the dorsal vein of the penis. The labelling of secretory proteins was determined in the liver and plasma at various time periods after label injection. Ethanol treatment decreased the secretion of both leucine- and fucose-labeled proteins into the plasma. This inhibition of secretion was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled immunoprecipitable secretory proteins. At the time of maximum inhibition of secretion, leucine labeled secretory proteins located in the Golgi apparatus represented about 50% of the accumulated secretory proteins in the livers of the ethanol-treated rats, whereas the remainder was essentially equally divided among the rough and smooth endoplasmic reticulum and cytosol. Because fucose is incorporated into secretory proteins almost exclusively in the Golgi complex, fucose-labeled proteins accumulated in the livers of the ethanol-treated rats mainly in the Golgi apparatus, with the remainder located in the cytosol. These results show that ethanol administration causes an impaired movement of secretory proteins along the secretory pathway, and that secretory proteins accumulate mainly, but not exclusively, in the Golgi apparatus.

  5. Immunodominant antigens in Naegleria fowleri excretory--secretory proteins were potential pathogenic factors.

    PubMed

    Kim, Jong-Hyun; Yang, Ae-Hee; Sohn, Hae-Jin; Kim, Daesik; Song, Kyoung-Ju; Shin, Ho-Joon

    2009-11-01

    Naegleria fowleri, a ubiquitous pathogenic free-living amoeba, is the most virulent species and causes primary amoebic meningoencephalitis in laboratory animals and humans. The parasite secretes various inducing molecules as biological responses, which are thought to be involved in pathophysiological and immunological events during infection. To investigate what molecules of N. fowleri excretory-secretory proteins (ESPs) are related with amoebic pathogenicity, N. fowleri ESPs fractionated by two-dimensional electrophoresis were reacted with N. fowleri infection or immune sera. To identify immunodominant ESPs, six major protein spots were selected and analyzed by N-terminal sequencing. Finally, six proteins, 58, 40, 24, 21, 18, and 16 kDa of molecular weight, were partially cloned and matched with reference proteins as follow: 58 kDa of exendin-3 precursor, 40 kDa of secretory lipase, 24 kDa of cathepsin B-like proteases and cysteine protease, 21 kDa of cathepsin B, 18 kDa of peroxiredoxin, and 16 kDa of thrombin receptor, respectively. These results suggest that N. fowleri ESPs contained important proteins, which may play an important role in the pathogenicity of N. fowleri.

  6. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

    PubMed

    Gullón, Sonia; Marín, Silvia; Mellado, Rafael P

    2015-01-01

    Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

  7. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans

    PubMed Central

    Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

    2015-01-01

    Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients’ depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host. PMID:26200356

  8. Secretion of sulfated and nonsulfated forms of parathyroid chromogranin A (secretory protein-I)

    SciTech Connect

    Gorr, S.U.; Cohn, D.V. )

    1990-02-25

    Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and (3H)Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues.

  9. New class of cargo protein in Tetrahymena thermophila dense core secretory granules.

    PubMed

    Haddad, Alex; Bowman, Grant R; Turkewitz, Aaron P

    2002-08-01

    Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.

  10. Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

    SciTech Connect

    Kierszenbaum, A.L.; Crowell, J.A.; Shabanowitz, R.B.; DePhilip, R.M.; Tres, L.L.

    1986-08-01

    An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates.

  11. Cobra venom contains a pool of cysteine-rich secretory proteins.

    PubMed

    Osipov, Alexey V; Levashov, Mikhail Yu; Tsetlin, Victor I; Utkin, Yuri N

    2005-03-04

    A large family of cysteine-rich secretory proteins (CRISPs) includes proteins of different origin, the function of the majority of CRISPs being unknown. For CRISPs isolated from snake venom, two types of activities were found: two proteins blocked cyclic nucleotide-gated ion channels, several others blocked potassium-stimulated smooth muscle contraction. Thus, snake CRISPs represent potentially valuable tools for studies of ion channels, which makes promising a search for new CRISPs. Here we report on the isolation of several novel CRISPs from the venoms of Asian cobra Naja kaouthia and African cobra Naja haje using a combination of different types of liquid chromatography. Four CRISP variants were identified in N. kaouthia venom and three proteins, one of them acidic, were found in N. haje venom. Acidic CRISP was found in a reptilian venom for the first time. Our data suggest that each cobra venom contains a pool of different CRISPs.

  12. Expression of Active Fluorophore Proteins in the Milk of Transgenic Pigs Bypassing the Secretory Pathway

    PubMed Central

    Mukherjee, Ayan; Garrels, Wiebke; Talluri, Thirumala R.; Tiedemann, Daniela; Bősze, Zsuzsanna; Ivics, Zoltán; Kues, Wilfried A.

    2016-01-01

    We describe the expression of recombinant fluorescent proteins in the milk of two lines of transgenic pigs generated by Sleeping Beauty transposon-mediated genetic engineering. The Sleeping Beauty transposon consisted of an ubiquitously active CAGGS promoter driving a fluorophore cDNA, encoding either Venus or mCherry. Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found. Unexpectedly, milk samples from lactating sows contained high levels of bioactive Venus or mCherry fluorophores. A detailed analysis suggested that exfoliated cells of the mammary epithelium carried the recombinant proteins passively into the milk. This is the first description of reporter fluorophore expression in the milk of livestock, and the findings may contribute to the development of an alternative concept for the production of bioactive recombinant proteins in the udder. PMID:27086548

  13. A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles.

    PubMed

    Matthiesen, S H; Shenoy, S M; Kim, K; Singer, R H; Satir, B H

    2001-12-01

    We cloned a gene, PRPI, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at M, 63 x 10(3) and 68 x 10(3), respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.

  14. Expression of transcripts for cysteine-rich secretory proteins (CRISPs) in the murine lacrimal gland.

    PubMed

    Haendler, B; Toda, I; Sullivan, D A; Schleuning, W D

    1999-03-01

    Cysteine-rich secretory proteins (CRISPs) represent a family of evolutionarily conserved proteins which may play a role in the innate immune system and are transcriptionally regulated by androgens in several tissues. Transcripts for all three members of the CRISP family have now been identified in the murine lacrimal gland. RT-PCR using primers able to discriminate between the related CRISP forms allowed the amplification of fragments with the expected length. DNA sequencing revealed a complete identity with the hitherto characterized epididymal CRISP-1, testicular CRISP-2, and salivary gland CRISP-3. An analysis of several mouse strains indicated that all expressed the three CRISP forms, but in differing amounts. RT-PCR analysis of RNA isolated from acinar cells of lacrimal glands revealed that they expressed CRISP-1 and CRISP-2. Semiquantitative and quantitative analyses furthermore showed higher CRISP-1 and CRISP-3 mRNA levels in the lacrimal glands of male BALB/c and NOD mice when compared to females. Testosterone treatment of C3H/HeJ female mice was followed by an upregulation of the steady-state CRISP-1 but not CRISP-2 transcript levels. A comparable stimulation was observed for the mRNAs coding for parotid secretory protein (PSP), a factor previously shown to exhibit sexual dimorphism in the murine lacrimal gland. The expression of CRISP transcripts in the lacrimal gland is consistent with a function in the innate immune system.

  15. Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters.

    PubMed

    Hahn, Heinz P; von Specht, Bernd Ulrich

    2003-07-15

    Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.

  16. SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

    SciTech Connect

    Kandaswamy, Krishna Kumar; Pugalenthi, Ganesan; Hartmann, Enno; Kalies, Kai-Uwe; Moeller, Steffen; Suganthan, P.N.; Martinetz, Thomas

    2010-01-15

    Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at (http://www.inb.uni-luebeck.de/tools-demos/spred/spred).

  17. The role of cysteine-rich secretory proteins in male fertility.

    PubMed

    Koppers, Adam J; Reddy, Thulasimala; O'Bryan, Moira K

    2011-01-01

    The cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and Pr-1 (CAP) protein superfamily, and are found only in vertebrates. They show a strong expression bias to the mammalian male reproductive tract and the venom of poisonous reptiles. Within the male reproductive tract CRISPs have been implicated in many aspects of male germ cell biology spanning haploid germ cell development, epididymal maturation, capacitation, motility and the actual processes of fertilization. At a structural level, CRISPs are composed of two domains, a CAP domain, which has been implicated in cell-cell adhesion, and a CRISP domain, which has been shown to regulate several classes of ion channels across multiple species. Herein, we will review the current literature on the role of CRISPs in male fertility, and by inference to related non-mammalian protein, infer potential biochemical functions.

  18. The role of cysteine-rich secretory proteins in male fertility

    PubMed Central

    Koppers, Adam J; Reddy, Thulasimala; O'Bryan, Moira K

    2011-01-01

    The cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and Pr-1 (CAP) protein superfamily, and are found only in vertebrates. They show a strong expression bias to the mammalian male reproductive tract and the venom of poisonous reptiles. Within the male reproductive tract CRISPs have been implicated in many aspects of male germ cell biology spanning haploid germ cell development, epididymal maturation, capacitation, motility and the actual processes of fertilization. At a structural level, CRISPs are composed of two domains, a CAP domain, which has been implicated in cell–cell adhesion, and a CRISP domain, which has been shown to regulate several classes of ion channels across multiple species. Herein, we will review the current literature on the role of CRISPs in male fertility, and by inference to related non-mammalian protein, infer potential biochemical functions. PMID:20972450

  19. Glucosidase II and MRH-domain containing proteins in the secretory pathway

    PubMed Central

    D’Alessio, Cecilia; Dahms, Nancy M.

    2015-01-01

    N -glycosylation in the endoplasmic reticulum (ER) consists of the transfer of a pre-assembled glycan conserved among species (Glc3Man9GlcNAc2) from a lipid donor to a consensus sequence within a nascent protein that is entering the ER. The protein-linked glycans are then processed by glycosidases and glycosyltransferases in the ER producing specific structures that serve as signalling molecules for the fate of the folding glycoprotein: to stay in the ER during the folding process, to be retrotranslocated to the cytosol for proteasomal degradation if irreversibly misfolded, or to pursue transit through the secretory pathway as a mature glycoprotein. In the ER, each glycan signalling structure is recognized by a specific lectin. A domain similar to that of the mannose 6-phosphate receptors (MPRs) has been identified in several proteins of the secretory pathway. These include the beta subunit of glucosidase II (GII), a key enzyme in the early processing of the transferred glycan that removes middle and innermost glucoses and is involved in quality control of glycoprotein folding in the ER (QC), the lectins OS-9 and XTP3-B, proteins involved in the delivery of ER misfolded proteins to degradation (ERAD), the gamma subunit of the Golgi GlcNAc-1-phosphotransferase, an enzyme involved in generating the mannose 6-phosphate (M6P) signal for sorting acidic hydrolases to lysosomes, and finally the MPRs that deliver those hydrolytic enzymes to the lysosome. Each of the MRH-containing proteins recognizes a different signalling N-glycan structure. Three-dimensional structures of some of the MRH domains have been solved, providing the basis to understand recognition mechanisms. PMID:25692846

  20. Identification of Secretory Proteins in Mycobacterium tuberculosis Using Pseudo Amino Acid Composition

    PubMed Central

    Yang, Huan; Tang, Hua; Chen, Xin-Xin; Zhang, Chang-Jian; Zhu, Pan-Pan; Ding, Hui

    2016-01-01

    Tuberculosis is killing millions of lives every year and on the blacklist of the most appalling public health problems. Recent findings suggest that secretory protein of Mycobacterium tuberculosis may serve the purpose of developing specific vaccines and drugs due to their antigenicity. Responding to global infectious disease, we focused on the identification of secretory proteins in Mycobacterium tuberculosis. A novel method called MycoSec was designed by incorporating g-gap dipeptide compositions into pseudo amino acid composition. Analysis of variance-based technique was applied in the process of feature selection and a total of 374 optimal features were obtained and used for constructing the final predicting model. In the jackknife test, MycoSec yielded a good performance with the area under the receiver operating characteristic curve of 0.93, demonstrating that the proposed system is powerful and robust. For user's convenience, the web server MycoSec was established and an obliging manual on how to use it was provided for getting around any trouble unnecessary. PMID:27597968

  1. A pH-regulated quality control cycle for surveillance of secretory protein assembly.

    PubMed

    Vavassori, Stefano; Cortini, Margherita; Masui, Shoji; Sannino, Sara; Anelli, Tiziana; Caserta, Imma R; Fagioli, Claudio; Mossuto, Maria F; Fornili, Arianna; van Anken, Eelco; Degano, Massimo; Inaba, Kenji; Sitia, Roberto

    2013-06-27

    To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44's active cysteine, simultaneously unmask the substrate binding site and -RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles.

  2. A pH-Regulated Quality Control Cycle for Surveillance of Secretory Protein Assembly

    PubMed Central

    Vavassori, Stefano; Cortini, Margherita; Masui, Shoji; Sannino, Sara; Anelli, Tiziana; Caserta, Imma R.; Fagioli, Claudio; Mossuto, Maria F.; Fornili, Arianna; van Anken, Eelco; Degano, Massimo; Inaba, Kenji; Sitia, Roberto

    2013-01-01

    Summary To warrant the quality of the secretory proteome, stringent control systems operate at the endoplasmic reticulum (ER)-Golgi interface, preventing the release of nonnative products. Incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly. Here we unveil how ERp44 cycles between cisGolgi and ER in a pH-regulated manner, patrolling assembly of disulfide-linked oligomers such as IgM and adiponectin. At neutral, ER-equivalent pH, the ERp44 carboxy-terminal tail occludes the substrate-binding site. At the lower pH of the cisGolgi, conformational rearrangements of this peptide, likely involving protonation of ERp44’s active cysteine, simultaneously unmask the substrate binding site and −RDEL motif, allowing capture of orphan secretory protein subunits and ER retrieval via KDEL receptors. The ERp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin/calreticulin and BiP-dependent quality control cycles. PMID:23685074

  3. Secretory fluorescent protein, a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system.

    PubMed

    Teng, Chao-Yi; Wu, Tzong-Yuan

    2007-07-01

    The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.

  4. Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

    PubMed Central

    Higgins, S J; Burchell, J M; Mainwaring, W I

    1976-01-01

    1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug. Images PLATE 1(a) PLATES 1(b), 1(c) AND 1(d) PLATE 2 PMID:985427

  5. Immunoproteomic Analysis of the Excretory-Secretory Proteins from Spirometra mansoni Sparganum

    PubMed Central

    HU, Dan Dan; CUI, Jing; WANG, Li; LIU, Li Na; WEI, Tong; WANG, Zhong Quan

    2013-01-01

    Background Sparganosis is caused by the invasion of Spirometra sparganum into various tissues/organs. Subcutaneous sparganosis can be diagnosed by biopsy, while visceral/cerebral sparganosis is not easy to be diagnosed. The diagnosis depends largely on the detection of specific anti-sparganum antibodies. The specificity of the ELISA could be increased by using S. mansoni sparganum excretory–secretory (ES) antigens, but it also had the cross-reactions with sera of patients with cysticercosis or paragonimiasis. The aim of this study was to identify early specific diagnostic antigens in S. mansoni sparganum ES proteins. Methods The sparganum ES proteins were analyzed by two-dimensional electrophoresis (2-DE) and Western blot probed with early sera from infected mice at 14 days post-infection. The immunoreactive protein spots were characterized by MALDI-TOF/ TOF-MS. Results A total of approximately 149 proteins spots were detected with isoelectric point (pI) varying from 3 to 7.5 and molecular weight from 20 to 115 kDa and seven protein spots with molecular weight of 23-31 kDa were recognized by the infection sera. Three of seven spots were successfully identified and characterized as the same S. mansoni protein (cysteine protease), and the proteins of other 4 spots were not included in the databases. Conclusion The cysteine protease from S. mansoni ES proteins recognized by early infection sera might be the early diagnostic antigens for sparganosis. PMID:24454434

  6. A Dynamic Study of Protein Secretion and Aggregation in the Secretory Pathway

    PubMed Central

    Mossuto, Maria Francesca; Sannino, Sara; Mazza, Davide; Fagioli, Claudio; Vitale, Milena; Yoboue, Edgar Djaha; 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. PMID:25279560

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

    PubMed

    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.

  8. Persistent dominant follicle alters pattern of oviductal secretory proteins from cows at estrus.

    PubMed

    Binelli, M; Hampton, J; Buhi, W C; Thatcher, W W

    1999-07-01

    The experimental objective was to compare synthesis of oviductal secretory proteins of dairy cows bearing a persistent dominant follicle (PDF) versus a fresh dominant follicle (FDF) at estrus. On Day 7 after synchronized estrus (Day 0), cows received an intravaginal progesterone device and injection of prostaglandin F2alpha (PGF2alpha). On Day 9, cows received an injection of a GnRH agonist (FDF group; n = 3) or received no injection (PDF group, n = 3). On Day 16, all cows received PGF2alpha, and progesterone devices were removed. At slaughter on Day 18 or Day 19, oviducts ipsilateral and contralateral to the dominant follicle were divided into infundibulum, ampulla, and isthmus regions. Explants from oviductal regions were cultured in minimal essential medium supplemented with [3H]leucine for 24 h. Two-dimensional fluorographs of proteins in conditioned media were analyzed by densitometry. Rate of incorporation of [3H]leucine into macromolecules was greater in the infundibulum, ampulla, and isthmus of FDF cows (p < 0.01). Overall, intensities of radiolabeled secretory protein (P) 2 and P13 were greater for FDF than for PDF. In the ampulla, P14 was more intense for FDF while P7 was more intense for PDF. Abundance of P1 in the isthmus was greater for PDF cows. Across regions, P5, P6, P8, P9, and P11 were more intense for PDF than for FDF in the ipsilateral side. In the contralateral side, P19 was more intense for PDF than for FDF, whereas P6, P8, P9, and P11 were more intense for FDF. Differences in biosynthetic activity and in secreted oviductal proteins from cows bearing a PDF may contribute to the decrease in fertility associated with a PDF.

  9. A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence.

    PubMed Central

    Rubartelli, A; Cozzolino, F; Talio, M; Sitia, R

    1990-01-01

    Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:2328723

  10. Haemonchus contortus excretory and secretory proteins (HcESPs) suppress functions of goat PBMCs in vitro.

    PubMed

    Gadahi, Javaid Ali; Yongqian, Bu; Ehsan, Muhammad; Zhang, Zhen Chao; Wang, Shuai; Yan, Ruo Feng; Song, Xiao Kai; Xu, Li Xin; Li, Xiang Rui

    2016-06-14

    Excretory and secretory products (ESPs) of nematode contain various proteins which are capable of inducing the instigation or depression of the host immune response and are involved in the pathogenesis of the worms. In the present study, Haemonchus contortus excretory and secretory products (HcESPs) were collected from the adult worms. Binding of HcESPs to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immune-fluorescence assay. Effects of the HcESPs on cytokine production, cell proliferation, cell migration and nitric oxide (NO) production of PBMCs were checked by co-incubation of HcESPs with goat PBMCs. The results indicated that the production of IL-4 and IFN-γ were significantly decreased by HcESPs in dose dependent manner. On the contrary, the production of IL-10 and IL-17 were increased. Cell migration was significantly enhanced by HcESPs, whereas, HcESPs treatment significantly suppressed the cell proliferation and NO production. These results indicated that the HcESPs played important suppressive regulatory roles on PBMCs and provided highlights to the understanding of the host-parasite interactions.

  11. Proteomic analysis of Trichinella spiralis adult worm excretory-secretory proteins recognized by early infection sera.

    PubMed

    Liu, Ruo Dan; Qi, Xin; Sun, Ge Ge; Jiang, Peng; Zhang, Xi; Wang, Li Ang; Liu, Xiao Lin; Wang, Zhong Quan; Cui, Jing

    2016-11-15

    At the intestinal stage of a Trichinella spiralis (T. spiralis) infection, the excretory-secretory (ES) antigens produced by adult worms (AWs) result in an early exposure to the host's immune system and elicit the production of specific antibodies; the AW ES proteins might provide early diagnostic markers of trichinellosis. The aim of this study was to identify early serodiagnostic markers from T. spiralis AW ES antigens. T. spiralis AWs were collected at 72h post infection, and their ES antigens were analysed by SDS-PAGE and Western blot. Then, the immunoreactive bands were subjected to shotgun LC-MS/MS and bioinformatics analyses. Our results showed that only one protein band (33kDa) was recognized by the sera of mice infected with T. spiralis at 8 days after infection. The shotgun LC-MS/MS analysis identified 23 proteins that were then clustered into 10 types; these proteins had molecular weights of 28.13-71.62kDa and pI 5.05-9.20. Certain enzymes (e.g., serine protease, adult-specific deoxyribonuclease [DNase] II, peptidase S1A subfamily, and multi cystatin-like domain protein) were found to be highly represented. The functions of the 10 proteins were further analysed: of the 6 annotated proteins, 3 had serine hydrolase activity and 2 had DNase II activity. These results provide a valuable basis for identifying early diagnostic antigens and vaccine candidates for trichinellosis.

  12. Purification of Toxoplasma dense granule proteins reveals that they are in complexes throughout the secretory pathway.

    PubMed

    Braun, Laurence; Travier, Laetitia; Kieffer, Sylvie; Musset, Karine; Garin, Jérôme; Mercier, Corinne; Cesbron-Delauw, Marie-France

    2008-01-01

    Dense granules are Apicomplexa specific secretory organelles. In Toxoplasma gondii, the dense granules proteins, named GRA proteins, are massively secreted into the parasitophorous vacuole (PV) shortly after invasion. Despite the presence of hydrophobic membrane segments, they are stored as both soluble and aggregated forms within the dense granules and are secreted as soluble forms into the vacuolar space where they further stably associate with PV membranes. In this study, we explored the unusual biochemical behavior of GRA proteins during their trafficking. Conventional chromatography indicated that the GRA proteins form high globular weight complexes within the parasite. To confirm these results, DeltaGRA knocked-out parasites were stably complemented with their respective HA-FLAG tagged GRA2 or GRA5. Purification of the tagged proteins by affinity chromatography showed that within the parasite and the PV soluble fraction, both the soluble GRA2-HA-FLAG and GRA5-HA-FLAG associate with several GRA proteins, the major ones being GRA3, GRA6 and GRA7. Following their insertion into the PV membranes, GRA2-HA-FLAG associated with GRA5 and GRA7 while GRA5-HA-FLAG associated with GRA7 only. Taken together, these data suggest that the GRA proteins form oligomeric complexes that may explain their solubility within the dense granules and the vacuolar matrix by sequestering their hydrophobic domains within the interior of the complex. Insertion into the PV membranes correlates with the decrease of the GRA partners number.

  13. The novel secretory protein CGREF1 inhibits the activation of AP-1 transcriptional activity and cell proliferation.

    PubMed

    Deng, Weiwei; Wang, Lan; Xiong, Ying; Li, Jing; Wang, Ying; Shi, Taiping; Ma, Dalong

    2015-08-01

    The transcription factor AP-1 plays an important role in inflammation and cell survival. Using a dual-luciferase reporter assay system and a library of 940 candidate human secretory protein cDNA clones, we identified that CGREF1 can inhibit the transcriptional activity of AP-1. We demonstrated that CGREF1 is secreted via the classical secretory pathway through the ER-to-Golgi apparatus. Functional investigations revealed that overexpression of CGREF1 can significantly inhibit the phosphorylation of ERK and p38 MAPK, and suppress the proliferation of HEK293T and HCT116 cells. Conversely, specific siRNAs against CGREF1 can increase the transcriptional activity of AP-1. These results clearly indicated that CGREF1 is a novel secretory protein, and plays an important role in regulation of AP-1 transcriptional activity and cell proliferation.

  14. Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway

    SciTech Connect

    Ortego, Javier; Ceriani, Juan E.; Patino, Cristina; Plana, Juan; Enjuanes, Luis

    2007-11-25

    A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-{delta}E) has been engineered. This deletion mutant only grows in cells expressing E protein (E{sup +} cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-{delta}E infected BHK-pAPN-E{sup -} cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E{sup -} cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-{delta}E virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-{delta}E subcellular localization by confocal and immunoelectron microscopy in infected E{sup -} cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation.

  15. Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins

    PubMed Central

    Farkaš, Robert; Ďatková, Zuzana; Mentelová, Lucia; Löw, Péter; Beňová-Liszeková, Denisa; Beňo, Milan; Sass, Miklós; Řehulka, Pavel; Řehulková, Helena; Raška, Otakar; Kováčik, Lubomír; Šmigová, Jana; Raška, Ivan; Mechler, Bernard M.

    2014-01-01

    In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal β-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity. PMID:24732043

  16. Determination of the electrophoretic pattern of somatic and excretory-secretory proteins of Ligula intestinalis parasite in spirlin (Alburnoides bipunctatus).

    PubMed

    Youssefi, M R; Hosseinifard, S M; Halimi, M; Kordafshari, S

    2012-12-01

    Ligula intestinalis parasite is a cestodes that causes remarkable damages to fish. It is also of prime importance in economic and hygienic aspects. SDS-PAGE and western blotting are the methods that can be used to determine the electerophoretic pattern of somatic and excretory-secretory proteins of parasites. In this study, after obtaining the plerocercoidal stage of this parasite from the spirlin (Alburnoides bipunctatus), its somatic proteins were prepared using ultrasonicae, and excretory-secretory proteins were prepared using the PBS solution. After protein assay, which included using the Bradford method and then SDS-PAGE on these two antigenic solutions, 5 protein bands of 26, 33, 38, 58, 70kDa in somatic antigens, and 7 bands of 25, 28, 33, 43, 49, 60, 70kDa in excretory-secretory antigens were observed. After western blotting on both antigens and adding the primary antibody (the sera of infected fish) and then the secondary antibody (Rabbit Anti-fish Polyclonal Antibody Conjugated from Abnova Corporation) no band was seen in excretory-secretory antigen. And only in the 55kDa band of somatic antigen, a positive response, in comparison of fish positive serum was observed.

  17. Two secretory protein genes in Chironomus tentans have arisen by gene duplication and exhibit different developmental expression patterns.

    PubMed

    Galli, J; Wieslander, L

    1993-05-20

    The salivary gland cells in the dipteran Chironomus tentans produce approximately 15 different secretory proteins, with relative molecular masses ranging between 1 x 10(4) and 1 x 10(6). Together these proteins form two types of extra corporal tubes, a larval protective housing and feeding tube or a pupation tube. The developmental change in tube formation is accompanied by a switch in production from one combination of secretory proteins to another. Here we characterize two genes, the sp38-40.A and B genes, which encode secretory proteins with relative molecular masses of 38,000 to 40,000. The two genes are located 346 base-pairs apart in the same orientation and have presumably arisen by gene duplication as the result of an illegitimate recombination event. Both genes contain two regions with cysteine codons, surrounded by regions with short repeats coding for proline and charged amino acid residues. The two genes and alleles of the genes differ in their number of repeats. This structure resembles the structure of the Balbiani ring (BR) genes, which encode the four largest salivary gland secretory proteins. The sp38-40.A and B genes are therefore likely to belong to a BR multigene family containing all or most of the 15 salivary gland secretory protein genes. The expression of the sp38-40.A and B genes are different: the A gene is expressed throughout the larval fourth instar but considerably less in the prepupal stage, while the B gene shows the opposite expression pattern. The developmental regulation of the expression of the two genes has therefore diverged after the gene duplication event.

  18. Structural divergence of cysteine-rich secretory proteins in snake venoms.

    PubMed

    Matsunaga, Yukiko; Yamazaki, Yasuo; Hyodo, Fumiko; Sugiyama, Yusuke; Nozaki, Masatoshi; Morita, Takashi

    2009-03-01

    Cysteine-rich secretory proteins (CRISPs) are expressed in spermatocytes and granules of neutrophils in mammals, and are associated with sperm maturation and host defense. Related proteins have recently been recovered in snake venoms, and some of the snake venom-derived CRISPs exhibit ion channel blocking activity. Here we isolated and identified two novel CRISPs (kaouthin-1 and kaouthin-2) from the venom of Naja kaouthia (Elapidae), and cloned the encoding cDNAs. Kaouthin-1 and kaouthin-2 were classified into two broad sister groups of Elapidae, the Asian species and the marine/Australian species, respectively. Sequence comparisons reveal that the high-frequency variable regions among snake venom CRISPs define a continuous line on the molecular surface of the N-terminal pathogenesis-related protein-1 (PR-1) domain and the C-terminal cysteine-rich domain (CRD). Snake venom proteins generally display efficient molecular diversity around functionally key regions, suggesting that the PR-1 domain of CRISPs is important for the recognition of target molecules.

  19. Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom.

    PubMed

    Peichoto, María E; Mackessy, Stephen P; Teibler, Pamela; Tavares, Flávio L; Burckhardt, Paula L; Breno, María C; Acosta, Ofelia; Santoro, Marcelo L

    2009-07-01

    Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.

  20. Tailoring Escherichia coli for the l-Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins.

    PubMed

    Hjelm, Anna; Karyolaimos, Alexandros; Zhang, Zhe; Rujas, Edurne; Vikström, David; Slotboom, Dirk Jan; de Gier, Jan-Willem

    2017-03-03

    Membrane and secretory protein production in Escherichia coli requires precisely controlled production rates to avoid the deleterious saturation of their biogenesis pathways. On the basis of this requirement, the E. coli l-rhamnose PBAD promoter (PrhaBAD) is often used for membrane and secretory protein production since PrhaBAD is thought to regulate protein production rates in an l-rhamnose concentration-dependent manner. By monitoring protein production in real-time in E. coli wild-type and an l-rhamnose catabolism deficient mutant, we demonstrate that the l-rhamnose concentration-dependent tunability of PrhaBAD-mediated protein production is actually due to l-rhamnose consumption rather than regulating production rates. Using this information, a RhaT-mediated l-rhamnose transport and l-rhamnose catabolism deficient double mutant was constructed. We show that this mutant enables the regulation of PrhaBAD-based protein production rates in an l-rhamnose concentration-dependent manner and that this is critical to optimize membrane and secretory protein production yields. The high precision of protein production rates provided by the PrhaBAD promoter in an l-rhamnose transport and catabolism deficient background could also benefit other applications in synthetic biology.

  1. Single GDP-dissociation Inhibitor Protein regulates endocytic and secretory pathways in Leishmania

    PubMed Central

    Shanmugam, Senthil kumar; Kumar, Kamal; Singh, Pawan Kishor; Rastogi, Ruchir; Mukhopadhyay, Amitabha

    2016-01-01

    The role of GDP dissociation inhibitor (GDI) protein in regulation of Rab cycle in Leishmania is not known. Here, we have cloned and characterized the functions of GDI homologue in vivo in Leishmania. Our results have shown that LdGDI:WT along with GDP removes the Rab5 from purified endosomes and inhibits the homotypic fusion between early endosomes. Whereas, LdGDI:R239A, a dominant negative mutant of GDI, under the same condition neither removes the Rab5 from endosome nor inhibits fusion. To determine the role of Ld-GDI in vivo, transgenic parasites overexpressing GFP-LdGDI:WT or GFP-LdGDI:R239A, are co-expressed with RFP-LdRab5:WT, RFP-LdRab7:WT or RFP-LdRab1:WT. Our results have shown that overexpression of GFP-LdGDI:WT extracts the RFP-LdRab5, RFP-LdRab7 or RFP-LdRab1 from their discrete endomembrane predominantly into cytosol. No change in the distribution of indicated Rabs is detected with overexpression of GFP-LdGDI:R239A. To determine the functional significance, we have used hemoglobin as an endocytic marker and gp63 as a marker for secretory pathway. We have found that overexpression of GFP-LdGDI:WT enhances the lysosomal targeting of internalized hemoglobin and the secretion of gp63 in the parasites possibly by triggering Rab cycle. This is the first demonstration of a single GDI ubiquitously regulating both endocytic and secretory pathways in Leishmania. PMID:27841328

  2. Analysis of mRNA With Microsomal Fractionation Using a SAGE-Based DNA Microarray System Facilitates Identification of the Genes Encoding Secretory Proteins

    PubMed Central

    Toyoda , Nobuaki; Nagai, Shigenori; Terashima, Yuya; Motomura, Kazushi; Haino, Makoto; Hashimoto, Shin-ichi; Takizawa, Hajime; Matsushima, Kouji

    2003-01-01

    In the regulation of host defense responses such as inflammation and immunity, the secretory proteins, including membrane proteins, play central roles. Although many secretory proteins have been identified by using methods such as differential display, random screening, or the signal sequence trap method, each method suffers from poor reproducibility, low sensitivity, or time-consuming or laborious work. Therefore, the strategy for facilitating the selection of the genes encoding the secretory proteins is desired. In this paper, we describe a system for isolating the genes encoding secretory proteins by analyzing mRNAs with microsomal fractionation on serial analysis of gene expression (SAGE)–based DNA microarray system. This system succeeded in discriminating the genes encoding secretory proteins from ones encoding nonsecretory proteins with 80% accuracy. We applied this system to human T lymphocytes. As a result, we were able to identify the genes that are not only encoding secretory proteins but also expressing selectively in a specific subset of T lymphocytes. The SAGE-based DNA microarray system is a promising system to identify the genes encoding specific secretory proteins. PMID:12805275

  3. Serodiagnosis of fasciolosis by fast protein liquid chromatography-fractionated excretory/secretory antigens.

    PubMed

    Mokhtarian, Kobra; Akhlaghi, Lame; Meamar, Ahmad Reza; Razmjou, Elham; Manouchehri Naeini, Kourosh; Gholami, Samaneh; Najafi Samei, Masoomeh; Falak, Reza

    2016-08-01

    In several studies, different antigenic preparations and diverse immunological tests were applied for serodiagnosis of Fasciola hepatica infections. Most of these preparations showed cross-reactivity with proteins of other parasites. Application of purified antigens might reduce these cross-reactivities. Here, we used fast protein liquid chromatography (FPLC)-fractionated extracts of F. hepatica excretory/secretory antigens (E/S Ags) for serodiagnosis of human and sheep fasciolosis. To develop an improved diagnostic method, we fractionated F. hepatica E/S Ags by anion exchange chromatography on a Sepharose CL-6B column and then tested the serodiagnostic values of the fractions. We used sera from F. hepatica-infected human and sheep as positive controls. Sera from patients with hydatidosis and strongyloidiasis were used for cross-reactivity studies. Enzyme-linked immunosorbent assays (ELISA) of the second FPLC peak, containing 20, 25, and 70 kDa proteins, discriminated between F. hepatica-infected and uninfected human and sheep samples. Fractionation of F. hepatica E/S Ags by FPLC is a fast and reproducible way of obtaining antigens useful for serodiagnosis of human and sheep fasciolosis with acceptable sensitivity and specificity. Graphical abstract ᅟ.

  4. Immune response to Candida albicans is preserved despite defect in O-mannosylation of secretory proteins.

    PubMed

    Corbucci, Cristina; Cenci, Elio; Skrzypek, Franck; Gabrielli, Elena; Mosci, Paolo; Ernst, Joachim F; Bistoni, Francesco; Vecchiarelli, Anna

    2007-12-01

    The PMT gene family in Candida albicans encodes five isoforms of the protein mannosyltransferases that initiate O-mannosylation of secretory proteins. Mutations at the Pmt level have been associated with differences in pathogenicity, e.g. in contrast to pmt5/pmt5, pmt2/PMT2 mutants showed poor virulence. Our objective was to determine whether these differences were related to the capacity of pmt2/PMT2 and pmt5/pmt5 to (i) express differences in selected virulence factors, and (ii) stimulate the natural immune system. The results show that pmt mutants (i) form hyphae in serum, (ii) show defective production of proteases but not of phospholipases with respect to the parental strain, (iii) undergo mycelial transition in the kidneys of hematogenously infected animals, (iv) are phagocytosed and killed by macrophages similar to the parental strain, although neutrophils are unable to destroy pmt5/pmt5, (v) engage TLR4 and stimulate MyD88 leading to NF-kappaB activation, and (vi) stimulate cytokine production by macrophages. Collectively our findings suggest that the defect in protein O-mannosylation in C. albicans cause attenuation of the virulence although the antigenic factors that retain the capacity to stimulate an efficient immune response are preserved.

  5. The human homolog of the JE gene encodes a monocyte secretory protein.

    PubMed Central

    Rollins, B J; Stier, P; Ernst, T; Wong, G G

    1989-01-01

    The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein. Images PMID:2513477

  6. Secretory Leukocyte Protease Inhibitor Binds to Neisseria gonorrhoeae Outer Membrane Opacity Protein and is Bactericidal

    PubMed Central

    Cooper, Morris D.; Roberts, Melissa H.; Barauskas, Ona L.; Jarvis, Gary A.

    2012-01-01

    Problem Secretory leukocyte protease inhibitor (SLPI) is an innate immune peptide present on the genitourinary tract mucosa which has antimicrobial activity. In this study, we investigated the interaction of SLPI with Neisseria gonorrhoeae. Method of study ELISA and far-western blots were used to analyze binding of SLPI to gonococci. The binding site for SLPI was identified by tryptic digests and mass spectrometry. Antimicrobial activity of SLPI for gonococci was determined using bactericidal assays. SLPI protein levels in cell supernatants were measured by ELISA, and SLPI mRNA levels were assessed by quantitative RT-PCR. Results SLPI bound directly to the gonococcal Opa protein and was bactericidal. Epithelial cells from the reproductive tract constitutively expressed SLPI at different levels. Gonococcal infection of cells did not affect SLPI expression. Conclusion We conclude that SLPI is bactericidal for gonococci and is expressed by reproductive tract epithelial cells and thus is likely to play a role in the pathogenesis of gonococcal infection. PMID:22537232

  7. Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV-2 Microglial Cells.

    PubMed

    Lee, Jinyoung; Kang, Jung-Mi; Kim, Tae Im; Kim, Jong-Hyun; Sohn, Hae-Jin; Na, Byoung-Kuk; Shin, Ho-Joon

    2017-03-01

    Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.

  8. Secretory Carrier Membrane Protein (SCAMP) deficiency influences behavior of adult flies

    PubMed Central

    Zheng, JiaLin C.; Tham, Chook Teng; Keatings, Kathleen; Fan, Steven; Liou, Angela Yen-Chun; Numata, Yuka; Allan, Douglas; Numata, Masayuki

    2014-01-01

    Secretory Carrier Membrane Proteins (SCAMPs) are a group of tetraspanning integral membrane proteins evolutionarily conserved from insects to mammals and plants. Mammalian genomes contain five SCAMP genes SCAMP1-SCAMP5 that regulate membrane dynamics, most prominently membrane-depolarization and Ca2+-induced regulated secretion, a key mechanism for neuronal and neuroendocrine signaling. However, the biological role of SCAMPs has remained poorly understood primarily owing to the lack of appropriate model organisms and behavior assays. Here we generate Drosophila Scamp null mutants and show that they exhibit reduced lifespan and behavioral abnormalities including impaired climbing, deficiency in odor associated long-term memory, and a susceptibility to heat-induced seizures. Neuron-specific restoration of Drosophila Scamp rescues all Scamp null behavioral phenotypes, indicating that the phenotypes are due to loss of neuronal Scamp. Remarkably, neuronal expression of human SCAMP genes rescues selected behavioral phenotypes of the mutants, suggesting the conserved function of SCAMPs across species. The newly developed Drosophila mutants present the first evidence that genetic depletion of SCAMP at the organismal level leads to varied behavioral abnormalities, and the obtained results indicate the importance of membrane dynamics in neuronal functions in vivo. PMID:25478561

  9. Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

    PubMed

    Dias, Felipe F; Amaral, Kátia B; Carmo, Lívia A S; Shamri, Revital; Dvorak, Ann M; Weller, Peter F; Melo, Rossana C N

    2014-06-01

    Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

  10. Secretory Carrier Membrane Protein (SCAMP) deficiency influences behavior of adult flies.

    PubMed

    Zheng, JiaLin C; Tham, Chook Teng; Keatings, Kathleen; Fan, Steven; Liou, Angela Yen-Chun; Numata, Yuka; Allan, Douglas; Numata, Masayuki

    2014-01-01

    Secretory Carrier Membrane Proteins (SCAMPs) are a group of tetraspanning integral membrane proteins evolutionarily conserved from insects to mammals and plants. Mammalian genomes contain five SCAMP genes SCAMP1-SCAMP5 that regulate membrane dynamics, most prominently membrane-depolarization and Ca(2+)-induced regulated secretion, a key mechanism for neuronal and neuroendocrine signaling. However, the biological role of SCAMPs has remained poorly understood primarily owing to the lack of appropriate model organisms and behavior assays. Here we generate Drosophila Scamp null mutants and show that they exhibit reduced lifespan and behavioral abnormalities including impaired climbing, deficiency in odor associated long-term memory, and a susceptibility to heat-induced seizures. Neuron-specific restoration of Drosophila Scamp rescues all Scamp null behavioral phenotypes, indicating that the phenotypes are due to loss of neuronal Scamp. Remarkably, neuronal expression of human SCAMP genes rescues selected behavioral phenotypes of the mutants, suggesting the conserved function of SCAMPs across species. The newly developed Drosophila mutants present the first evidence that genetic depletion of SCAMP at the organismal level leads to varied behavioral abnormalities, and the obtained results indicate the importance of membrane dynamics in neuronal functions in vivo.

  11. A Dynamic Analysis of Secretory Granules Containing Proteins Involved In Learning

    NASA Astrophysics Data System (ADS)

    Prahl, Louis; Simon, Alex; Jacobs, Conor; Fulwiler, Audrey; Hilken, Lindsay; Scalettar, Bethe; Lochner, Janis

    2010-10-01

    Formation and encoding of long-term memories requires a series of structural changes at synapses, or sites of neuronal communication, in the hippocampus; these changes are mediated by neuromodulatory proteins and serve to strengthen synapses to improve communication. Two prominent neuromodulators, tissue plasminogen activator (tPA) and brain-derived neurotrophic factor (BDNF), are copackaged into secretory granules (SGs) in the body of nerve cells and are transported to distal synapses by motor proteins. At synapses, particularly presynaptic sites, the fate of tPA and BDNF is largely unknown. Motivated by this, and by recent data implicating presynaptic BDNF in early phases of learning, we used fluorescence microscopy to elucidate dynamic properties of presynaptic tPA and BDNF. We find that presynaptic SGs containing tPA and/or BDNF undergo Brownian and anomalous diffusive motion that, in 75% of cases, is so slow that it typically would be classified as immobility. These results suggest that tPA and BDNF are retained at presynaptic sites to facilitate their corelease and role in learning.

  12. The effect of total starvation and very low energy diet in lean men on kinetics of whole body protein and five hepatic secretory proteins.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It is unclear whether the rate of weight loss, independent of magnitude, affects whole body protein metabolism and the synthesis and plasma concentrations of specific hepatic secretory proteins. We examined 1) whether lean men losing weight rapidly (starvation) show greater changes in whole body pro...

  13. Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

    PubMed Central

    Brown, H; Larsson, O; Bränström, R; Yang, S N; Leibiger, B; Leibiger, I; Fried, G; Moede, T; Deeney, J T; Brown, G R; Jacobsson, G; Rhodes, C J; Braun, J E; Scheller, R H; Corkey, B E; Berggren, P O; Meister, B

    1998-01-01

    Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels. PMID:9724640

  14. Age-dependent modifications of the human salivary secretory protein complex.

    PubMed

    Cabras, Tiziana; Pisano, Elisabetta; Boi, Roberto; Olianas, Alessandra; Manconi, Barbara; Inzitari, Rosanna; Fanali, Chiara; Giardina, Bruno; Castagnola, Massimo; Messana, Irene

    2009-08-01

    Physiological variability of the naturally occurring, human salivary secretory peptidome was studied as a function of age. The qualitative and quantitative changes occurring in the secretion of proteins/peptides specific to the oral cavity (i.e., basic salivary proline-rich proteins, salivary acidic proline-rich phosphoproteins, statherin, proline-rich peptide P-B, salivary cystatins, and histatins) were investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry in 67 subjects aged between 3 and 44 years. Subjects were divided into five age groups: group A, 8 donors, 3-5 years; group B, 11 donors, 6-9 years; group C, 20 donors, 10-12 years; group D, 15 donors, 13-17 years; group E, 13 donors, 24-44 years. Basic salivary proline-rich proteins, almost undetectable in the 3-5 and 6-9 years groups, reached salivary levels comparable to that of adults (24-44 years) around puberty. Levels of peptide P-D, basic peptide P-F, peptide P-H, peptide P-J (a new basic salivary proline-rich protein characterized in this study), and basic proline-rich peptide IB-1 were significantly higher in the 10-12-year-old group than in the 3-5-year-old group, whereas the increase of proline-rich peptide II-2 was significant only after the age of 12 years. The concentration of salivary acidic proline-rich phosphoproteins, histatin-3 1/24, histatin-3 1/25, and monophosphorylated and diphosphorylated cystatin S showed a minimum in the 6-9-year-old group. Finally, the histatin-1 concentration was significantly higher in the youngest subjects (3-5 years) than in the other groups.

  15. Purification and assay of secretory lithostathine in human pancreatic juice by fast protein liquid chromatography.

    PubMed Central

    Mariani, A; Mezzi, G; Malesci, A

    1995-01-01

    Impaired secretion of lithostathine, a pancreatic glycoprotein capable of inhibiting the growth of CaCO3 crystals, has been reported in chronic calcifying pancreatitis. Controversial results were obtained, however, using immunoassays with different antibodies. The aim of this study was to purify and to measure juice lithostathine by a non-immunological method. Fast protein liquid chromatography (FPLC) on a cation exchange column eluted by a sodium chloride gradient, was used. The conditions appropriate to separate secretory (S) from hydrolysed (H) isoforms of immunopurified lithostathine were also used for juice analysis. Pancreatic juice was collected by endoscopic cannulation of the major pancreatic duct, after secretin stimulation, from eight patients with chronic pancreatitis (CP) and from eight controls. In all samples, S-isoforms of lithostathine (ranging from 16 to 19 Mr at SDS-PAGE) were the only constituent of two of the 15 peaks in which FPLC resolved the pancreatic proteins. The nature of these two peaks was confirmed by their coelution with immunopurified S-lithostathine and by immunoblot analysis with polyclonal anti-lithostathine antibodies. The ratio between the area of S-lithostathine peaks and the total area of proteic eluates, was always lower in CP patients (5.3 micrograms/mg of protein, median value; 0.2-15.4, range) than in controls (35.2 micrograms/mg; 16.6-55.9). It is concluded that lithostathine can be purified and measured in pancreatic juice by FPLC. Our results with a nonimmunological assay confirm a reduced secretion of lithostathine in patients with CP. Images Figure 2 Figure 4 Figure 5 PMID:7737574

  16. Pseudomonas aeruginosa and tumor necrosis factor-alpha attenuate Clara cell secretory protein promoter function.

    PubMed

    Harrod, Kevin S; Jaramillo, Richard J

    2002-02-01

    The Clara cell secretory protein (CCSP, also CC-10/uterglobin) is a 16-kD homodimeric protein abundantly expressed in the airways of mammals. Although the molecular function is unknown, gene-targeting studies indicate CCSP as a regulator of lung inflammation following acute respiratory infection or injury. CCSP is decreased in the lungs of mice following acute Pseudomonas aeruginosa (P.a.) infection. In the present study, the role of decreased promoter function in the regulation of CCSP by P.a. was assessed using an in vitro co-culture system and in vivo studies of transgenic mice. CCSP promoter activity in lung epithelial cells was markedly decreased by P.a. or tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner. Regulation of CCSP promoter function by either P.a. or TNF-alpha was localized to the proximal 166 bp flanking region of the CCSP promoter activity. Decreased regulation of the CCSP promoter by P.a. or TNF-alpha was specific to CCSP, as human surfactant protein D (SP-D) promoter activity was unaffected or increased by P.a. or TNF-alpha, respectively. A neutralizing antibody against human TNF-alpha was able to reverse both the TNF-alpha- mediated as well as P.a.-mediated decrease in CCSP promoter function in lung epithelial cells. TNF-alpha secretion by lung epithelial cells coincided with the decrease in CCSP promoter function following P.a. administration. Using a transgenic mouse model, P.a. administration to the lung markedly attenuated CCSP promoter-conferred gene expression in vivo. The attenuation of CCSP promoter activity in lung epithelial cells by P.a. involves, in part, autocrine/paracrine secretion of TNF-alpha, which in turn regulates CCSP transcription through cis-active elements in the proximal promoter region.

  17. Highly Sensitive Quenched Fluorescent Substrate of Legionella Major Secretory Protein (Msp) Based on Its Structural Analysis*

    PubMed Central

    Poras, Hervé; Duquesnoy, Sophie; Dange, Emilie; Pinon, Anthony; Vialette, Michèle; Fournié-Zaluski, Marie-Claude; Ouimet, Tanja

    2012-01-01

    Legionella pneumophila has been shown to secrete a protease termed major secretory protein (Msp). This protease belongs to the M4 family of metalloproteases and shares 62.9% sequence similarity with pseudolysin (EC 3.4.24.26). With the aim of developing a specific enzymatic assay for the detection and quantification of Msp, the Fluofast substrate library was screened using both enzymes in parallel. Moreover, based on the crystal structure of pseudolysin, a model of the Msp structure was built. Screening of the peptide library identified a lead substrate specifically cleaved by Msp that was subsequently optimized by rational design. The proposed model for Msp is consistent with the enzymatic characteristics of the studied peptide substrates and provides new structural information useful for the characterization of the protease. This study leads to the identification of the first selective and high affinity substrate for Msp that is able to detect picomolar concentrations of the purified enzyme. The identified substrate could be useful for the development of a novel method for the rapid detection of Legionella. PMID:22528499

  18. Secretory proteins characteristic of environmental changes in cellular signal transduction: Expression in oral fluid

    NASA Astrophysics Data System (ADS)

    Mednieks, M. I.; Burke, J. C.; Sivakumar, T. P.; Hand, A. R.; Grindeland, R. E.

    2000-01-01

    Past studies have shown that both hypo- and hyper-gravity have significant consequences on a variety of tissues and organ systems. It is not known if the effects of environmental stimuli such as altered gravity are beneficial or detrimental, and if the effects can be prevented or reversed. Animal experiments from the Space Lab and Cosmos missions indicate that events that are mediated by cyclic AMP, such as cellular responses to catecholamine and peptide hormone action, are significantly altered in a number of tissues as a consequence of space flight. A secretory cyclic AMP-receptor protein (cARP), is present in saliva, and can serve as an indicator of individual responses to physiologic and environmental stress. Animal experiments have shown that the hypergravity component of space flight is a significant stress factor. In humans, cARP levels in each individual are constant under normal conditions, but elevated after acute stress. Additionally, the levels of cARP in secreted saliva can be compared to those in gingival crevicular fluid (GCF), which reflects the protein composition of serum. The ratio of cARP in saliva to that in GCF can be used as a measure of basal compared to hyper-or hypo-gravity values. An ultimate goal is to test hyper and zero G responses in human saliva to determine if cARP is a suitable index of acute and chronic stress. A miniaturized test kit for saliva collection has been designed. Samples can be collected and stored till analyses are carried out that will distinguish the effects of increased gravity from those of one and zero G. Such tests can serve as an individualized monitoring system for physiologic responses either in space or on earth. .

  19. Corticotropin-releasing factor binding protein enters the regulated secretory pathway in neuroendocrine cells and cortical neurons.

    PubMed

    Blanco, Elías H; Zúñiga, Juan Pablo; Andrés, María Estela; Alvarez, Alejandra R; Gysling, Katia

    2011-08-01

    Corticotropin releasing factor binding protein (CRF-BP) is a 37kDa glycoprotein that binds CRF with high affinity. CRF-BP controls CRF levels within plasma during human pregnancy. It has also been shown that CRF-BP is expressed in various brain nuclei. Main actions that have been proposed for brain CRF-BP are either decreasing available CRF or facilitating CRF ligand-induced activation of CRF-R2 receptors. For both actions, it is necessary the release of CRF-BP from CRF-BP expressing neurons. However, the secretion mode of CRF-BP is currently unknown. We used heterologous expression of CRF-BP-Flag in PC12 cells and in primary culture of rat cortical neurons to study CRF-BP secretion mode. We observed that CRF-BP-Flag immunoreactivity presents the typical cytoplasmatic punctuate pattern that has been described for neuropeptides and proteins that enter the regulated secretory pathway in PC12 cells. Quantitative analysis of double immunofluorescence confocal images showed that CRF-BP-Flag colocalizes with secretogranin II, marker of secretory granules, both in PC12 and in primary-cultured rat neurons. Furthermore, CRF-BP-Flag is released from PC12 cells upon high K(+)-depolarization. Thus, our results show that CRF-BP is efficiently sorted to the regulated secretory pathway in two cellular contexts, suggesting that the extracellular levels of CRF-BP in the central nervous system depends on neuronal activity.

  20. Secretory proteins are delivered to the septin-organized penetration interface during root infection by Verticillium dahliae

    PubMed Central

    Zhou, Ting-Ting; Zhao, Yun-Long

    2017-01-01

    Successful infection of the host requires secretion of effector proteins to evade or suppress plant immunity. Secretion of effectors in root-infecting fungal pathogens, however, remains unexplored. We previously reported that Verticillium dahliae, a root-infecting phytopathogenic fungus, develops a penetration peg from a hyphopodium to infect cotton roots. In this study, we report that a septin ring, requiring VdSep5, partitions the hyphopodium and the invasive hypha and form the specialized fungus-host interface. The mutant strain, VdΔnoxb, in which NADPH oxidase B (VdNoxB) is deleted, impaired formation of the septin ring at the hyphal neck, indicating that NADPH oxidases regulate septin ring organization. Using GFP tagging and live cell imaging, we observed that several signal peptide containing secreted proteins showed ring signal accumulation/secretion at the penetration interface surrounding the hyphal neck. Targeted mutation for VdSep5 reduced the delivery rate of secretory proteins to the penetration interface. Blocking the secretory pathway by disrupting the vesicular trafficking factors, VdSec22 and VdSyn8, or the exocyst subunit, VdExo70, also arrested delivery of the secreted proteins inside the hyphopodium. Reduced virulence was observed when cotton roots were infected with VdΔsep5, VdΔsec22, VdΔsyn8 and VdΔexo70 mutants compared to infection with the isogenic wild-type V592. Taken together, our data demonstrate that the hyphal neck is an important site for protein secretion during plant root infection, and that the multiple secretory routes are involved in the secretion. PMID:28282450

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

    PubMed

    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.

  2. Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein.

    PubMed Central

    Montalto, G; Bonicel, J; Multigner, L; Rovery, M; Sarles, H; De Caro, A

    1986-01-01

    Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences. Images Fig. 1. PMID:3541906

  3. Mutant torsinA interferes with protein processing through the secretory pathway in DYT1 dystonia cells

    PubMed Central

    Hewett, Jeffrey W.; Tannous, Bakhos; Niland, Brian P.; Nery, Flavia C.; Zeng, Juan; Li, Yuqing; Breakefield, Xandra O.

    2007-01-01

    TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope responsible for early onset torsion dystonia (DYT1). Most cases of this dominantly inherited movement disorder are caused by deletion of a glutamic acid in the carboxyl terminal region of torsinA. We used a sensitive reporter, Gaussia luciferase (Gluc) to evaluate the role of torsinA in processing proteins through the ER. In primary fibroblasts from controls and DYT1 patients most Gluc activity (95%) was released into the media and processed through the secretory pathway, as confirmed by inhibition with brefeldinA and nocodazole. Fusion of Gluc to a fluorescent protein revealed coalignment and fractionation with ER proteins and association of Gluc with torsinA. Notably, fibroblasts from DYT1 patients were found to secrete markedly less Gluc activity as compared with control fibroblasts. This decrease in processing of Gluc in DYT1 cells appear to arise, at least in part, from a loss of torsinA activity, because mouse embryonic fibroblasts lacking torsinA also had reduced secretion as compared with control cells. These studies demonstrate the exquisite sensitivity of this reporter system for quantitation of processing through the secretory pathway and support a role for torsinA as an ER chaperone protein. PMID:17428918

  4. Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins.

    PubMed

    Medraño-Fernandez, Iria; Fagioli, Claudio; Mezghrani, Alexandre; Otsu, Mieko; Sitia, Roberto

    2014-04-01

    To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-μ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

  5. α1-antitrypsin Deficiency: A Misfolded Secretory Protein Variant with Unique Effects on the Endoplasmic Reticulum

    PubMed Central

    Perlmutter, David H

    2016-01-01

    In the classical form of α1-antitrypsin deficiency (ATD) a point mutation leads to accumulation of a misfolded secretory glycoprotein in the endoplasmic reticulum (ER) of liver cells and so ATD has come to be considered a prototypical ER storage disease. It is associated with two major types of clinical disorders, chronic obstructive pulmonary disease (COPD) by loss-of-function mechanisms and hepatic cirrhosis and carcinogenesis by gain-of-function mechanisms. The lung disease predominantly results from proteolytic damage to the pulmonary connective tissue matrix because of reduced levels of protease inhibitor activity of α1-anitrypsin (AT) in the circulating blood and body fluids. Cigarette smoking is a powerful disease-promoting modifier but other modifiers are known to exist because variation in the lung disease phenotype is still found in smoking and non-smoking homozygotes. The liver disease is highly likely to be caused by the proteotoxic effects of intracellular misfolded protein accumulation and a high degree of variation in the hepatic phenotype among affected homozygotes has been hypothetically attributed to genetic and environmental modifiers that alter proteostasis responses. Liver biopsies of homozygotes show intrahepatocytic inclusions with dilation and expansion of the ER and recent studies of iPS-derived hepatocyte-like cells from individuals with ATD indicate that the changes in the ER directly vary with the hepatic phenotype i.e there is much lesser alteration in the ER in cells derived from homozygotes that do not have clinically significant liver disease. From a signaling perspective, studies in mammalian cell line and animal models expressing the classical α1-antitrypsin Z variant (ATZ) have found that ER signaling is perturbed in a relatively unique way with powerful activation of autophagy and the NFκB pathway but relatively limited, if any, UPR signaling. It is still not known how much these unique structural and functional changes and

  6. Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

    PubMed Central

    Titorenko, V I; Ogrydziak, D M; Rachubinski, R A

    1997-01-01

    We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum. PMID:9271399

  7. The Emerging Roles of Early Protein Folding Events in the Secretory Pathway in the Development of Neurodegenerative Maladies

    PubMed Central

    Dubnikov, Tatyana; Cohen, Ehud

    2017-01-01

    Although, protein aggregation and deposition are unifying features of various neurodegenerative disorders, recent studies indicate that different mechanisms can lead to the development of the same malady. Among these, failure in early protein folding and maturation emerge as key mechanistic events that lead to the manifestation of a myriad of illnesses including Alzheimer's disease and prion disorders. Here we delineate the cascade of maturation steps that nascent polypeptides undergo in the secretory pathway to become functional proteins, and the chaperones that supervise and assist this process, focusing on the subgroup of proline cis/trans isomerases. We also describe the chaperones whose failure was found to be an underlying event that initiates the run-up toward neurodegeneration as well as chaperones whose activity impairs protein homeostasis (proteostasis) and thus, promotes the manifestation of these maladies. Finally, we discuss the roles of aggregate deposition sites in the cellular attempt to maintain proteostasis and point at potential targets for therapeutic interventions. PMID:28223916

  8. The Emerging Roles of Early Protein Folding Events in the Secretory Pathway in the Development of Neurodegenerative Maladies.

    PubMed

    Dubnikov, Tatyana; Cohen, Ehud

    2017-01-01

    Although, protein aggregation and deposition are unifying features of various neurodegenerative disorders, recent studies indicate that different mechanisms can lead to the development of the same malady. Among these, failure in early protein folding and maturation emerge as key mechanistic events that lead to the manifestation of a myriad of illnesses including Alzheimer's disease and prion disorders. Here we delineate the cascade of maturation steps that nascent polypeptides undergo in the secretory pathway to become functional proteins, and the chaperones that supervise and assist this process, focusing on the subgroup of proline cis/trans isomerases. We also describe the chaperones whose failure was found to be an underlying event that initiates the run-up toward neurodegeneration as well as chaperones whose activity impairs protein homeostasis (proteostasis) and thus, promotes the manifestation of these maladies. Finally, we discuss the roles of aggregate deposition sites in the cellular attempt to maintain proteostasis and point at potential targets for therapeutic interventions.

  9. The Tobacco etch virus P3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments.

    PubMed

    Cui, Xiaoyan; Wei, Taiyun; Chowda-Reddy, R V; Sun, Guangyu; Wang, Aiming

    2010-02-05

    Plant potyviruses encode two membrane proteins, 6K and P3. The 6K protein has been shown to induce virus replication vesicles. However, the function of P3 remains unclear. In this study, subcellular localization of the Tobacco etch virus (TEV) P3 protein was investigated in Nicotiana benthamiana leaf cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway. The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments and colocalize with the 6K-containing replication vesicles. Taken together, these data support previous suggestions that P3 may play dual roles in virus movement and replication.

  10. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    SciTech Connect

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-07-01

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary lambdagt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/..beta..-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of /sup 125/I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene.

  11. A 24,500 Da protein derived from rat germ cells is associated with sertoli cell secretory function.

    PubMed

    Onoda, M; Djakiew, D

    1993-12-15

    A function and identify of a 24,500 Da protein derived from round spermatids of the rat testis was investigated with a specific polyclonal antiserum raised against RSP-24.5. The proteins released from cultured round spermatids significantly stimulated the secretion of de novo synthesized protein from cultured immature rat Sertoli cells 2.4-fold above control levels. Immunoprecipitation of RSP-24.5 from round spermatid protein further enhanced the stimulation of Sertoli cell protein secretion up to 3.1-fold above control levels, indicating that RSP-24.5 plays a role in the down regulation of Sertoli cell secretion. The antiserum recognized the 24,500 Da protein in Western blots of round spermatid protein, pachytene spermatoctye protein, Sertoli cell lysate and peritubular myoid cell lysate. A 40 amino acid sequence of a cyanogen bromide cleaved internal fragment of RSP-24.5 showed 80.5% homology to a phosphatidylethanolamine binding protein. These results suggest that phosphatidylethanolamine binding protein participates in the negative regulation of Sertoli cell secretory function during spermatogenesis.

  12. High abundance of Serine/Threonine-rich regions predicted to be hyper-O-glycosylated in the secretory proteins coded by eight fungal genomes

    PubMed Central

    2012-01-01

    Background O-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae) in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk). Results By comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length. Conclusions About one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends. PMID:22994653

  13. Antibody and cytokine responses to Giardia excretory/secretory proteins in Giardia intestinalis-infected BALB/c mice.

    PubMed

    Jiménez, Juan C; Fontaine, Josette; Creusy, Colette; Fleurisse, Laurence; Grzych, Jean-Marie; Capron, Monique; Dei-Cas, Eduardo

    2014-07-01

    The humoral and cellular responses against excretory/secretory proteins and soluble extracts of Giardia intestinalis were evaluated in the course of experimental G. intestinalis infection in BALB/c mice. Production of IgG1, IgG2a, IgA, and IgE antibodies against excreted/secreted proteins and soluble extract was detected after infection by G. intestinalis. Specific IgA antibody against E/S proteins and soluble extract form intestinal fluids in infected mice was detected by ELISA. The Western blotting identified proteins of 30, 58, 63, and 83 kDa for IgA and IgG, respectively. High proliferation rate in vitro of spleen cell and secretion of interleukin-4 (IL-4) at 21 days p.i. after stimulation with excreted/secreted proteins and low proliferative response in the presence of soluble extract in infected BALB/c mice was observed. High production of interferon gamma (IFN-γ) and interleukin-5 (IL-5) at the time of decreasing cyst output (14-21 days p.i.) in infected mice was recorded, suggesting the important role of these cytokines in the control of the infection. Interestingly, progressive and gradual increase of the interleukin-10 after stimulation with both preparations was recorded from 7 days until 28 days after infection, indicating the possible regulatory effect of these antigens on the immune response during Giardia infection. Therefore, the infection by Giardia duodenalis stimulates a mixed response Th1 and Th2, mainly stimulated by excretory/secretory antigens. The immunogenicity of these antigens may be a suitable for identification of the proteins related with the effective immune response in the course of infection by G. duodenalsis.

  14. Maurer's clefts of Plasmodium falciparum are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte

    PubMed Central

    Bhattacharjee, Souvik; van Ooij, Christiaan; Balu, Bharath; Adams, John H.

    2008-01-01

    In blood-stage infection by the human malaria parasite Plasmodium falciparum, export of proteins from the intracellular parasite to the erythrocyte is key to virulence. This export is mediated by a host-targeting (HT) signal present on a “secretome” of hundreds of parasite proteins engaged in remodeling the erythrocyte. However, the route of HT-mediated export is poorly understood. Here we show that minimal soluble and membrane protein reporters that contain the HT motif and mimic export of endogenous P falciparum proteins are detected in the lumen of “cleft” structures synthesized by the pathogen. Clefts are efficiently targeted by the HT signal. Furthermore, the HT signal does not directly translocate across the parasitophorous vacuolar membrane (PVM) surrounding the parasite to deliver protein to the erythrocyte cytoplasm, as suggested by current models of parasite protein trafficking to the erythrocyte. Rather, it is a lumenal signal that sorts protein into clefts, which then are exported beyond the PVM. These data suggest that Maurer's clefts, which are unique to the virulent P falciparum species, are pathogen-induced secretory organelles that concentrate HT-containing soluble and membrane parasite proteins in their lumen for delivery to the host erythrocyte. PMID:18057226

  15. Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

    PubMed

    Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

    2015-09-01

    Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.

  16. Immunoprotection in sheep against Haemonchus contortus using its thiol-purified excretory/secretory proteins.

    PubMed

    Arunkumar, Selvarayar

    2012-01-01

    Excretory/Secretory antigen was prepared by culturing live adult worms of Haemonchus contortus in RPMI 1640 medium at a concentration of 50 worms per mL in a culture-flask at 37 ˚C for 24 hr and the culture supernatant was used as antigen. The E/S antigen was purified by thiol-sepharose affinity chromatography. On western blot analysis, it was demonstrated that thiol-purified antigen showed a single reactive band at 66 kDa. In immunization trial, sheep were administered intramuscularly with 500 µg of thiol-purified excretory/secretory antigen along with montanide as adjuvant on day 0, 30 and 60. On ELISA, it was observed that the mean absorbance values were significantly (p ≤ 0.01) higher up to 20 weeks post immunization in Group-I (purified antigen) compared to Group- II (unimmunized control). Further, the mean EPG values was lower in Group I (200.00 ± 40.82 to 400.00 ± 91.29) than Group II (2200.00 ± 108.01 to 5100.00 ± 169.56) and the percentage reduction in mean fecal egg counts was 88.50%. Similarly, the mean abomasal worm counts was lower in Group I (808.33 ± 78.29) than Group II (3280.00 ± 147.19) and the percentage reduction in mean abomasal worm count was 75.40%.

  17. Secretory mechanism of fibroin, a silk protein, in the posterior silk gland cells of Bombyx mori.

    PubMed

    Sasaki, S; Nakagaki, I

    1980-01-01

    There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intracellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.

  18. Secretory protein decondensation as a distinct, Ca2+-mediated event during the final steps of exocytosis in Paramecium cells

    PubMed Central

    1981-01-01

    The contents of secretory vesicles ("trichocysts") were isolated in the condensed state from Paramecium cells. It is well known that the majority portion of trichocysts perform a rapid decondensation process during exocytosis, which is visible in the light microscope. We have analyzed this condensed leads to decondensed transition in vitro and determined some relevant parameters. In the condensed state, free phosphate (and possibly magnesium) ions screen local surplus charges. This is supported by x-ray spectra recorded from individual trichocysts (prepared by physical methods) in a scanning transmission electron microscope. Calcium, as well as other ions that eliminate phosphate by precipitation, produces decondensation in vitro. Under in vivo conditions, Ca2+ enters the vesicle lumen from the outside medium, once an exocytic opening has been formed. Consequently, within the intact cell, membrane fusion and protein decondensation take place with optimal timing. Ca2+ might then trigger decondensation in the same way by precipitating phosphate ions (as it does in vitro) and, indeed, such precipitates (again yielding Ca and P signals in x-ray spectra) can be recognized in situ under trigger conditions. As decondensation is a unidirectional, rapid process in Paramecium cells, it would contribute to drive the discharge of the secretory contents to the outside. Further implications on the energetics of exocytosis are discussed. PMID:7204486

  19. Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein

    SciTech Connect

    Plopper, C.G. . E-mail: cgplopper@ucdavis.edu; Mango, G.W.; Hatch, G.E.; Wong, V.J.; Toskala, E.; Reynolds, S.D.; Tarkington, B.K.; Stripp, B.R.

    2006-05-15

    Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O{sub 3}). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

  20. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR).

    PubMed

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J; Laclette, Juan P; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-05-19

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest.

  1. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins

    PubMed Central

    Mehnert, Martin; Sommermeyer, Franziska; Berger, Maren; Kumar Lakshmipathy, Sathish; Gauss, Robert; Aebi, Markus; Jarosch, Ernst; Sommer, Thomas

    2015-01-01

    Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1. PMID:25428985

  2. Dirofilaria immitis exhibits sex- and stage-specific differences in excretory/secretory miRNA and protein profiles.

    PubMed

    Tritten, Lucienne; Clarke, Damian; Timmins, Scott; McTier, Tom; Geary, Timothy G

    2016-12-15

    The canine heartworm Dirofilaria immitis releases excretory/secretory molecules into its host and in culture. We report analyses of the types, amounts and stage-dependence of microRNAs and proteins found in D. immitis culture media recovered after incubating 800,000 microfilariae for 6days, 500L3 and 500L4 for 7days, as well as 40 adult females and 40 adult males for 48h, all separately. In addition, the presence of exosome-like particles was established by nanoparticle tracking analysis. Our results are in concordance with the D. immitis molecules previously detected in dog blood and in culture medium, but add additional insight into the sex- and stage-specificity of these processes. Of 131 miRNA candidates analyzed, none of the most abundant sequences was exclusively associated with one stage. Several isoforms of the nematode miR-100 family, miR-279, miR-71, were highly represented and overlapped substantially with the profile of heartworm miRNAs described from infected dog blood. lin-4 was primarily associated with males. We also report 4, 27 and 72 proteins in media from microfilariae, females and males, respectively. The only protein in common to all samples was actin, and only 9/88 proteins with a gene ontology description had not been reported in other studies of filarial secretomes. Exosomal proteins were well represented, dominated by cytoskeletal proteins, metabolic enzymes, zeta polypeptide, and chaperones.

  3. The effect of total starvation and very low energy diet in lean men on kinetics of whole body protein and five hepatic secretory proteins.

    PubMed

    Afolabi, Paul R; Jahoor, Farook; Jackson, Alan A; Stubbs, James; Johnstone, Alexander M; Faber, Peter; Lobley, Gerald; Gibney, Eileen; Elia, Marinos

    2007-12-01

    It is unclear whether the rate of weight loss, independent of magnitude, affects whole body protein metabolism and the synthesis and plasma concentrations of specific hepatic secretory proteins. We examined 1) whether lean men losing weight rapidly (starvation) show greater changes in whole body protein kinetics, synthesis, and circulating concentrations of selected hepatic secretory proteins than those losing the same amount of weight more slowly [very low energy diet (VLED)]; and 2) whether plasma concentrations and synthetic rates of these proteins are related. Whole body protein kinetics were measured using [1-(13)C]leucine in 11 lean men (6 starvation, 5 VLED). Fractional and absolute synthetic rates of HDL-apolipoprotein A1 (apoA1), retinol binding protein, transthyretin, alpha(1)-antitrypsin (alpha(1)-AT), and transferrin were measured using a prime-constant intravenous infusion of [(13)C(2)]glycine. Compared with VLED group, the starvation group showed greater increases (at a 5% weight loss) in whole body protein oxidation (P < 0.05); fractional synthetic rates of HDL-apoA1 (25.3 vs. -1.52%; P = 0.003) and retinol binding protein (30.6 vs. 7.1%; P = 0.007); absolute synthetic rates of HDL-apoA1 (7.1 vs. -3.8 mg.kg(-1).day(-1); P = 0.003) and alpha(1)-AT (17.8 vs. 3.6 mg.kg(-1).day(-1); P = 0.02); and plasma concentration of alpha(1)-AT (P = 0.025). Relationships between synthetic rates and plasma concentrations varied between the secreted proteins. It is concluded that synthetic rates of hepatic secreted proteins in lean men are more closely related to the rate than the magnitude of weight loss. Changes in concentration of these secreted proteins can occur independently of changes in synthetic rates, and vice versa.

  4. Changes in protein expression after treatment with Ancylostoma caninum excretory/secretory products in a mouse model of colitis.

    PubMed

    Sotillo, Javier; Ferreira, Ivana; Potriquet, Jeremy; Laha, Thewarach; Navarro, Severine; Loukas, Alex; Mulvenna, Jason

    2017-02-13

    Different reports have highlighted the potential use of helminths and their secretions in the treatment of inflammatory bowel disease (IBD) conditions; however, no reports have investigated their effects at a proteome level. Herein, we characterise the protein expression changes that occur in lamina propria (LP) and the intestinal epithelial cells (IEC) of mice with dextran sulfate sodium (DSS)-induced colitis treated with Ancylostoma caninum excretory/secretory (ES) products using a quantitative proteomic approach. We have shown how parasite products can significantly alter the expression of proteins involved in immune responses, cell death and with an antioxidant activity. Interestingly, significant changes in the expression levels of different mucins were observed in this study. MUC13, a mucin implicated in gastrointestinal homeostasis, was upregulated in the LP of mice with DSS-induced colitis treated with ES, while MUC2, a major component of mucus, was upregulated in the IEC. In addition, A. caninum proteins have an important effect on proteins with antioxidant functions and proteins involved in intestinal homeostasis and tissue integrity and regeneration. Understanding how parasites can ameliorate IBD pathogenesis can help us design novel treatments for autoimmune diseases.

  5. Changes in protein expression after treatment with Ancylostoma caninum excretory/secretory products in a mouse model of colitis

    PubMed Central

    Sotillo, Javier; Ferreira, Ivana; Potriquet, Jeremy; Laha, Thewarach; Navarro, Severine; Loukas, Alex; Mulvenna, Jason

    2017-01-01

    Different reports have highlighted the potential use of helminths and their secretions in the treatment of inflammatory bowel disease (IBD) conditions; however, no reports have investigated their effects at a proteome level. Herein, we characterise the protein expression changes that occur in lamina propria (LP) and the intestinal epithelial cells (IEC) of mice with dextran sulfate sodium (DSS)-induced colitis treated with Ancylostoma caninum excretory/secretory (ES) products using a quantitative proteomic approach. We have shown how parasite products can significantly alter the expression of proteins involved in immune responses, cell death and with an antioxidant activity. Interestingly, significant changes in the expression levels of different mucins were observed in this study. MUC13, a mucin implicated in gastrointestinal homeostasis, was upregulated in the LP of mice with DSS-induced colitis treated with ES, while MUC2, a major component of mucus, was upregulated in the IEC. In addition, A. caninum proteins have an important effect on proteins with antioxidant functions and proteins involved in intestinal homeostasis and tissue integrity and regeneration. Understanding how parasites can ameliorate IBD pathogenesis can help us design novel treatments for autoimmune diseases. PMID:28191818

  6. A computational method for prediction of saliva-secretory proteins and its application to identification of head and neck cancer biomarkers for salivary diagnosis.

    PubMed

    Sun, Ying; Du, Wei; Zhou, Chunguang; Zhou, You; Cao, Zhongbo; Tian, Yuan; Wang, Yan

    2015-03-01

    Human saliva is rich in proteins, which have been used for disease detection such as oral diseases and systematic diseases. In this paper, we present a computational method for predicting secretory proteins in human saliva based on two sets of human proteins from published literatures and public databases. One set contains known proteins which can be secreted into saliva, and the other contains the proteins that are deemed to be not extracellular secretion. The protein features with discerning power between two sets were firstly gathered. Then a classifier was trained based on the identified features to predict whether a protein was saliva-secretory one or not. The average values of the sensitivity, specificity, precision, accuracy, and Matthews correlation coefficient value by 10-fold cross validation repeated 100 times were 80.67%, 90.56%, 90.09%, 85.53%, and 0.7168, respectively. These results indicated that our selected features are informative. We applied the classifier for prediction saliva-secretory proteins out of all human proteins, if a known biomarker was likely to enter into saliva, and the potential salivary biomarkers for head and neck squamous cell carcinoma. We also compared the top 1000 proteins predicted by computational methods in different kind of fluids. This work provided a useful tool for effectively identifying the salivary biomarkers for various human diseases and facilitate the development of salivary diagnosis.

  7. Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*

    PubMed Central

    Heard, William; Sklenář, Jan; Tomé, Daniel F. A.; Robatzek, Silke; Jones, Alexandra M. E.

    2015-01-01

    The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein

  8. Protein Domain of Unknown Function 3233 is a Translocation Domain of Autotransporter Secretory Mechanism in Gamma proteobacteria

    PubMed Central

    Prakash, Ananth; Yogeeshwari, S.; Sircar, Sanchari; Agrawal, Shipra

    2011-01-01

    Vibrio cholerae, the enteropathogenic gram negative bacteria is one of the main causative agents of waterborne diseases like cholera. About 1/3rd of the organism's genome is uncharacterised with many protein coding genes lacking structure and functional information. These proteins form significant fraction of the genome and are crucial in understanding the organism's complete functional makeup. In this study we report the general structure and function of a family of hypothetical proteins, Domain of Unknown Function 3233 (DUF3233), which are conserved across gram negative gammaproteobacteria (especially in Vibrio sp. and similar bacteria). Profile and HMM based sequence search methods were used to screen homologues of DUF3233. The I-TASSER fold recognition method was used to build a three dimensional structural model of the domain. The structure resembles the transmembrane beta-barrel with an axial N-terminal helix and twelve antiparallel beta-strands. Using a combination of amphipathy and discrimination analysis we analysed the potential transmembrane beta-barrel forming properties of DUF3233. Sequence, structure and phylogenetic analysis of DUF3233 indicates that this gram negative bacterial hypothetical protein resembles the beta-barrel translocation unit of autotransporter Va secretory mechanism with a gene organisation that differs from the conventional Va system. PMID:22073138

  9. iTRAQ-based comparative proteomic analysis of excretory-secretory proteins of schistosomula and adult worms of Schistosoma japonicum.

    PubMed

    Cao, Xiaodan; Fu, Zhiqiang; Zhang, Min; Han, Yanhui; Han, Hongxiao; Han, Qian; Lu, Ke; Hong, Yang; Lin, Jiaojiao

    2016-04-14

    Schistosomiasis remains a serious public health problem with 200 million people infected and 779 million people at risk worldwide. The schistosomulum and adult worm are two stages of the complex lifecycle of Schistosoma japonicum and excretory/secretory proteins (ESPs) play a major role in host-parasite interactions. In this study, iTRAQ-coupled LC-MS/MS was used to investigate the proteome of ESPs obtained from schistosomula and adult worms of S. japonicum, and 298 differential ESPs were identified. Bioinformatics analysis of differential ESPs in the two developmental stages showed that 161 ESPs upregulated in schistosomula were associated with stress responses, carbohydrate metabolism and protein degradation, whereas ESPs upregulated in adult worms were mainly related to immunoregulation and purine metabolism. Recombinant heat shock protein 70 (HSP70) and thioredoxin peroxidase (TPx), two differential proteins identified in this study, were expressed. Further studies showed that rSjHSP70 and rSjTPx stimulated macrophages expressing high levels of the anti-inflammatory factors TGF-β, IL-10 and Arg-1, and suppressed the expression of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and iNOS in LPS-induced macrophages. This study provides new insights into the survival and development of schistosomes in the final host and helps identify vaccine candidates or new diagnostic reagents for schistosomiasis.

  10. Brugia malayi soluble and excretory-secretory proteins attenuate development of streptozotocin-induced type 1 diabetes in mice.

    PubMed

    Amdare, N; Khatri, V; Yadav, R S P; Tarnekar, A; Goswami, K; Reddy, M V R

    2015-12-01

    Understanding the modulation of the host-immune system by pathogens-like filarial parasites offers an alternate approach to prevent autoimmune diseases. In this study, we have shown that treatment with filarial proteins prior to or after the clinical onset of streptozotocin-induced type-1 diabetes (T1D) can ameliorate the severity of disease in BALB/c mice. Pre-treatment with Brugia malayi adult soluble (Bm A S) or microfilarial excretory-secretory (Bm mf ES) or microfilarial soluble (Bm mf S) antigens followed by induction of diabetes led to lowering of fasting blood glucose levels with as many as 57.5-62.5% of mice remaining nondiabetic. These proteins were more effective when they were used to treat the mice with established T1D as 62.5-71.5% of the mice turned to be nondiabetic. Histopathological examination of pancreas of treated mice showed minor inflammatory changes in pancreatic islet cell architecture. The therapeutic effect was found to be associated with the decreased production of cytokines TNF-α & IFN-γ and increased production of IL-10 in the culture supernatants of splenocytes of treated mice. A switch in the production of anti-insulin antibodies from IgG2a to IgG1 isotype was also seen. Together these results provide a proof towards utilizing the filarial derived proteins as novel anti-diabetic therapeutics.

  11. Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

    PubMed

    Hansson, Karin; Kjellberg, Margareta; Fernlund, Per

    2009-08-01

    BETA-microseminoprotein (MSP), a 10 kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is.

  12. Neuroendocrine secretory protein 55 (NESP55) immunoreactivity in male and female rat superior cervical ganglion and other sympathetic ganglia.

    PubMed

    Li, Yongling; Wang, Zhanyou; Dahlström, Annica

    2007-03-30

    Neuroendocrine secretory protein 55 (NESP55) is a soluble, acidic and heat-stable protein, belonging to the class of chromogranins. It is expressed specifically in endocrine cells and the nervous system, and is probably involved in both constitutive and regulated secretion. In the present study, we investigated the distribution of NESP55 in various rat sympathetic ganglia by immunohistochemistry. The expression of NESP55-IR was detected in a subpopulation of principal neurons in the rat SCG, which was also TH positive, and, thus, adrenergic. In the rat stellate ganglion, more than two thirds of NESP55 positive neurons were adrenergic. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP) in cholinergic neurons was also observed. In the rat thoracic chain, however, the majority of NESP55 positive neurons appeared to lack TH. No detectable NESP55-IR was found in the mouse SCG. Furthermore, in the sexually dimorphic SCG, it was demonstrated that, 80% of the NESP55 positive principal neurons were also NPY positive in the male rat, while a slightly higher, but statistically significant proportion, 87%, was found in the female. Whether or not this small difference is physiologically significant is unknown. The present data provide basic knowledge about the expression of NESP55 in the sympathetic autonomic nervous system of rat, which may further our understanding of the functional significance of NESP55.

  13. Analysis of Translocation-Competent Secretory Proteins by HDX-MS.

    PubMed

    Tsirigotaki, A; Papanastasiou, M; Trelle, M B; Jørgensen, T J D; Economou, A

    2017-01-01

    Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR. Here, we present an alternative approach for the in vitro analysis of nonfolded translocation-competent protein states and their comparison with their native states. We make use of hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS), a method based on differentiated isotope exchange rates in structured vs unstructured protein states/regions, and highly dynamic vs more rigid regions. We present a complete structural characterization pipeline, starting from the preparation of the polypeptides to data analysis and interpretation. Proteolysis and mass spectrometric conditions for the analysis of the labeled proteins are discussed, followed by the analysis and interpretation of HDX-MS data. We highlight the suitability of HDX-MS for identifying short structured regions within otherwise highly flexible protein states, as illustrated by an exported protein example, experimentally tested in our lab. Finally, we discuss statistical analysis in comparative HDX-MS. The protocol is applicable to any protein and protein size, exhibiting slow or fast loss of translocation competence. It could be easily adapted to more complex assemblies, such as the interaction of chaperones with nonnative protein states.

  14. Independent transport and sorting of functionally distinct protein families in Tetrahymena thermophila dense core secretory granules.

    PubMed

    Rahaman, Abdur; Miao, Wei; Turkewitz, Aaron P

    2009-10-01

    Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, DeltaGRT1 DeltaGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in DeltaGRT1 DeltaGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from DeltaGRT1 DeltaGRT2 cells appear less adhesive than those from the wild type.

  15. Characterization of toxins from the broad-banded water snake Helicops angulatus (Linnaeus, 1758): isolation of a cysteine-rich secretory protein, Helicopsin.

    PubMed

    Estrella, Amalid; Sánchez, Elda E; Galán, Jacob A; Tao, W Andy; Guerrero, Belsy; Navarrete, Luis F; Rodríguez-Acosta, Alexis

    2011-04-01

    Helicops angulatus (broad-banded water snake) according to recent proposals is presently cited in the family Dipsadidae, subfamily Xenodontinae, forming the tribe Hydropsini along with the genera Hydrops and Pseudoeryx. The current work characterizes the proteolytic and neurotoxic activities of H. angulatus crude toxins from salivary excretion (SE) and describes the isolation and identification of a cysteine-rich secretory protein (CRISP) called helicopsin. The SE lethal dose (LD₅₀) was 5.3 mg/kg; however, the SE did not contain hemorrhagic activity. Helicopsin was purified using activity-guided, Superose 12 10/300 GL molecular exclusion, Mono Q10 ion exchange, and Protein Pak 60 molecular exclusion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a highly purified band of approximately 20 kDa. The minimal lethal dose for helicopsin was 0.4 mg/kg. Liquid chromatography mass spectrometry (LC-MS/MS) analysis identified 2 unique peptides MEWYPEAAANAER and YTQIVWYK, representing a protein sequence (deleted homology) belonging to cysteine-rich secretory proteins, which are conserved in snake venoms (CRISPs). CRISPs are a large family of cysteine-rich secretory proteins found in various organisms and participate in diverse biological processes. Helicopsin exhibited robust neurotoxic activity as evidenced by immediate death (~8 min) due to respiratory paralysis in NIH mice. These observations for helicopsin purified from H. angulatus provide further evidence of the extensive distribution of highly potent neurotoxins in the Colubroidea superfamily of snakes than previously described.

  16. Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro.

    PubMed

    Reyes-Moreno, Carlos; Boilard, Mathieu; Sullivan, Robert; Sirard, Marc-André

    2002-12-01

    Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture

  17. Design of a covalently linked human interleukin-10 fusion protein and its secretory expression in Escherichia coli.

    PubMed

    Guggenbichler, Florian; Büttner, Carolin; Rudolph, Wolfram; Zimmermann, Kurt; Gunzer, Florian; Pöhlmann, Christoph

    2016-12-01

    Wild-type human interleukin-10 (hIL-10) is a non-covalent homodimer with a short half-life, thus limiting its therapeutic applications in vivo. To avoid loss of function due to dimer dissociation, we designed a synthetic hIL-10 analog by bridging both monomers via a 15 amino acid-long peptide spacer in a C-terminal to N-terminal fashion. For secretory expression in Escherichia coli, a 1156 bp fragment was generated from template vector pAZ1 by fusion PCR encoding a T7 promoter region and the signal sequence of the E. coli outer membrane protein F fused in frame to two tandem E. coli codon-optimized mature hIL-10 genes connected via a 45 nucleotide linker sequence. The construct was cloned into pUC19 for high-level expression in E. coli BL21 (DE3). The mean concentrations of hIL-10 fusion protein in the periplasm and supernatant of E. coli at 37 °C growth temperature were 130 ± 40 and 2 ± 1 ng/ml, respectively. The molecular mass of the recombinant protein was assessed via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, indicating correct processing of the signaling sequence in E. coli. In vitro biological activity was shown by phosphorylation of signal transducer and activator of transcription protein 3 and suppression of tumor necrosis factor α secretion in lipopolysaccharide-stimulated macrophages.

  18. Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and Leishmania

    PubMed Central

    Adade, Camila M.; Carvalho, Ana Lúcia O.; Tomaz, Marcelo A.; Costa, Tatiana F. R.; Godinho, Joseane L.; Melo, Paulo A.; Lima, Ana Paula C. A.; Rodrigues, Juliany C. F.; Zingali, Russolina B.; Souto-Padrón, Thaïs

    2014-01-01

    Background The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Methodology/Principal Findings Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10–2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. Conclusions This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases. PMID:25330220

  19. Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1.

    PubMed

    Perttilä, Julia; Spuul, Pirjo; Ahola, Tero

    2013-04-01

    Hepatitis E virus (HEV) is a positive-strand RNA virus and a major causative agent of acute sporadic and epidemic hepatitis. HEV replication protein is encoded by ORF1 and contains the predicted domains of methyltransferase (MT), protease, macro domain, helicase (HEL) and polymerase (POL). In this study, the full-length protein pORF1 (1693 aa) and six truncated variants were expressed by in vitro translation and in human HeLa and hepatic Huh-7 cells by using several vector systems. The proteins were visualized by three specific antisera directed against the MT, HEL and POL domains. In vitro translation of full-length pORF1 yielded smaller quantities of two fragments. However, these fragments were not observed after pORF1 expression and pulse-chase studies in human cells, and their production was not dependent on the predicted protease domain in pORF1. The weight of evidence supports the proposition that pORF1 is not subjected to specific proteolytic processing, which is unusual among animal positive-strand RNA viruses but common for plant viruses. pORF1 was membrane associated in cells and localized to a perinuclear region, where it partially overlapped with localization of the endoplasmic reticulum (ER) marker BAP31 and was closely interspersed with staining of the ER-Golgi intermediate compartment marker protein ERGIC-53. Co-localization with BAP31 was enhanced by treatment with brefeldin A. Therefore, HEV may utilize modified early secretory pathway membranes for replication.

  20. Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.

    PubMed

    Claes, Katrien; Vandewalle, Kristof; Laukens, Bram; Laeremans, Toon; Vosters, Olivier; Langer, Ingrid; Parmentier, Marc; Steyaert, Jan; Callewaert, Nico

    2016-10-21

    Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies.

  1. Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway.

    PubMed Central

    Sanderson, C M; McQueen, N L; Nayak, D P

    1993-01-01

    We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins. Images PMID:8380460

  2. Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

    SciTech Connect

    Vogel, Lotte K. . E-mail: vogel@imbg.ku.dk; Larsen, Jakob E.; Hansen, Martin; Truffer, Renato

    2005-05-13

    Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity of secretion is determined by conformational sensitive sorting signals.

  3. Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

    PubMed Central

    Labunskyy, Vyacheslav M.; Gerashchenko, Maxim V.; Delaney, Joe R.; Kaya, Alaattin; Kennedy, Brian K.; Kaeberlein, Matt; Gladyshev, Vadim N.

    2014-01-01

    Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity. PMID:24391512

  4. βIII Spectrin Regulates the Structural Integrity and the Secretory Protein Transport of the Golgi Complex*

    PubMed Central

    Salcedo-Sicilia, Laia; Granell, Susana; Jovic, Marko; Sicart, Adrià; Mato, Eugenia; Johannes, Ludger; Balla, Tamas; Egea, Gustavo

    2013-01-01

    A spectrin-based cytoskeleton is associated with endomembranes, including the Golgi complex and cytoplasmic vesicles, but its role remains poorly understood. Using new generated antibodies to specific peptide sequences of the human βIII spectrin, we here show its distribution in the Golgi complex, where it is enriched in the trans-Golgi and trans-Golgi network. The use of a drug-inducible enzymatic assay that depletes the Golgi-associated pool of PI4P as well as the expression of PH domains of Golgi proteins that specifically recognize this phosphoinositide both displaced βIII spectrin from the Golgi. However, the interference with actin dynamics using actin toxins did not affect the localization of βIII spectrin to Golgi membranes. Depletion of βIII spectrin using siRNA technology and the microinjection of anti-βIII spectrin antibodies into the cytoplasm lead to the fragmentation of the Golgi. At ultrastructural level, Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of protein transport assays, we show that the endoplasmic reticulum-to-Golgi and post-Golgi protein transports were impaired in βIII spectrin-depleted cells. However, the internalization of the Shiga toxin subunit B to the endoplasmic reticulum was unaffected. We state that βIII spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain its structural integrity and secretory activity, and unlike actin, PI4P appears to be highly relevant for the association of βIII spectrin the Golgi complex. PMID:23233669

  5. Genome-Wide Comparison of Magnaporthe Species Reveals a Host-Specific Pattern of Secretory Proteins and Transposable Elements

    PubMed Central

    Gowda, Malali

    2016-01-01

    Blast disease caused by the Magnaporthe species is a major factor affecting the productivity of rice, wheat and millets. This study was aimed at generating genomic information for rice and non-rice Magnaporthe isolates to understand the extent of genetic variation. We have sequenced the whole genome of the Magnaporthe isolates, infecting rice (leaf and neck), finger millet (leaf and neck), foxtail millet (leaf) and buffel grass (leaf). Rice and finger millet isolates infecting both leaf and neck tissues were sequenced, since the damage and yield loss caused due to neck blast is much higher as compared to leaf blast. The genome-wide comparison was carried out to study the variability in gene content, candidate effectors, repeat element distribution, genes involved in carbohydrate metabolism and SNPs. The analysis of repeat element footprints revealed some genes such as naringenin, 2-oxoglutarate 3-dioxygenase being targeted by Pot2 and Occan, in isolates from different host species. Some repeat insertions were host-specific while other insertions were randomly shared between isolates. The distributions of repeat elements, secretory proteins, CAZymes and SNPs showed significant variation across host-specific lineages of Magnaporthe indicating an independent genome evolution orchestrated by multiple genomic factors. PMID:27658241

  6. Human epididymis protein 4 and secretory leukocyte protease inhibitor in vaginal fluid: relation to vaginal components and bacterial composition.

    PubMed

    Orfanelli, Theofano; Jayaram, Aswathi; Doulaveris, Georgios; Forney, Larry J; Ledger, William J; Witkin, Steven S

    2014-04-01

    Human epididymis protein 4 (HE4) is a protease inhibitor and a recently identified serum biomarker for ovarian cancer. Properties of HE4 in the genital tract of healthy women have not been evaluated. We evaluated associations between HE4 and a second vaginal protease inhibitor, secretory leukocyte protease inhibitor (SLPI), with vaginal concentrations of innate immune mediators or proteases and with the types of vaginal bacterial communities. Vaginal secretions were collected from 18 healthy reproductive age women and assayed by enzyme-linked immunosorbent assay for concentrations of HE4, SLPI, kallikrein 5, cathepsin B, interleukin 1β (IL-1), IL-1 receptor antagonist (IL-1 ra), mannose-binding lectin (MBL), the inducible 70-kDa heat shock protein, and matrix metalloproteinase (MMP)-8. The species composition of vaginal bacterial communities in 16 women was characterized by sequencing amplicons derived from 16S bacterial ribosomal RNA genes. Correlations between any 2 assays were analyzed by the Spearman rank correlation tests. Differences in the concentrations of HE4 and SLPI, and between soluble components and vaginal community types, were analyzed by the Mann-Whitney U tests. Vaginal HE4 concentrations, but not SLPI levels, were positively correlated with levels of IL-1β (P = .0152), IL-1ra (P = .0061), MBL (P = .0100), and MMP-8 (P = .0315). The median vaginal HE4 level, as well as concentrations of MBL, IL-1β, IL-1ra, and MMP-8, was highest when Gardnerella vaginalis dominated a vaginal community. The association between HE4, elevated levels of proteases, immune mediators and high proportions of G vaginalis strongly suggests that HE4 is a component of the proinflammatory immune response in the female genital tract.

  7. Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway.

    PubMed Central

    Harter, C; Pavel, J; Coccia, F; Draken, E; Wegehingel, S; Tschochner, H; Wieland, F

    1996-01-01

    Coatomer, a cytosolic heterooligomeric protein complex that consists of seven subunits [alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP (nonclathrin coat protein)], has been shown to interact with dilysine motifs typically found in the cytoplasmic domains of various endoplasmic-reticulum-resident membrane proteins [Cosson, P. & Letourneur, F. (1994) Science 263, 1629-1631]. We have used a photo-cross-linking approach to identify the site of coatomer that is involved in binding to the dilysine motifs. An octapeptide corresponding to the C-terminal tail of Wbp1p, a component of the yeast N-oligosaccharyltransferase complex, has been synthesized with a photoreactive phenylalanine at position -5 and was radioactively labeled with [125I]iodine at a tyrosine residue introduced at the N terminus of the peptide. Photolysis of isolated coatomer in the presence of this peptide and immunoprecipitation of coatomer from photo-cross-linked cell lysates reveal that gamma-COP is the predominantly labeled protein. From these results, we conclude that coatomer is able to bind to the cytoplasmic dilysine motifs of membrane proteins of the early secretory pathway via its gamma-COP subunit, whose complete cDNA-derived amino acid sequence is also presented. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8700856

  8. Identification of Pathways in Liver Repair Potentially Targeted by Secretory Proteins from Human Mesenchymal Stem Cells

    PubMed Central

    Winkler, Sandra; Hempel, Madlen; Brückner, Sandra; Tautenhahn, Hans-Michael; Kaufmann, Roland; Christ, Bruno

    2016-01-01

    Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor β (TGF-β) and hypoxia-inducible factor 1-α (HIF1-α) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration. PMID:27409608

  9. Cloning of human epidermal growth factor as a bacterial secretory protein, its properties and mutagenesis

    SciTech Connect

    Engler, D.A.; Matsunami, R.K.; Campion, S.R.; Foote, R.S.; Mural, R.J.; Larimer, F.W.; Stevens, A.; Niyogi, S.K.

    1987-05-01

    A chimeric gene, containing the DNA coding for the human epidermal growth factor (EGF) and that for the signal peptide of E. coli alkaline phosphatase, was constructed by the annealing and subsequent ligation of appropriate DNA oligonucleotides synthesized in an automated DNA synthesizer. The gene was then cloned into a bacterial plasmid under the transcriptional control of the E. coli trp-lac (tac) promoter, and then transformed into E. coli. Following induction with isopropylthiogalactoside, the secretion of EGF into the E. coli periplasmic space and some into the growth medium was confirmed by its specific binding to the EGF receptor and stimulation of the EGF receptor tyrosine kinase activity. The size and physicochemical properties of the purified protein mimicked those of authentic human EGF. Studies of structure/function relationships by specific alterations of targeted amino acid residues in the EGF molecule have been initiated by utilizing site-directed mutagenesis.

  10. Ancylostoma ceylanicum Excretory-Secretory Protein 2 Adopts a Netrin-Like Fold and Defines a Novel Family of Nematode Proteins

    SciTech Connect

    K Kucera; L Harrison; M Cappello; Y Modis

    2011-12-31

    Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 {angstrom} resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-likefold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.

  11. Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

    PubMed Central

    Chapman, Nora M.; Kim, Kyung-Soo; Tracy, Steven; Jackson, John; Höfling, Katja; Leser, J. Smith; Malone, James; Kolbeck, Peter

    2000-01-01

    We cloned the sequence encoding murine interleukin-4 (mIL-4), including the secretory signal, into the genome of CVB3/0, an artificially attenuated strain of coxsackievirus B3, at the junction of the capsid protein 1D and the viral protease 2Apro. Two strains of chimeric CVB3 were constructed using, in one case, identical sequences to encode 2Apro cleavage sites (CVB3/0-mIL4/47) on either side of the inserted coding sequence and, in the other case, nonidentical sequences that varied at the nucleotide level without changing the amino acid sequences (CVB3-PL2-mIL4/46). Transfection of HeLa cells yielded progeny viruses that replicated with rates similar to that of the parental CVB3/0 strain, although yields of mIL-4-expressing strains were approximately 10-fold lower than those of the parental virus. Western blot analysis of viral proteins isolated from HeLa cells inoculated with either strain of chimeric virus demonstrated that the chimeric viruses synthesized capsid protein 1D at approximately twofold-higher levels than the parental virus. mIL-4 protein was detected by enzyme-linked immunosorbent assay (ELISA) in HeLa cells inoculated with either strain of chimeric virus. Lysates of HeLa cells inoculated with either chimeric virus induced the proliferation of the mIL-4-requiring murine MC-9 cell line, demonstrating biological activity of the CVB3-expressed mIL-4. Reverse transcription (RT)-PCR analysis of viral RNA derived from sequential passaging of CVB3/0-mIL4/47 in HeLa cells demonstrated deletion of the mIL-4 coding sequence occurring by the fourth passage, while similar analysis of CVB3-PL2-mIL4/46 RNA demonstrated detection of the mIL-4 coding sequence in the virus population through 10 generations in HeLa cells. mIL-4 protein levels determined by ELISA were consistent with the stability and loss data determined by RT-PCR analysis of the passaged viral genomes. Studies of insert stability of CVB3-PL2-mIL4/46 during replication in mice showed the presence of

  12. Recombinant Haemonchus contortus 24 kDa excretory/secretory protein (rHcES-24) modulate the immune functions of goat PBMCs in vitro.

    PubMed

    Gadahi, Javaid Ali; Li, Baojie; Ehsan, Muhammad; Wang, Shuai; Zhang, Zhenchao; Wang, Yujian; Hasan, Muhammad Waqqas; Yan, Ruofeng; Song, Xiaokai; Xu, Lixin; Li, Xiangrui

    2016-12-20

    A 24 kDa protein is one of the important components in Haemonchus contortus (barber pole worm) excretory/secretory products (HcESPs), which was shown to have important antigenic function. However, little is known about the immunomodulatory effects of this proteinon host cell. In the present study gene encoding 24kDa excretory/secretory protein (HcES-24) was cloned. The recombinant protein of HcES-24 (rHcES-24) was expressed in a histidine-tagged fusion protein soluble form in Escherichia coli. Binding activity of rHcES-24 to goat PBMCs was confirmed by immunofluorescence assay (IFA) and its immunomudulatory effect on cytokine secretion, cell proliferation, cell migration and nitric oxide production were observed by co-incubation of rHcES-24. IFA results revealed that rHcES-24 could bind to the PBMCs. The interaction of rHcES-24 increased the production of IL4, IL10, IL17 and cell migration in dose dependent manner. However, rHcES-24 treatment significantly suppressed the production of IFNγ, proliferation of the PBMC and Nitric oxide (NO) production. Our findings showed that the rHcES-24 played important regulatory effects on the goat PBMCs.

  13. Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

    PubMed

    Yan, X; Gonzales, R A; Wagner, G J

    1997-11-01

    Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.

  14. Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

    PubMed Central

    Yan, X; Gonzales, R A; Wagner, G J

    1997-01-01

    Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM. PMID:9390428

  15. Proteomic Analysis of the Excretory and Secretory Proteins of Haemonchus contortus (HcESP) Binding to Goat PBMCs In Vivo Revealed Stage-Specific Binding Profiles

    PubMed Central

    Gadahi, Javaid Ali; Wang, Shuai; Bo, Gao; Ehsan, Muhammad; Yan, RuoFeng; Song, XiaoKai; Xu, LiXin; Li, XiangRui

    2016-01-01

    Haemonchus contortus is a parasitic gastrointestinal nematode, and its excretory and secretory products (HcESPs) interact extensively with the host cells. In this study, we report the interaction of proteins from HcESPs at different developmental stages to goat peripheral blood mononuclear cells (PBMCs) in vivo using liquid chromatography-tandem mass spectrometry. A total of 407 HcESPs that interacted with goat PBMCs at different time points were identified from a H. contortus protein database using SEQUEST searches. The L4 and L5 stages of H. contortus represented a higher proportion of the identified proteins compared with the early and late adult stages. Both stage-specific interacting proteins and proteins that were common to multiple stages were identified. Forty-seven interacting proteins were shared among all stages. The gene ontology (GO) distributions of the identified goat PBMC-interacting proteins were nearly identical among all developmental stages, with high representation of binding and catalytic activity. Cellular, metabolic and single-organism processes were also annotated as major biological processes, but interestingly, more proteins were annotated as localization processes at the L5 stage than at the L4 and adult stages. Based on the clustering of homologous proteins, we improved the functional annotations of un-annotated proteins identified at different developmental stages. Some unnamed H. contortus ATP-binding cassette proteins, including ADP-ribosylation factor and P-glycoprotein-9, were identified by STRING protein clustering analysis. PMID:27467391

  16. Better Than Nothing? Limitations of the Prediction Tool SecretomeP in the Search for Leaderless Secretory Proteins (LSPs) in Plants.

    PubMed

    Lonsdale, Andrew; Davis, Melissa J; Doblin, Monika S; Bacic, Antony

    2016-01-01

    In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a plant-specific tool for predicting LSPs, the mammalian-trained SecretomeP has been applied to plant proteins in multiple studies to identify the most likely LSPs. This study investigates the effectiveness of using SecretomeP on plant proteins, identifies its limitations and provides a benchmark for its use. In the absence of experimentally verified LSPs we exploit the common-feature hypothesis behind SecretomeP and use known classically secreted proteins (CSPs) of plants as a proxy to evaluate its accuracy. We show that, contrary to the common-feature hypothesis, plant CSPs are a poor proxy for evaluating LSP detection due to variation in the SecretomeP prediction scores when the signal peptide (SP) is modified. Removing the SP region from CSPs and comparing the predictive performance against non-secretory proteins indicates that commonly used threshold scores of 0.5 and 0.6 result in false-positive rates in excess of 0.3 when applied to plants proteins. Setting the false-positive rate to 0.05, consistent with the original mammalian performance of SecretomeP, yields only a marginally higher true positive rate compared to false positives. Therefore the use of SecretomeP on plant proteins is not recommended. This study investigates the trade-offs of using SecretomeP on plant proteins and provides insights into predictive features for future development of plant-specific common-feature tools.

  17. Better Than Nothing? Limitations of the Prediction Tool SecretomeP in the Search for Leaderless Secretory Proteins (LSPs) in Plants

    PubMed Central

    Lonsdale, Andrew; Davis, Melissa J.; Doblin, Monika S.; Bacic, Antony

    2016-01-01

    In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a plant-specific tool for predicting LSPs, the mammalian-trained SecretomeP has been applied to plant proteins in multiple studies to identify the most likely LSPs. This study investigates the effectiveness of using SecretomeP on plant proteins, identifies its limitations and provides a benchmark for its use. In the absence of experimentally verified LSPs we exploit the common-feature hypothesis behind SecretomeP and use known classically secreted proteins (CSPs) of plants as a proxy to evaluate its accuracy. We show that, contrary to the common-feature hypothesis, plant CSPs are a poor proxy for evaluating LSP detection due to variation in the SecretomeP prediction scores when the signal peptide (SP) is modified. Removing the SP region from CSPs and comparing the predictive performance against non-secretory proteins indicates that commonly used threshold scores of 0.5 and 0.6 result in false-positive rates in excess of 0.3 when applied to plants proteins. Setting the false-positive rate to 0.05, consistent with the original mammalian performance of SecretomeP, yields only a marginally higher true positive rate compared to false positives. Therefore the use of SecretomeP on plant proteins is not recommended. This study investigates the trade-offs of using SecretomeP on plant proteins and provides insights into predictive features for future development of plant-specific common-feature tools. PMID:27729919

  18. DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana Contributes to Potyvirus Movement and Transports to Plasmodesmata via the Early Secretory Pathway and the Actomyosin System1[OPEN

    PubMed Central

    Geng, Chao; Cong, Qian-Qian; Li, Xiang-Dong; Mou, An-Li; Gao, Rui; Liu, Jin-Liang; Tian, Yan-Ping

    2015-01-01

    The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 5′-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement. PMID:25540331

  19. DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana contributes to potyvirus movement and transports to plasmodesmata via the early secretory pathway and the actomyosin system.

    PubMed

    Geng, Chao; Cong, Qian-Qian; Li, Xiang-Dong; Mou, An-Li; Gao, Rui; Liu, Jin-Liang; Tian, Yan-Ping

    2015-02-01

    The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 5'-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement.

  20. Transport of proteins to the plant vacuole is not by bulk flow through the secretory system, and requires positive sorting information.

    PubMed

    Dorel, C; Voelker, T A; Herman, E M; Chrispeels, M J

    1989-02-01

    Plant cells, like other eukaryotic cells, use the secretory pathway to target proteins to the vacuolar/lysosomal compartment and to the extracellular space. We wished to determine whether the presence of a hydrophobic signal peptide would result in the transport of a reporter protein to vacuoles by bulk flow; to investigate this question, we expressed a chimeric gene in transgenic tobacco. The chimeric gene, Phalb, used for this study consists of the 1,188-bp 5' upstream sequence and the hydrophobic signal sequence of a vacuolar seed protein phytohemagglutinin, and the coding sequence of a cytosolic seed albumin (PA2). The chimeric protein PHALB cross-reacted with antibodies to PA2 and was found in the seeds of the transgenic plants (approximately 0.7% of total protein), but not in the leaves, roots, or flowers. Immunoblot analyses of seed extracts revealed four glycosylated polypeptides ranging in molecular weight from 29,000 to 32,000. The four polypeptides are glycoforms of a single polypeptide of Mr 27,000, and the heterogeneity is due to the presence of high mannose and endoglycosidase H-resistant glycans. The PHALB products reacted with an antiserum specific for complex plant glycans indicating that the glycans had been modified in the Golgi apparatus. Subcellular fractionation of glycerol extracts of mature seeds showed that only small amounts of PHALB accumulated in the protein storage vacuoles of the tobacco seeds. In homogenates made in an isotonic medium, very little PHALB was associated with the organelle fraction containing the endoplasmic reticulum and Golgi apparatus; most of it was in the soluble fraction. We conclude that PHALB passed through the Golgi apparatus, but did not arrive in the vacuoles. Transport to vacuoles is not by a bulk-flow mechanism, once proteins have entered the secretory system, and requires information beyond that provided by a hydrophobic signal peptide.

  1. The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

    PubMed Central

    Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

    1995-01-01

    The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626033

  2. Secretory granule biogenesis: rafting to the SNARE.

    PubMed

    Tooze, S A; Martens, G J; Huttner, W B

    2001-03-01

    Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.

  3. Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland

    PubMed Central

    Cunnick, Jess M; Kim, Stephanie; Hadsell, James; Collins, Stephen; Cerra, Carmine; Reiser, Patti; Flynn, Daniel C; Cho, Youngjin

    2014-01-01

    Actin filament-associated protein 1 (AFAP1) is an adaptor protein of cSrc that binds to filamentous actin and regulates the activity of this tyrosine kinase to affect changes to the organization of the actin cytoskeleton. In breast and prostate cancer cells, AFAP1 has been shown to regulate cellular responses requiring actin cytoskeletal changes such as adhesion, invadopodia formation and invasion. However, a normal physiological role for AFAP1 has remained elusive. In this study, we generated an AFAP1 knockout mouse model that establishes a novel physiological role for AFAP1 in lactation. Specifically, these animals displayed a defect in lactation that resulted in an inability to efficiently nurse. Histologically, the mammary glands of the lactating knockout mice were distinguished by the accumulation of large cytoplasmic lipid droplets in the alveolar epithelial cells. There was a reduction in lipid synthesis and the expression of lipogenic genes without a corresponding reduction in the production of beta-casein, a milk protein. Furthermore, these defects were associated with histological and biochemical signs of precocious involution. This study also demonstrated that AFAP1 responds to prolactin, a lactogenic hormone, by forming a complex with cSrc and becoming tyrosine phosphorylated. Together, these observations pointed to a defect in secretory activation. Certain characteristics of this phenotype mirrored the defect in secretory activation in the cSrc knockout mouse, but most importantly, the activity of cSrc in the mammary gland was reduced during early lactation in the AFAP1 null mouse and the localization of active cSrc at the apical surface of luminal epithelial cells during lactation was selectively lost in the absence of AFAP1. These data define, for the first time, the requirement of AFAP1 for the spatial and temporal regulation of cSrc activity in the normal breast, specifically for milk production. PMID:25043309

  4. The Secretory System of Arabidopsis

    PubMed Central

    Bassham, Diane C.; Brandizzi, Federica; Otegui, Marisa S.; Sanderfoot, Anton A.

    2008-01-01

    Over the past few years, a vast amount of research has illuminated the workings of the secretory system of eukaryotic cells. The bulk of this work has been focused on the yeast Saccharomyces cerevisiae, or on mammalian cells. At a superficial level, plants are typical eukaryotes with respect to the operation of the secretory system; however, important differences emerge in the function and appearance of endomembrane organelles. In particular, the plant secretory system has specialized in several ways to support the synthesis of many components of the complex cell wall, and specialized kinds of vacuole have taken on a protein storage role—a role that is intended to support the growing seedling, but has been co-opted to support human life in the seeds of many crop plants. In the past, most research on the plant secretory system has been guided by results in mammalian or fungal systems but recently plants have begun to stand on their own as models for understanding complex trafficking events within the eukaryotic endomembrane system. PMID:22303241

  5. Genome-wide analysis of Excretory/Secretory proteins in root-knot nematode, Meloidogyne incognita provides potential targets for parasite control.

    PubMed

    Gahoi, Shachi; Gautam, Budhayash

    2017-04-01

    The root-knot nematode, Meloidogyne incognita causes significant damage to various economically important crops. Infection is associated with secretion of effector proteins into host cytoplasm and interference with host innate immunity. To combat this infection, the identification and functional annotations of Excretory/Secretory (ES) proteins serve as a key to produce durable control measures. The identification of ES proteins through experimental methods are expensive and time consuming while bioinformatics approaches are cost-effective by prioritizing the experimental analysis of potential drug targets for parasitic diseases. In this study, we predicted and functionally annotated the 1889 ES proteins in M. incognita genome using integration of several bioinformatics tools. Of these 1889 ES proteins, 473 (25%) had orthologues in free living nematode Caenorhabditis elegans, 825(67.8%) in parasitic nematodes whereas 561 (29.7%) appeared to be novel and M. incognita specific molecules. Of the C. elegans homologues, 17 ES proteins had "loss of function phenotype" by RNA interference and could represent potential drug targets for parasite intervention and control. We could functionally annotate 429 (22.7%) ES proteins using Gene Ontology (GO) terms, 672 (35.5%) proteins to protein domains and established pathway associations for 223 (11.8%) sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG). The 162 (8.5%) ES proteins were also mapped to several important plant cell-wall degrading CAZyme families including chitinase, cellulase, xylanase, pectate lyase and endo-β-1,4-xylanase. Our comprehensive analysis of M. incognita secretome provides functional information for further experimental study.

  6. Secretory leukocyte protease inhibitor reverses inhibition by CNS myelin, promotes regeneration in the optic nerve, and suppresses expression of the TGFβ signaling protein Smad2

    PubMed Central

    Hannila, Sari S.; Siddiq, Mustafa M.; Carmel, Jason B.; Hou, Jianwei; Chaudhry, Nagarathnamma; Bradley, Peter M.J.; Hilaire, Melissa; Richman, Erica L.; Hart, Ronald P.; Filbin, Marie T.

    2013-01-01

    Following CNS injury, axonal regeneration is limited by myelin-associated inhibitors; however, this can be overcome through elevation of intracellular cyclic AMP, as occurs with conditioning lesions of the sciatic nerve. This study reports that expression of secretory leukocyte protease inhibitor (SLPI) is strongly upregulated in response to elevation of cyclic AMP. We also show that SLPI can overcome inhibition by CNS myelin and significantly enhance regeneration of transected retinal ganglion cell axons in rats. Furthermore, regeneration of dorsal column axons does not occur after a conditioning lesion in SLPI null mutant mice, indicating that expression of SLPI is required for the conditioning lesion effect. Mechanistically, we demonstrate that SLPI localizes to the nuclei of neurons, binds to the Smad2 promoter, and reduces levels of Smad2 protein. Adenoviral overexpression of Smad2 also blocked SLPI-induced axonal regeneration. SLPI and Smad2 may therefore represent new targets for therapeutic intervention in CNS injury. PMID:23516280

  7. Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands*

    PubMed Central

    Shridas, Preetha; Bailey, William M.; Boyanovsky, Boris B.; Oslund, Rob C.; Gelb, Michael H.; Webb, Nancy R.

    2010-01-01

    We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A2 (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A2 (sPLA2), but not a catalytically inactive mutant form of GX sPLA2, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA2 inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA2-overexpressing Y1 cells, ruling out a role for this sPLA2 receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA2. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA2 antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA2 is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression. PMID:20421306

  8. Group X secretory phospholipase A2 regulates the expression of steroidogenic acute regulatory protein (StAR) in mouse adrenal glands.

    PubMed

    Shridas, Preetha; Bailey, William M; Boyanovsky, Boris B; Oslund, Rob C; Gelb, Michael H; Webb, Nancy R

    2010-06-25

    We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A(2) (GX KO). These mice have approximately 80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A(2) (sPLA(2)), but not a catalytically inactive mutant form of GX sPLA(2), significantly reduced steroid production 30-40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA(2) inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA(2)-overexpressing Y1 cells, ruling out a role for this sPLA(2) receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was approximately 2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA(2). Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA(2) antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA(2) is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.

  9. High ω-3:ω-6 fatty acids ratio increases fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in human ectopic endometrial cells

    PubMed Central

    Khanaki, Korosh; Sadeghi, Mohammad Reza; Akhondi, Mohammad Mehdi; Darabi, Masoud; Mehdizadeh, Amir; Shabani, Mahdi; Rahimipour, Ali; Nouri, Mohammad

    2014-01-01

    Background: Endometriosis, a common chronic inflammatory disorder, is defined by the atypical growth of endometrium- like tissue outside of the uterus. Secretory phospholipase A2 group IIa (sPLA2-IIa) and fatty acid binding protein4 (FABP4) play several important roles in the inflammatory diseases. Objective: Due to reported potential anti-inflammatory effects of ω-3 and ω-6 fatty acids, the purpose of the present study was to investigate the effects of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) on fatty acid binding protein 4 and extracellular secretory phospholipase A2IIa in cultured endometrial cells. Materials and Methods: Ectopic and eutopic endometrial tissues obtained from 15 women were snap frozen. After thawing and tissue digestion, primary mixed stromal and endometrial epithelial cell culture was performed for 8 days in culture mediums supplemented with normal and high ratios of ω-3 and ω-6 PUFA. sPLA2-IIa in the culture medium and FABP4 level was determined using enzyme immuno assay (EIA) technique. Results: Within ectopic endometrial cells group, the level of cellular FABP4 and extracellular sPLA2-IIa were remarkably increased under high ω-3 PUFA exposure compared with control condition (p=0.014 and p=0.04 respectively). Conclusion: ω-3 PUFAs may increase the level of cellular FABP4 and extracellular sPLA2-IIa in ectopic endometrial cells, since sPLAIIa and FABP4 may affect endometriosis via several mechanisms, more relevant studies are encouraged to know the potential effect of increased cellular FABP4 and extracellular sPLA2-IIa on endometriosis. PMID:25709631

  10. Calelectrin, a calcium-dependent membrane-binding protein associated with secretory granules in Torpedo cholinergic electromotor nerve endings and rat adrenal medulla.

    PubMed

    Walker, J H; Obrocki, J; Südhof, T C

    1983-07-01

    Calelectrin, a calcium-dependent membrane-binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine beta-hydroxylase, although the two antisera did not cross-react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vesicles and also self aggregates in a calcium-dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30-80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.

  11. Immunosuppressive PAS-1 is an excretory/secretory protein released by larval and adult worms of the ascarid nematode Ascaris suum.

    PubMed

    Antunes, M F P; Titz, T O; Batista, I F C; Marques-Porto, R; Oliveira, C F; Alves de Araujo, C A; Macedo-Soares, M F

    2015-05-01

    Helminths use several strategies to evade and/or modify the host immune response, including suppression or inactivation of the host antigen-specific response. Several helminth immunomodulatory molecules have been identified. Our studies have focused on immunosuppression induced by the roundworm Ascaris suum and an A. suum-derived protein named protein 1 from A. suum (PAS-1). Here we assessed whether PAS-1 is an excretory/secretory (E/S) protein and whether it can suppress lipopolysaccharide-induced inflammation. Larvae from infective eggs were cultured in unsupplemented Dulbecco's modified Eagle medium (DMEM) for 2 weeks. PAS-1 was then measured in the culture supernatants and in adult A. suum body fluid at different time points by enzyme-linked immunosorbent assay (ELISA) with the monoclonal antibody MAIP-1. Secreted PAS-1 was detected in both larval culture supernatant and adult body fluid. It suppressed lipopolysaccharide (LPS)-induced leucocyte migration and pro-inflammatory cytokine production, and stimulated interleukin (IL)-10 secretion, indicating that larval and adult secreted PAS-1 suppresses inflammation in this model. Moreover, the anti-inflammatory activity of PAS-1 was abolished by treatment with MAIP-1, a PAS-1-specific monoclonal antibody, confirming the crucial role of PAS-1 in suppressing LPS-induced inflammation. These findings demonstrate that PAS-1 is an E/S protein with anti-inflammatory properties likely to be attributable to IL-10 production.

  12. Engineering the cellular protein secretory pathway for enhancement of recombinant tissue plasminogen activator expression in Chinese hamster ovary cells: effects of CERT and XBP1s genes.

    PubMed

    Rahimpour, Azam; Vaziri, Behrouz; Moazzami, Reza; Nematollahi, Leila; Barkhordari, Farzaneh; Kokabee, Leila; Adeli, Ahmad; Mahboudi, Fereidoun

    2013-08-01

    Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

  13. Histochemical Analysis of Plant Secretory Structures.

    PubMed

    Demarco, Diego

    2017-01-01

    Histochemical analysis is essential for the study of plant secretory structures whose classification is based, at least partially, on the composition of their secretion. As each gland may produce one or more types of substances, a correct analysis of its secretion should be done using various histochemical tests to detect metabolites of different chemical classes. Here I describe some of the most used methods to detect carbohydrates, proteins, lipids, phenolic compounds, and alkaloids in the secretory structures.

  14. A functional cyclic AMP response element plays a crucial role in neuroendocrine cell type-specific expression of the secretory granule protein chromogranin A.

    PubMed Central

    Wu, H; Mahata, S K; Mahata, M; Webster, N J; Parmer, R J; O'Connor, D T

    1995-01-01

    Chromogranin A, a soluble acidic protein, is a ubiquitous component of secretory vesicles throughout the neuroendocrine system. We reported previously the cloning and initial characterization of the mouse chromogranin A gene promoter, which showed that the promoter contains both positive and negative domains and that a proximal promoter spanning nucleotides -147 to +42 bp relative to the transcriptional start site is sufficient for neuroendocrine cell type-specific expression. The current study was undertaken to identify the particular elements within this proximal promoter that control tissue-specific expression. We found that deletion or point mutations in the potential cAMP response element (CRE) site at -68 bp virtually abolished promoter activity specifically in neuroendocrine (PC12 chromaffin or AtT20 corticotrope) cells, with little effect on activity in control (NIH3T3 fibroblast) cells; thus, the CRE box is necessary for neuroendocrine cell type-specific activity of the chromogranin A promoter. Furthermore, the effect of the CRE site is enhanced in the context of intact (wild-type) promoter sequences between -147 and -100 bp. DNase I footprint analysis showed that these regions (including the CRE box) bind nuclear proteins present in both neuroendocrine (AtT20) and control (NIH3T3) cells. In AtT20 cells, electrophoretic mobility shift assays and factor-specific antibody supershifts showed that an oligonucleotide containing the chromogranin A CRE site formed a single, homogeneous protein-DNA complex containing the CRE-binding protein CREB. However, in control NIH3T3 cells we found evidence for an additional immunologically unrelated protein in this complex. A single copy of this oligonucleotide was able to confer neuroendocrine-specific expression to a heterologous (thymidine kinase) promoter, albeit with less fold selectivity than the full proximal chromogranin A promoter. Hence, the CRE site was partially sufficient to explain the neuroendocrine cell type

  15. Toxoplasma secretory granules: one population or more?

    PubMed

    Mercier, Corinne; Cesbron-Delauw, Marie-France

    2015-02-01

    In Toxoplasma gondii, dense granules are known as the storage secretory organelles of the so-called GRA proteins (for dense granule proteins), which are destined to the parasitophorous vacuole (PV) and the PV-derived cyst wall. Recently, newly annotated GRA proteins targeted to the host cell nucleus have enlarged this view. Here we provide an update on the latest developments on the Toxoplasma secreted proteins, which to date have been mainly studied at both the tachyzoite and bradyzoite stages, and we point out that recent discoveries could open the issue of a possible, yet uncharacterized, distinct secretory pathway in Toxoplasma.

  16. Blocking effect and crystal structure of natrin toxin, a cysteine-rich secretory protein from Naja atra venom that targets the BKCa channel.

    PubMed

    Wang, Jing; Shen, Bing; Guo, Min; Lou, Xiaohua; Duan, Yuanyuan; Cheng, Xin Ping; Teng, Maikun; Niu, Liwen; Liu, Qun; Huang, Qingqiu; Hao, Quan

    2005-08-02

    Cysteine-rich secretory proteins (CRISPs) are widespread in snake venoms. Some members of these CRISPs recently have been found to block L-type Ca(2+) channels or cyclic nucleotide-gated ion (CNG) channels. Here, natrin purified from Naja atra venom, a member of the CRISP family, can induce a further contractile response in the endothelium-denuded thoracic aorta of mouse which has been contracted by a high-K(+) solution. Further experiments show it can block the high-conductance calcium-activated potassium (BK(Ca)) channel in a concentration-dependent manner with an IC(50) of 34.4 nM and a Hill coefficient of 1.02, which suggests that only a single natrin molecule is required to bind an ion channel to block BK(Ca) current. The crystal structure of natrin displaying two domains in tandem shows its cysteine-rich domain (CRD) has relatively independent flexibility, especially for the C-terminal long loop (loop I) of CRD to participate in the interface of two domains. On the basis of previous studies of CNG channel and L-Ca(2+) channel blockers, and the sequence and structural comparison of natrin and stecrisp, the deviation of the vital loop I of CRD is suggested to contribute to different effects of some CRISPs in protein-protein interaction.

  17. Investigating possible biological targets of Bj-CRP, the first cysteine-rich secretory protein (CRISP) isolated from Bothrops jararaca snake venom.

    PubMed

    Lodovicho, Marina E; Costa, Tássia R; Bernardes, Carolina P; Menaldo, Danilo L; Zoccal, Karina F; Carone, Sante E; Rosa, José C; Pucca, Manuela B; Cerni, Felipe A; Arantes, Eliane C; Tytgat, Jan; Faccioli, Lúcia H; Pereira-Crott, Luciana S; Sampaio, Suely V

    2017-01-04

    Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions.

  18. Secretory diarrhoea: mechanisms and emerging therapies.

    PubMed

    Thiagarajah, Jay R; Donowitz, Mark; Verkman, Alan S

    2015-08-01

    Diarrhoeal disease remains a major health burden worldwide. Secretory diarrhoeas are caused by certain bacterial and viral infections, inflammatory processes, drugs and genetic disorders. Fluid secretion across the intestinal epithelium in secretory diarrhoeas involves multiple ion and solute transporters, as well as activation of cyclic nucleotide and Ca(2+) signalling pathways. In many secretory diarrhoeas, activation of Cl(-) channels in the apical membrane of enterocytes, including the cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, increases fluid secretion, while inhibition of Na(+) transport reduces fluid absorption. Current treatment of diarrhoea includes replacement of fluid and electrolyte losses using oral rehydration solutions, and drugs targeting intestinal motility or fluid secretion. Therapeutics in the development pipeline target intestinal ion channels and transporters, regulatory proteins and cell surface receptors. This Review describes pathogenic mechanisms of secretory diarrhoea, current and emerging therapeutics, and the challenges in developing antidiarrhoeal therapeutics.

  19. Secretory diarrhoea: mechanisms and emerging therapies

    PubMed Central

    Thiagarajah, Jay R.; Donowitz, Mark; Verkman, Alan S.

    2016-01-01

    Diarrhoeal disease remains a major health burden worldwide. Secretory diarrhoeas are caused by certain bacterial and viral infections, inflammatory processes, drugs and genetic disorders. Fluid secretion across the intestinal epithelium in secretory diarrhoeas involves multiple ion and solute transporters, as well as activation of cyclic nucleotide and Ca2+ signalling pathways. In many secretory diarrhoeas, activation of Cl− channels in the apical membrane of enterocytes, including the cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl− channels, increases fluid secretion, while inhibition of Na+ transport reduces fluid absorption. Current treatment of diarrhoea includes replacement of fluid and electrolyte losses using oral rehydration solutions, and drugs targeting intestinal motility or fluid secretion. Therapeutics in the development pipeline target intestinal ion channels and transporters, regulatory proteins and cell surface receptors. This Review describes pathogenic mechanisms of secretory diarrhoea, current and emerging therapeutics, and the challenges in developing antidiarrhoeal therapeutics. PMID:26122478

  20. The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator miR-27b in the spermatozoa of patients with asthenozoospermia.

    PubMed

    Zhou, Jun-Hao; Zhou, Qi-Zhao; Lyu, Xiao-Ming; Zhu, Ting; Chen, Zi-Jian; Chen, Ming-Kun; Xia, Hui; Wang, Chun-Yan; Qi, Tao; Li, Xin; Liu, Cun-Dong

    2015-01-01

    Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia. Initially, through analyzing CRISP2 expression and methylation on CRISP2 promoter activity in sperm, we observed a decreased expression of CRISP2 protein rather than its mRNA in the ejaculated spermatozoa from asthenozoospermic patients and no methylation in the CRISP2 promoter, suggesting CRISP2 expression may be regulated in the sperm at the posttranscriptional level. Subsequently, we found that microRNA 27b (miR-27b), predicted as a candidate regulator of CRISP2 using bioinformatics, was highly expressed in the ejaculated spermatozoa from asthenozoospermic patients. Luciferase reporter assay and transfection experiments disclosed that this microRNA could target CRISP2 by specifically binding its 3' untranslated region, suppressing CRISP2 expression. Extended clinical observation further confirmed a highly expressed miR-27b and its obviously negative correlation with CRISP2 protein expression in ejaculated spermatozoa samples from asthenozoospermic patients. Finally, we conducted a retrospective follow-up study to support that either high miR-27b expression or low CRISP2 protein expression was significantly associated with low sperm progressive motility, abnormal morphology, and infertility. Thus, this study provides the first preliminary insight into the mechanism leading to the reduced CRISP2 expression in asthenozoospermia, offering a potential therapeutic target for treating male infertility or for male contraception.

  1. Quality control in the secretory assembly line.

    PubMed Central

    Helenius, A

    2001-01-01

    As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway. PMID:11260794

  2. Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

    PubMed Central

    Gibbs, Gerard M.; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J.; Martínez-López, Pablo; Luis de la Vega-Beltràn, José; Lo, Jennifer C. Y.; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K.

    2011-01-01

    The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function. PMID:21482758

  3. Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function.

    PubMed

    Gibbs, Gerard M; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J; Martínez-López, Pablo; de la Vega-Beltràn, José Luis; Lo, Jennifer C Y; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K

    2011-04-26

    The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

  4. Fate of secretory proteins trapped in oocytes of Xenopus laevis by disruption of the cytoskeleton or by imbalanced subunit synthesis

    PubMed Central

    1981-01-01

    The effects of imbalanced subunit synthesis, temperature, colchicine, and cytochalasin on the secretion from Xenopus laevis oocytes of a variety of avian and mammalian proteins were investigated; these proteins were encoded by microinjected messenger RNA. Cytochalasin and colchicine together severely reduced secretion in a temperature- independent manner, the exact reduction varying among the different proteins. In contrast cytochalasin alone had no effect, whereas colchicine alone caused a smaller, temperature-dependent reduction. The synthesis and subcellular compartmentation of these proteins were unaffected by the drug treatments; however, the proteins did not accumulate in the drug-treated oocytes but were degraded. The rate of degradation of each protein was similar to its rate of exocytosis from untreated oocytes. A similar result was obtained without recourse to drugs by studying the fate of immunoglobulin light chains trapped in oocytes by a deficiency in heavy chain synthesis. These results are discussed in terms of the disruptive effects, as revealed by electron microscopy, of the drug treatments on the cytoskeleton of the oocyte. PMID:6173386

  5. The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

    PubMed

    Hook, V Y; Sei, C; Yasothornsrikul, S; Toneff, T; Kang, Y H; Efthimiopoulos, S; Robakis, N K; Van Nostrand, W

    1999-01-29

    Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

  6. Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy

    PubMed Central

    Horne, A.W.; Duncan, W.C.; King, A.E.; Burgess, S.; Lourenco, P.C.; Cornes, P.; Ghazal, P.; Williams, A.R.; Udby, L.; Critchley, H.O.D.

    2009-01-01

    Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT–PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic

  7. Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy.

    PubMed

    Horne, A W; Duncan, W C; King, A E; Burgess, S; Lourenco, P C; Cornes, P; Ghazal, P; Williams, A R; Udby, L; Critchley, H O D

    2009-05-01

    Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT-PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic

  8. Sphingolipids Containing Very-Long-Chain Fatty Acids Define a Secretory Pathway for Specific Polar Plasma Membrane Protein Targeting in Arabidopsis[W

    PubMed Central

    Markham, Jonathan E.; Molino, Diana; Gissot, Lionel; Bellec, Yannick; Hématy, Kian; Marion, Jessica; Belcram, Katia; Palauqui, Jean-Christophe; Satiat-JeuneMaître, Béatrice; Faure, Jean-Denis

    2011-01-01

    Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2a– and Rab-A1e–labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes. PMID:21666002

  9. Construction of a shuttle vector for protein secretory expression in Bacillus subtilis and the application of the mannanase functional heterologous expression.

    PubMed

    Guo, Su; Tang, Jia-jie; Wei, Dong-zhi; Wei, Wei

    2014-04-01

    We report the construction of two Bacillus subtilis expression vectors, pBNS1/pBNS2. Both vectors are based on the strong promoter P43 and the ampicillin resistance gene expression cassette. Additionally, a fragment with the Shine-Dalgarno sequence and a multiple cloning site (BamHI, SalI, SacI, XhoI, PstI, SphI) were inserted. The coding region for the amyQ (encoding an amylase) signal peptide was fused to the promoter P43 of pBNS1 to construct the secreted expression vector pBNS2. The applicability of vectors was tested by first generating the expression vectors pBNS1-GFP/pBNS2-GFP and then detecting for green fluorescent protein gene expression. Next, the mannanase gene from B. pumilus Nsic-2 was fused to vector pBNS2 and we measured the mannanase activity in the supernatant. The mannanase total enzyme activity was 8.65 U/ml, which was 6 times higher than that of the parent strain. Our work provides a feasible way to achieve an effective transformation system for gene expression in B. subtilis and is the first report to achieve B. pumilus mannanase secretory expression in B. subtilis.

  10. AB035. The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator mir-27b in the spermatozoa of patients with asthenozoospermia

    PubMed Central

    Zhou, Jun-Hao; Zhou, Qi-Zhao; Lyu, Xiao-Ming; Zhu, Ting; Chen, Zi-Jian; Chen, Ming-Kun; Xia, Hui; Wang, Chun-Yan; Qi, Tao; Li, Xin; Liu, Cun-Dong

    2016-01-01

    Background Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia Methods Spermatozoa were isolated from 90 study subjects’ ejaculated semen samples. DNA methylation was evaluated using bisulfite-sequencing PCR and methylation-specific PCR. The CRISP2 mRNA and protein expression levels were examined in the ejaculated spermatozoa by qRT-PCR and Western blot respectively. miRNA expression was detected by qRT-PCR. The direct regulatory effect of miR-27b on CRISP2 was predicted computationally and validated via luciferase reporter assay and in vitro experiments in which miR-27b mimic or inhibitor was transfected into 293T cells. Respective correlations of miR-27b and CRISP2 protein expression with clinical features were analyzed using Spearman’s correlation coefficient. Results Initially, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic males. Meanwhile, methylation was not found in CRISP2 promoter. These data suggest a possible post-transcriptional regulation of CRISP2 in asthenozoospermia. Subsequently, bioinformatics prediction, luciferase reporter assay and miR-27b transfection experiments revealed that miR-27b could specifically target CRISP2 by binding its 3’-UTR, suppressing CRISP2 expression post-transcriptionally. Further evidence was provided by the clinical observation of a high miR-27b expression in the ejaculated spermatozoa from asthenozoospermic patients and a negative correlation between miR-27b and CRISP2 protein expression. Finally, a retrospective

  11. Variations in concentrations of the major endometrial secretory proteins (placental protein 14 and insulin-like growth factor binding protein-1) in assisted conception regimes.

    PubMed

    Arthur, I D; Anthony, F W; Chard, T; Masson, G M; Thomas, E J

    1995-03-01

    We have previously shown that placental protein 14 (PP14) concentrations were depressed in two pregnancies that followed down-regulation of the anterior pituitary and exogenous hormone support prior to a frozen-thawed embryo transfer. We now report on a more comprehensive series of pregnancies following this form of treatment, in-vitro fertilization (IVF) and natural cycle frozen-thawed embryo transfer. Serum specimens were analysed for PP14 and insulin-like growth factor binding protein-1 12 days after embryo transfer and at 7 weeks gestation. At 12 days after embryo transfer, the mean serum PP14 concentrations in the IVF and natural cycle were significantly higher in those who conceived than those who did not (82 versus 23 and 107 versus 39 micrograms/l respectively, P < 0.001). Although the mean PP14 concentration in the hormone-supported pregnant patients was higher than in the non-pregnant patients, this had not reached statistical significance 12 days after embryo transfer (49 versus 31 micrograms/l). By 7 weeks gestation the PP14 concentrations in the hormone-supported pregnant patients were significantly higher than in the non-pregnant patients (152 versus 31 micrograms/l, P < 0.001). However, the PP14 concentrations for hormone-supported pregnant patients were significantly lower (P < 0.001) than those for pregnant IVF or natural cycle patients at 7 weeks gestation (152, 777 and 660 micrograms/l respectively). The PP14 concentrations in the pregnant patients, although lower than those in IVF and natural cycle pregnancies, were higher than those previously reported in ovarian failure and Turner's syndrome ovum donation cycles.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Proteoglycans from Boswellia serrata Roxb. and B. carteri Birdw. and identification of a proteolytic plant basic secretory protein.

    PubMed

    Herrmann, Andreas; König, Simone; Lechtenberg, Matthias; Sehlbach, Maria; Vakhrushev, Sergey Y; Peter-Katalinic, Jasna; Hensel, Andreas

    2012-11-01

    Water-soluble high molecular weight compounds were isolated in yields of 21-22% from the oleogum of Boswellia serrata and B. carteri. Using anion exchange chromatography and gel permeation chromatography, different proteoglycans were purified and characterized, leading to four principally different groups: (i) Hyp-/Ser-rich extensins with O-glycosidic attached arabinan side chains; (ii) Modified extensins, with arabinogalactosylated side chains containing GlA and 4-O-Me-GlcA; (iii) Glycoproteins with N-glycosidic side chains containing higher amounts of Fuc, Man and GluNH(2,) featuring a 200 kD metalloproteinase that has been de novo sequenced and is described for the first time; (iv) Type II arabinogalactans-proteins. Significant differences between the gums from the two species were observed in the protein content (6% vs 22%), offering the possibility of a quick differentiation of gums from both species for analytical quality control. The data also offer an insight into the plant response towards wound-closing by the formation of extensin and AGP-containing gum.

  13. Disruption of gene expression rhythms in mice lacking secretory vesicle proteins IA-2 and IA-2β.

    PubMed

    Punia, Sohan; Rumery, Kyle K; Yu, Elizabeth A; Lambert, Christopher M; Notkins, Abner L; Weaver, David R

    2012-09-15

    Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molecular rhythms in the hypothalamic suprachiasmatic nuclei (SCN) of wild-type (WT) and DKO mice. The results indicate significant disruption of molecular rhythmicity in the SCN, which serves as the central pacemaker regulating circadian behavior. We also used quantitative PCR to assess gene expression rhythms in peripheral tissues of DKO, single-knockout, and WT mice. The results indicate significant attenuation of gene expression rhythms in several peripheral tissues of DKO mice but not in either single knockout. To distinguish whether this reduction in rhythmicity reflects defective oscillatory function in peripheral tissues or lack of entrainment of peripheral tissues, animals were injected with dexamethasone daily for 15 days, and then molecular rhythms were assessed throughout the day after discontinuation of injections. Dexamethasone injections improved gene expression rhythms in liver and heart of DKO mice. These results are consistent with the hypothesis that peripheral tissues of DKO mice have a functioning circadian clockwork, but rhythmicity is greatly reduced in the absence of robust, rhythmic physiological signals originating from the SCN. Thus, IA-2 and IA-2β play an important role in the regulation of circadian rhythms, likely through their participation in neurochemical communication among SCN neurons.

  14. An Epididymis-Specific Secretory Protein HongrES1 Critically Regulates Sperm Capacitation and Male Fertility

    PubMed Central

    Zhou, Yuchuan; Zheng, Min; Shi, Qixian; Zhang, Li; Zhen, Wei; Chen, Wenying; Zhang, Yonglian

    2008-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilizion. Although progress had been made in understanding the physiology and biochemistry of capacitation, little is known about the potential roles of epididymal proteins during this process. Here we report that HongrES1, a new member of the SERPIN (serine proteinase inhibitor) family exclusively expressed in the rat cauda epididymis and up-regulated by androgen, is secreted into the lumen and covers the sperm head. Co-culture of caudal sperms with HongrES1 antibody in vitro resulted in a significant increase in the percentage of capacitated spermatozoa. Furthermore, the percentage of capacitated spermatozoa clearly increased in rats when HongrES1 was down-regulated by RNAi in vivo. Remarkably, knockdown of HongrES1 in vivo led to reduced fertility accompanied with deformed appearance of fetuses and pups. These results identify HongrES1 as a novel and critical molecule in the regulation of sperm capacitation and male fertility. PMID:19116669

  15. Secretory carrier membrane protein SCAMP2 and phosphatidylinositol 4,5-bisphosphate interactions in the regulation of dense core vesicle exocytosis.

    PubMed

    Liao, Haini; Ellena, Jeff; Liu, Lixia; Szabo, Gabor; Cafiso, David; Castle, David

    2007-09-25

    Secretory carrier membrane protein 2 (SCAMP2) functions in late steps of membrane fusion in calcium-dependent granule exocytosis. A basic/hydrophobic peptide segment within SCAMP2 (SCAMP2 E: CWYRPIYKAFR) has been implicated in this function and shown to bind and sequester phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 or PIP2] within membranes through an electrostatic mechanism. We now show that alanine substitution of tryptophan W2 within SCAMP2 E substantially weakens peptide binding to negatively charged liposomes; other substitutions for arginine R4 and lysine K8 have only limited effects on binding. Electron paramagnetic resonance analysis of liposomes containing spin-labeled PIP2 shows that R4 but not K8 is critical for SCAMP E binding to PIP2. The interfacial locations of SCAMP E and its structural variants within lipid bicelles measured by oxygen enhancement of nuclear relaxation are all similar. Corresponding point mutations within full-length SCAMP2 (SC2-R204A, SC2-K208A, and SC2-W202A) have been analyzed for biological effects on dense core vesicle exocytosis in neuroendocrine PC12 cells. With the same level of overexpression, SC2-R204A but not SC2-K208A inhibited secretion of cotransfected human growth hormone and of noradrenalin. Inhibition by SC2-R204A was the same as or greater than previously observed for SC2-W202A. Analysis of noradrenalin secretion by amperometry showed that inhibitory mutants of SCAMP2 decrease the probability of fusion pore opening and the stability of initially opened but not yet expanded fusion pores. The strong correlation between SCAMP2 E interactions with PIP2 and inhibition of exocytosis, particularly by SC2-R204A, led us to propose that SCAMP2 interaction with PIP2 within the membrane interface regulates fusion pore formation during exocytosis.

  16. Liver-Enriched Gene 1, a Glycosylated Secretory Protein, Binds to FGFR and Mediates an Anti-stress Pathway to Protect Liver Development in Zebrafish

    PubMed Central

    Zhang, Chunxia; Liu, Feng; Cui, Zongbin; Chen, Jun; Peng, Jinrong

    2016-01-01

    Unlike mammals and birds, teleost fish undergo external embryogenesis, and therefore their embryos are constantly challenged by stresses from their living environment. These stresses, when becoming too harsh, will cause arrest of cell proliferation, abnormal cell death or senescence. Such organisms have to evolve a sophisticated anti-stress mechanism to protect the process of embryogenesis/organogenesis. However, very few signaling molecule(s) mediating such activity have been identified. liver-enriched gene 1 (leg1) is an uncharacterized gene that encodes a novel secretory protein containing a single domain DUF781 (domain of unknown function 781) that is well conserved in vertebrates. In the zebrafish genome, there are two copies of leg1, namely leg1a and leg1b. leg1a and leg1b are closely linked on chromosome 20 and share high homology, but are differentially expressed. In this report, we generated two leg1a mutant alleles using the TALEN technique, then characterized liver development in the mutants. We show that a leg1a mutant exhibits a stress-dependent small liver phenotype that can be prevented by chemicals blocking the production of reactive oxygen species. Further studies reveal that Leg1a binds to FGFR3 and mediates a novel anti-stress pathway to protect liver development through enhancing Erk activity. More importantly, we show that the binding of Leg1a to FGFR relies on the glycosylation at the 70th asparagine (Asn70 or N70), and mutating the Asn70 to Ala70 compromised Leg1’s function in liver development. Therefore, Leg1 plays a unique role in protecting liver development under different stress conditions by serving as a secreted signaling molecule/modulator. PMID:26901320

  17. Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.

    PubMed

    Ohara-Imaizumi, Mica; Nakamichi, Yoko; Tanaka, Toshiaki; Katsuta, Hidenori; Ishida, Hitoshi; Nagamatsu, Shinya

    2002-04-01

    The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

  18. Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway.

    PubMed

    Marsolais, Frédéric; Pajak, Agnieszka; Yin, Fuqiang; Taylor, Meghan; Gabriel, Michelle; Merino, Diana M; Ma, Vanessa; Kameka, Alexander; Vijayan, Perumal; Pham, Hai; Huang, Shangzhi; Rivoal, Jean; Bett, Kirstin; Hernández-Sebastià, Cinta; Liu, Qiang; Bertrand, Annick; Chapman, Ralph

    2010-06-16

    A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed.

  19. Synaptic Control of Secretory Trafficking in Dendrites

    PubMed Central

    Hanus, Cyril; Kochen, Lisa; Dieck, Susanne tom; Racine, Victor; Sibarita, Jean-Baptiste; Schuman, Erin M.; Ehlers, Michael D.

    2016-01-01

    Summary Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER) from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. Here, we monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor KIF17 by Ca2+/calmodulin-dependent protein kinases (CaMK). Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport. PMID:24931613

  20. Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family.

    PubMed

    Foster, J A; Gerton, G L

    1996-06-01

    We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically.

  1. Mammary Analogue Secretory Carcinoma.

    PubMed

    Stevens, Todd M; Parekh, Vishwas

    2016-09-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor that shares the same histologic appearance and ETV6 gene (12p13) rearrangement as secretory carcinoma of the breast. Prior to its recognition, MASC cases were commonly labeled acinic cell carcinoma and adenocarcinoma, not otherwise specified. Despite distinctive histologic features, MASC may be difficult to distinguish from other salivary gland tumors, in particular zymogen-poor acinic cell carcinoma and low-grade salivary duct carcinoma. Although characteristic morphologic and immunohistochemical features form the basis of a diagnosis of MASC, the presence of an ETV6-NTRK3 gene fusion is confirmatory. Given its recent recognition the true prognostic import of MASC is not yet clearly defined.

  2. Isolation of a cDNA clone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway

    SciTech Connect

    Bernhard, W.R.; Thoma, S.; Botella, J.; Somerville, C.R. )

    1991-01-01

    A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles.

  3. Disorders in barrier protein mRNA expression and placenta secretory activity under the influence of polychlorinated biphenyls in vitro.

    PubMed

    Wojciechowska, A; Mlynarczuk, J; Kotwica, J

    2017-02-01

    Pregnancy disorders are often correlated with the presence of organic pollutants in the tissues of living bodies. The aim of this study was to investigate the effects (over 24 and 48 hours) of polychlorinated biphenyls (PCBs) 153, 126, and 77 at doses of 1, 10, and 100 ng/mL on barrier function and secretory activity in cow placentome sections collected during the second trimester of pregnancy. None of the PCBs affected the viability of the sections (P > 0.05). Polychlorinated biphenyl 153 decreased (P < 0.05) connexin 26 (Cx 26) mRNA expression, and all three PCBs reduced (P < 0.05) Cx 43 mRNA expression. Cx 32 mRNA expression showed a downward trend (P > 0.05) under the influence of PCBs 126 and 77. Moreover, PCBs 153 and 126 increased keratin 8 (KRT8) mRNA expression, whereas all PCBs decreased (P < 0.05) placenta specific protein 1 (PLAC-1) mRNA expression without changing (P > 0.05) hypoxia inducible factor 1α (HIF1α) mRNA expression. Concomitantly, PCBs 153 and 126 stimulated (P < 0.05) cyclooxygenase 2 (COX-2) mRNA expression, all PCBs increased (P < 0.05) prostaglandin E2 synthase (PGES) mRNA expression, and PCBs 126 and 77 increased prostaglandin E2 (PGE2) secretion. All three PCBs decreased (P < 0.05) prostaglandin F2α synthase (PGFS) mRNA expression and prostaglandin F2α (PGF2α) secretion. In addition, all three PCBs increased (P < 0.05) neurophysin I/oxytocin (NP-I/OT) mRNA expression and OT secretion but did not affect peptidyl-glycine-α-amidating monooxygenase (PGA) mRNA expression (P > 0.05). Moreover, the PCBs increased (P < 0.05) estradiol (E2) secretion, whereas progesterone (P4) secretion remained unchanged (P > 0.05). These changes could affect trophoblast invasion and uterine contractility and thus impact the course of gestation and/or fetal development in the cow.

  4. Muscle as a secretory organ.

    PubMed

    Pedersen, Bente K

    2013-07-01

    Skeletal muscle is the largest organ in the body. Skeletal muscles are primarily characterized by their mechanical activity required for posture, movement, and breathing, which depends on muscle fiber contractions. However, skeletal muscle is not just a component in our locomotor system. Recent evidence has identified skeletal muscle as a secretory organ. We have suggested that cytokines and other peptides that are produced, expressed, and released by muscle fibers and exert either autocrine, paracrine, or endocrine effects should be classified as "myokines." The muscle secretome consists of several hundred secreted peptides. This finding provides a conceptual basis and a whole new paradigm for understanding how muscles communicate with other organs such as adipose tissue, liver, pancreas, bones, and brain. In addition, several myokines exert their effects within the muscle itself. Many proteins produced by skeletal muscle are dependent upon contraction. Therefore, it is likely that myokines may contribute in the mediation of the health benefits of exercise.

  5. The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene.

    PubMed Central

    Toonen, R F; Gowan, S; Bingle, C D

    1996-01-01

    The mechanisms that direct expression of the Clara cell secretory protein (CCSP) gene to the bronchiolar epithelial cells of the lung remain to be elucidated. Previous studies have identified a number of proteins which bind to a functionally important region (Region 1) located -132 to -76 bp from the transcription start site in the rat CCSP gene. Subsequently we have shown that while Region 1 is an important positive regulator of CCSP gene expression, sequences 3' of this region (-75 to +38) are sufficient to confer tissue-specific expression of a reporter gene. In the present study we have used transient transfections with a deletion series of CCSP-CAT reporter plasmids (where CAT is chloramphenicol acetyltransferase) and gel mobility shift assays with a series of overlapping oligonucleotides covering the whole minimal promoter region to study protein-DNA interactions within this region. These studies have identified a conserved functional binding site for the lung and thyroid enriched homeodomain transcription factor TTF-1, located between positions -51 and -42 from the transcription start site. CCSP-CAT chimaeric reporters containing this region are specifically activated by TTF-1 in co-transfection assays, and nuclear extracts from cells which express TTF-1 bind to this region, as does in vitro translated rat TTF-1. Three additional conserved regions were identified, and in further gel mobility shift studies with an oligonucleotide spanning the conserved region immediately 5' to the TTF-1 site we identified a binding site for the ubiquitously expressed zinc-finger-containing proteins Sp1 and Sp3. These studies suggest that cell-type-restricted and ubiquitous nuclear proteins may play a combined role in the regulation of the CCSP gene within the bronchiolar epithelium by interacting with the minimal promoter region. PMID:8687389

  6. Parotid salivary secretory pattern in bulimia nervosa.

    PubMed

    Riad, M; Barton, J R; Wilson, J A; Freeman, C P; Maran, A G

    1991-01-01

    Parotid gland enlargement occurs in about 25% of patients with the binge eating syndrome of bulimia nervosa. The parotid salivary secretory patterns in 28 bulimics were determined in order to investigate the functional abnormality in the glands. Bulimia patients had a reduced resting flow rate. Bulimics who developed sialadenosis (4 patients) had reduced resting and stimulated flow rates. The salivary amylase activity was increased in both the resting and stimulated states in bulimics and the sialadenosis group. The resting total protein levels were greater in the bulimics. The electrolyte and immunoglobulin levels were within normal limits. The possibility of protein and enzymatic secretory disturbances due to autonomic nerve disorders as an explanation for the development of sialadenosis in bulimia nervosa is discussed.

  7. The secretory synapse: the secrets of a serial killer.

    PubMed

    Bossi, Giovanna; Trambas, Christina; Booth, Sarah; Clark, Richard; Stinchcombe, Jane; Griffiths, Gillian M

    2002-11-01

    Cytotoxic T lymphocytes (CTLs) destroy their targets by a process involving secretion of specialized granules. The interactions between CTLs and target can be very brief; nevertheless, adhesion and signaling proteins segregate into an immunological synapse. Secretion occurs in a specialized secretory domain. Use of live and fixed cell microscopy allows this secretory synapse to be visualized both temporally and spatially. The combined use of confocal and electron microscopy has produced some surprising findings, which suggest that the secretory synapse may be important both in delivering the lethal hit and in facilitating membrane transfer from target to CTL. Studies on the secretory synapse in wild-type and mutant CTLs have been used to identify proteins involved in secretion. Further clues as to the signals required for secretion are emerging from comparisons of inhibitory and activating synapses formed by natural killer cells.

  8. Ursodeoxycholic acid attenuates colonic epithelial secretory function

    PubMed Central

    Kelly, Orlaith B; Mroz, Magdalena S; Ward, Joseph B J; Colliva, Carolina; Scharl, Michael; Pellicciari, Roberto; Gilmer, John F; Fallon, Padraic G; Hofmann, Alan F; Roda, Aldo; Murray, Frank E; Keely, Stephen J

    2013-01-01

    Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl− secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl− secretory responses to the Ca2+ and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n= 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na+/K+-ATPase activity and basolateral K+ channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg−1) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50–200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea. PMID:23507881

  9. AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins.

    PubMed

    Foster, J A; Friday, B B; Maulit, M T; Blobel, C; Winfrey, V P; Olson, G E; Kim, K S; Gerton, G L

    1997-05-09

    The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in

  10. P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

    PubMed

    Kaur, Jasber; Cutler, Daniel F

    2002-03-22

    The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.

  11. Docking of Secretory Vesicles Is Syntaxin Dependent

    PubMed Central

    de Wit, Heidi; Cornelisse, L. Niels; Toonen, Ruud F.G.; Verhage, Matthijs

    2006-01-01

    Secretory vesicles dock at the plasma membrane before they undergo fusion. Molecular docking mechanisms are poorly defined but believed to be independent of SNARE proteins. Here, we challenged this hypothesis by acute deletion of the target SNARE, syntaxin, in vertebrate neurons and neuroendocrine cells. Deletion resulted in fusion arrest in both systems. No docking defects were observed in synapses, in line with previous observations. However, a drastic reduction in morphologically docked secretory vesicles was observed in chromaffin cells. Syntaxin-deficient chromaffin cells showed a small reduction in total and plasma membrane staining for the docking factor Munc18-1, which appears insufficient to explain the drastic reduction in docking. The sub-membrane cortical actin network was unaffected by syntaxin deletion. These observations expose a docking role for syntaxin in the neuroendocrine system. Additional layers of regulation may have evolved to make syntaxin redundant for docking in highly specialized systems like synaptic active zones. PMID:17205130

  12. [Estimation of secretory immunoglobulin A in serum of pregnant women (author's transl)].

    PubMed

    Briese, V; Lorenz, U; Brock, J; Straube, W

    1982-01-01

    Secretory immunoglobulin A (S-IgA) was measured by means of radial immunodiffusion, according to Mancini, with a self-made antiserum to the secretory component being used together with a S-IgA standard. S-IgA serum concentrations went up with significance in the course of pregnancy, which was probably attributable to increased production of secretory protein in the breasts in preparation for lactation.

  13. Secretory Carcinoma of the Breast

    PubMed Central

    Aktepe, Fatma; Sarsenov, Dauren; Özmen, Vahit

    2016-01-01

    Secretory carcinoma is a very rare subtype of breast carcinoma. These tumors are generally associated with a favorable prognosis, although having triple-negative phenotype (estrogen receptor (ER), progesterone receptor (PR) negative and c-erbB2 (HER2) negative). In this presentation, a rare secretory carcinoma of the breast in a woman aged 24 years is discussed and the literature is reviewed. PMID:28331758

  14. Label-free Quantitative Proteomics Reveals a Role for the Mycobacterium tuberculosis SecA2 Pathway in Exporting Solute Binding Proteins and Mce Transporters to the Cell Wall.

    PubMed

    Feltcher, Meghan E; Gunawardena, Harsha P; Zulauf, Katelyn E; Malik, Seidu; Griffin, Jennifer E; Sassetti, Christopher M; Chen, Xian; Braunstein, Miriam

    2015-06-01

    Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on solute binding proteins and Mce transporter export, our label-free quantitative analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology.

  15. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

    PubMed

    Godinho, Rodrigo Maciel da Costa; Matassoli, Flavio Lemos; Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J Thomas; Marques, Ernesto Torres de Azevedo; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  16. HID-1 is required for homotypic fusion of immature secretory granules during maturation.

    PubMed

    Du, Wen; Zhou, Maoge; Zhao, Wei; Cheng, Dongwan; Wang, Lifen; Lu, Jingze; Song, Eli; Feng, Wei; Xue, Yanhong; Xu, Pingyong; Xu, Tao

    2016-10-18

    Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules.

  17. An evolving paradigm for the secretory pathway?

    PubMed Central

    Lippincott-Schwartz, Jennifer

    2011-01-01

    The paradigm that the secretory pathway consists of a stable endoplasmic reticulum and Golgi apparatus, using discrete transport vesicles to exchange their contents, gained important support from groundbreaking biochemical and genetic studies during the 1980s. However, the subsequent development of new imaging technologies with green fluorescent protein introduced data on dynamic processes not fully accounted for by the paradigm. As a result, we may be seeing an example of how a paradigm is evolving to account for the results of new technologies and their new ways of describing cellular processes. PMID:22039065

  18. Secretory Defect and Cytotoxicity

    PubMed Central

    Li, Songhua; Yang, Zhihui; Hu, Jane; Gordon, William C.; Bazan, Nicolas G.; Haas, Arthur L.; Bok, Dean; Jin, Minghao

    2013-01-01

    Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival and function. Recently, a D1080N mutation in IRBP was found in patients with retinitis pigmentosa, a frequent cause of retinal degeneration. The molecular and cellular bases for pathogenicity of the mutation are unknown. Here, we show that the mutation abolishes secretion of IRBP and results in formation of insoluble high molecular weight complexes via disulfide bonds. Co-expression of protein disulfide isomerase A2 that regulates disulfide bond formation or introduction of double Cys-to-Ala substitutions at positions 304 and 1175 in D1080N IRBP promoted secretion of the mutated IRBP. D1080N IRBP was not transported to the Golgi apparatus, but accumulated in the endoplasmic reticulum (ER), bound with the ER-resident chaperone proteins such as BiP, protein disulfide isomerase, and heat shock proteins. Splicing of X-box-binding protein-1 mRNA, expression of activating transcription factor 4 (ATF4), and cleavage of ATF6 were significantly increased in cells expressing D1080N IRBP. Moreover, D1080N IRBP induced up-regulation and nuclear translocation of the C/EBP homologous protein, a proapoptotic transcription factor associated with the unfolded protein response. These results indicate that loss of normal function (nonsecretion) and gain of cytotoxic function (ER stress) are involved in the disease mechanisms of D1080N IRBP. Chemical chaperones and low temperature, which help proper folding of many mutated proteins, significantly rescued secretion of D1080N IRBP, suggesting that misfolding is the molecular basis for pathogenicity of D1080N substitution and that chemical chaperones are therapeutic candidates for the mutation-caused blinding disease. PMID:23486466

  19. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

    SciTech Connect

    Suzuki, Nobuhiro; Yamazaki, Yasuo; Brown, R. Lane; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

    2008-10-01

    The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.

  20. Crystallization and preliminary X-ray diffraction analyses of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels

    SciTech Connect

    Suzuki, Nobuhiro; Yamazaki, Yasuo; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

    2005-08-01

    Crystals of pseudechetoxin and pseudecin, potent peptidic inhibitors of cyclic nucleotide-gated ion channels, have been prepared and X-ray diffraction data have been collected to 2.25 and 1.90 Å resolution, respectively. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction of retinal and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins are structurally classified as cysteine-rich secretory proteins and exhibit structural features that are quite distinct from those of other known small peptidic channel blockers. This article describes the crystallization and preliminary X-ray diffraction analyses of these toxins. Crystals of PsTx belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.30, b = 61.59, c = 251.69 Å, and diffraction data were collected to 2.25 Å resolution. Crystals of Pdc also belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with similar unit-cell parameters a = 60.71, b = 61.67, c = 251.22 Å, and diffraction data were collected to 1.90 Å resolution.

  1. Some features of secretory systems in plants.

    PubMed

    Juniper, B E; Gilchrist, A J; Robins, R J

    1977-09-01

    Recent work on secretion in plants is reviewed, with emphasis on the anatomy and physiology of root cap cells in higher plants, the stalked glands of Drosera capensis, and the secretory mechanism of Dionaea muscipula. Cells of the root cap of higher plants switch from a geo-perceptive role to one of mucilage secretion at maturation. Features of this process, the role of the Golgi and the pathway for mucilage distribution are reviewed. In contrast, the stalked glands of the leaves of Drosera capensis are much longer lived and have a complex anatomy. The mechanisms for mucilage secretion, protein absorption and the role of the cell membranes in the internal secretion of the protein are described, using data from X-ray microscopv. The secretion of fluid and protein by Dionaea is stimulated by various nitrogen-containing compounds. Uric acid, often excreted by captured insects, is particularly effective in this respect.

  2. Secretion from Myeloid Cells: Secretory Lysosomes.

    PubMed

    Griffiths, Gillian M

    2016-08-01

    Many cells of the myeloid lineage use an unusual secretory organelle to deliver their effector mechanisms. In these cells, the lysosomal compartment is often modified not only to fulfill the degradative functions of a lysosome but also as a mechanism for secreting additional proteins that are found in the lysosomes of each specialized cell type. These extra proteins vary from one cell type to another according to the specialized function of the cell. For example, mast cells package histamine; cytotoxic T cells express perforin; azurophilic granules in neutrophils express antimicrobial peptides, and platelets von Willebrand factor. Upon release, these very different proteins can trigger inflammation, cell lysis, microbial death, and clotting, respectively, and hence deliver the very different effector mechanisms of these different myeloid cells.

  3. Detection of Secretory Immunoglobulin A in Human Colostrum as Mucosal Immune Response against Proteins of the Type Three Secretion System of Salmonella, Shigella and Enteropathogenic Escherichia Coli

    PubMed Central

    Durand, David; Ochoa, Theresa J.; Bellomo, Sicilia M. E.; Contreras, Carmen A.; Bustamante, Víctor H.; Ruiz, Joaquim; Cleary, Thomas G.

    2013-01-01

    Background Some enteropathogens use the type three secretion system (T3SS) to secrete proteins that allows them to interact with enterocytes and promote bacterial attachment or intracellular survival. These proteins are Salmonella invasion proteins (Sip), invasion plasmid antigens (Ipa) of Shigella and E. coli secreted proteins (Esp) of enteropathogenic E. coli (EPEC). There are no previous studies defining the presence of colostral sIgA against all these three major enteric pathogens. Objective To evaluate the presence of sIgA in colostrum against proteins of the T3SS of Salmonella, Shigella and EPEC. Methods We collected 76 colostrum samples from puerperal women in Lima, Peru. These samples were reacted with T3SS proteins extracted from bacterial culture supernatants and evaluated by Western Blot. Results Antibodies were detected against Salmonella antigens SipA in 75 samples (99%), SipC in 62 (82%) and SipB in 31 (41%); against Shigella antigens IpaC in 70 (92%), IpaB in 68 (89%), IpaA in 66 (87%) and IpaD in 41 (54%); and against EPEC EspC in 70 (92%), EspB-D in 65 (86%) and EspA in 41 (54%). 10% of samples had antibodies against all proteins evaluated; and 42% against all except one protein. There was no sample negative to all these proteins. Conclusions The extraordinarily high frequency of antibodies in colostrum of puerperal women detected in this study against these multiple enteric pathogens, shows evidence of immunological memory and prior exposure to these pathogens, in addition to its possible protective role against infection. PMID:23538526

  4. AB087. The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator miR-27b in the spermatozoa of patients with asthenozoospermia

    PubMed Central

    Zhou, Junhao; Zhou, Qizhao; Lyu, Xiaoming; Zhu, Ting; Chen, Zijian; Chen, Mingkun; Xia, Hui; Wang, Chunyan; Qi, Tao; Li, Xin; Liu, Cundong

    2016-01-01

    Objective Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia. Methods Spermatozoa were isolated from 90 study subjects’ ejaculated semen samples. DNA methylation was evaluated using bisulfite-sequencing PCR and methylation-specific PCR. The CRISP2 mRNA and protein expression levels were examined in the ejaculated spermatozoa by qRT-PCR and Western blot respectively. MiRNA expression was detected by qRT-PCR. The direct regulatory effect of miR-27b on CRISP2 was predicted computationally and validated via luciferase reporter assay and in vitro experiments in which miR-27b mimic or inhibitor was transfected into 293T cells. Respective correlations of miR-27b and CRISP2 protein expression with clinical features were analyzed using Spearman’s correlation coefficient. Results Initially, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic males. Meanwhile, methylation was not found in CRISP2 promoter. These data suggest a possible post-transcriptional regulation of CRISP2 in asthenozoospermia. Subsequently, bioinformatics prediction, luciferase reporter assay and miR-27b transfection experiments revealed that miR-27b could specifically target CRISP2 by binding its 3’-UTR, suppressing CRISP2 expression post-transcriptionally. Further evidence was provided by the clinical observation of a high miR-27b expression in the ejaculated spermatozoa from asthenozoospermic patients and a negative correlation between miR-27b and CRISP2 protein expression. Finally, a retrospective

  5. An uncleaved signal peptide directs the Malus xiaojinensis iron transporter protein Mx IRT1 into the ER for the PM secretory pathway.

    PubMed

    Zhang, Peng; Tan, Song; Berry, James O; Li, Peng; Ren, Na; Li, Shuang; Yang, Guang; Wang, Wei-Bing; Qi, Xiao-Ting; Yin, Li-Ping

    2014-11-07

    Malus xiaojinensis iron-regulated transporter 1 (Mx IRT1) is a highly effective inducible iron transporter in the iron efficient plant Malus xiaojinensis. As a multi-pass integral plasma membrane (PM) protein, Mx IRT1 is predicted to consist of eight transmembrane domains, with a putative N-terminal signal peptide (SP) of 1-29 amino acids. To explore the role of the putative SP, constructs expressing Mx IRT1 (with an intact SP) and Mx DsIRT1 (with a deleted SP) were prepared for expression in Arabidopsis and in yeast. Mx IRT1 could rescue the iron-deficiency phenotype of an Arabidopsis irt1 mutant, and complement the iron-limited growth defect of the yeast mutant DEY 1453 (fet3fet4). Furthermore, fluorescence analysis indicated that a chimeric Mx IRT1-eGFP (enhanced Green Fluorescent Protein) construct was translocated into the ER (Endoplasmic reticulum) for the PM sorting pathway. In contrast, the SP-deleted Mx DsIRT1 could not rescue either of the mutant phenotypes, nor direct transport of the GFP signal into the ER. Interestingly, immunoblot analysis indicated that the SP was not cleaved from the mature protein following transport into the ER. Taken together, data presented here provides strong evidence that an uncleaved SP determines ER-targeting of Mx IRT1 during the initial sorting stage, thereby enabling the subsequent transport and integration of this protein into the PM for its crucial role in iron uptake.

  6. An Uncleaved Signal Peptide Directs the Malus xiaojinensis Iron Transporter Protein Mx IRT1 into the ER for the PM Secretory Pathway

    PubMed Central

    Zhang, Peng; Tan, Song; Berry, James O.; Li, Peng; Ren, Na; Li, Shuang; Yang, Guang; Wang, Wei-Bing; Qi, Xiao-Ting; Yin, Li-Ping

    2014-01-01

    Malus xiaojinensis iron-regulated transporter 1 (Mx IRT1) is a highly effective inducible iron transporter in the iron efficient plant Malus xiaojinensis. As a multi-pass integral plasma membrane (PM) protein, Mx IRT1 is predicted to consist of eight transmembrane domains, with a putative N-terminal signal peptide (SP) of 1–29 amino acids. To explore the role of the putative SP, constructs expressing Mx IRT1 (with an intact SP) and Mx DsIRT1 (with a deleted SP) were prepared for expression in Arabidopsis and in yeast. Mx IRT1 could rescue the iron-deficiency phenotype of an Arabidopsis irt1 mutant, and complement the iron-limited growth defect of the yeast mutant DEY 1453 (fet3fet4). Furthermore, fluorescence analysis indicated that a chimeric Mx IRT1-eGFP (enhanced Green Fluorescent Protein) construct was translocated into the ER (Endoplasmic reticulum) for the PM sorting pathway. In contrast, the SP-deleted Mx DsIRT1 could not rescue either of the mutant phenotypes, nor direct transport of the GFP signal into the ER. Interestingly, immunoblot analysis indicated that the SP was not cleaved from the mature protein following transport into the ER. Taken together, data presented here provides strong evidence that an uncleaved SP determines ER-targeting of Mx IRT1 during the initial sorting stage, thereby enabling the subsequent transport and integration of this protein into the PM for its crucial role in iron uptake. PMID:25387073

  7. Secretory transport in Plasmodium.

    PubMed

    Elmendorf, H G; Haldar, K

    1993-03-01

    The asexual blood stage of the human malaria parasite Plasmodium falciparum resides within the mature erythrocyte - a cell that has no intracellular organelles and few biosynthetic activities. However, Plasmodium, as on actively growing and dividing cell, has numerous requirements for the uptake o f nutrients and expulsion of waste. Hence, the parasite must extensively remodel the erythrocyte to facilitate its survival, not only by exporting numerous proteins, but also by providing the requisite machinery for their .trafficking. In this review, Heidi Elmendorf and Kastun Haldar propose a model for secretion in P. falciparum.

  8. Formation of secretory granules by chromogranins.

    PubMed

    Inomoto, Chie; Osamura, Robert Yoshiyuki

    2009-12-01

    This review article covers the molecular mechanisms of secretory granule formation by chromogranin transfection. Recently, a few investigators have reported that the transfection of chromogranin A and B produces the structures of secretory granules. We used the GFP-chromogranin A transfection method to nonendocrine cells, COS-7 cells, which are not equipped with secretory granules. Despite the absence of endogenous secretory granules in nontransfected COS-7 cells, COS-7 cells transfected with chromogranin A contained granule-like structures in electron micrographs. The granules were composed of an outer limiting membrane with core structures that were interpreted as secretory granules. Human chromogranin A (CgA) labeled with 5-nm gold particles was present in several dense-core granules in our previous electron microscopy study. This review depicts the role of chromogranin A in the formation of secretory granules. It emphasizes the application of recently developed new technologies and the genesis of secretory granules.

  9. P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells

    PubMed Central

    1992-01-01

    P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P- selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P- selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules. PMID:1370497

  10. Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

    PubMed Central

    1992-01-01

    Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface. To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [35S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP. In the presence of GTP gamma S, the constitutive vesicles became significantly more dense, suggesting that a coated intermediate was stabilized. Larger immature regulated secretory granules, enriched in sulfated secretogranin II, also escaped from the permeabilized cells in vitro. During granule maturation, their density increased and the amount of cofractionating proteoglycans diminished. The data suggest that sorting continues during secretory granule maturation. PMID:1572894

  11. The evolution of plant secretory structures and emergence of terpenoid chemical diversity.

    PubMed

    Lange, Bernd Markus

    2015-01-01

    Secretory structures in terrestrial plants appear to have first emerged as intracellular oil bodies in liverworts. In vascular plants, internal secretory structures, such as resin ducts and laticifers, are usually found in conjunction with vascular bundles, whereas subepidermal secretory cavities and epidermal glandular trichomes generally have more complex tissue distribution patterns. The primary function of plant secretory structures is related to defense responses, both constitutive and induced, against herbivores and pathogens. The ability to sequester secondary (or specialized) metabolites and defense proteins in secretory structures was a critical adaptation that shaped plant-herbivore and plant-pathogen interactions. Although this review places particular emphasis on describing the evolution of pathways leading to terpenoids, it also assesses the emergence of other metabolite classes to outline the metabolic capabilities of different plant lineages.

  12. The secretory pathway of protists: spatial and functional organization and evolution.

    PubMed Central

    Becker, B; Melkonian, M

    1996-01-01

    All cells secrete a diversity of macromolecules to modify their environment or to protect themselves. Eukaryotic cells have evolved a complex secretory pathway consisting of several membrane-bound compartments which contain specific sets of proteins. Experimental work on the secretory pathway has focused mainly on mammalian cell lines or on yeasts. Now, some general principles of the secretory pathway have become clear, and most components of the secretory pathway are conserved between yeast cells and mammalian cells. However, the structure and function of the secretory system in protists have been less extensively studied. In this review, we summarize the current knowledge about the secretory pathway of five different groups of protists: Giardia lamblia, one of the earliest lines of eukaryotic evolution, kinetoplastids, the slime mold Dictyostelium discoideum, and two lineages within the "crown" of eukaryotic cell evolution, the alveolates (ciliates and Plasmodium species) and the green algae. Comparison of these systems with the mammalian and yeast system shows that most elements of the secretory pathway were presumably present in the earliest eukaryotic organisms. However, one element of the secretory pathway shows considerable variation: the presence of a Golgi stack and the number of cisternae within a stack. We suggest that the functional separation of the plasma membrane from the nucleus-endoplasmic reticulum system during evolution required a sorting compartment, which became the Golgi apparatus. Once a Golgi apparatus was established, it was adapted to the various needs of the different organisms. PMID:8987360

  13. Excretory/secretory products from in vitro-cultured Echinococcus granulosus protoscoleces.

    PubMed

    Virginio, Veridiana G; Monteiro, Karina M; Drumond, Fernanda; de Carvalho, Marcos O; Vargas, Daiani M; Zaha, Arnaldo; Ferreira, Henrique B

    2012-05-01

    Cystic hydatid disease (CHD) is caused by infection with Echinococcus granulosus metacestodes and affects humans and livestock. Proteins secreted or excreted by protoscoleces, pre-adult worms found in the metacestode, are thought to play fundamental roles in the host-parasite relationship. In this work, we performed an LC-MS/MS proteomic analysis of the excretory-secretory products obtained from the first 48 h of an in vitro culture of the protoscoleces. We identified 32 proteins, including 18 that were never detected previously in metacestode proteomic studies. Among the novel identified excretory-secretory products are antigenic proteins, such as EG19 and P-29 and a calpain protease. We also identified other important protoscolex excretory-secretory products, such as thioredoxin peroxidase and 14-3-3 proteins, which are potentially involved in evasion mechanisms adopted by parasites to establish infection. Several intracellular proteins were found in the excretory-secretory products, revealing a set of identified proteins not previously thought to be exposed at the host-parasite interface. Additionally, immunological analyses established the antigenic profiles of the newly identified excretory-secretory products and revealed, for the first time, the in vitro secretion of the B antigen by protoscoleces. Considering that the excretory-secretory products obtained in vitro might reflect the products released and exposed to the host in vivo, our results provide valuable information on parasite survival strategies in adverse host environments and on the molecular mechanisms underpinning CHD immunopathology.

  14. ECHIDNA protein impacts on male fertility in Arabidopsis by mediating trans-Golgi network secretory trafficking during anther and pollen development.

    PubMed

    Fan, Xinping; Yang, Caiyun; Klisch, Doris; Ferguson, Alison; Bhaellero, Rishi P; Niu, Xiwu; Wilson, Zoe A

    2014-03-01

    The trans-Golgi network (TGN) plays a central role in cellular secretion and has been implicated in sorting cargo destined for the plasma membrane. Previously, the Arabidopsis (Arabidopsis thaliana) echidna (ech) mutant was shown to exhibit a dwarf phenotype due to impaired cell expansion. However, ech also has a previously uncharacterized phenotype of reduced male fertility. This semisterility is due to decreased anther size and reduced amounts of pollen but also to decreased pollen viability, impaired anther opening, and pollen tube growth. An ECH translational fusion (ECHPro:ECH-yellow fluorescent protein) revealed developmentally regulated tissue-specific expression, with expression in the tapetum during early anther development and microspore release and subsequent expression in the pollen, pollen tube, and stylar tissues. Pollen viability and production, along with germination and pollen tube growth, were all impaired. The ech anther endothecium secondary wall thickening also appeared reduced and disorganized, resulting in incomplete anther opening. This did not appear to be due to anther secondary thickening regulatory genes but perhaps to altered secretion of wall materials through the TGN as a consequence of the absence of the ECH protein. ECH expression is critical for a variety of aspects of male reproduction, including the production of functional pollen grains, their effective release, germination, and tube formation. These stages of pollen development are fundamentally influenced by TGN trafficking of hormones and wall components. Overall, this suggests that the fertility defect is multifaceted, with the TGN trafficking playing a significant role in the process of both pollen formation and subsequent fertilization.

  15. Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory-secretory proteins and their biochemical properties.

    PubMed

    Lee, Jinyoung; Kim, Jong-Hyun; Sohn, Hae-Jin; Yang, Hee-Jong; Na, Byoung-Kuk; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

    2014-08-01

    Naegleria fowleri causes a lethal primary amoebic meningoencephalitis (PAM) in humans and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56 and 46 % identity to Naegleria gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 and 34 kDa bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using their respective antibodies. Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximum at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.

  16. Sorting and storage during secretory granule biogenesis: looking backward and looking forward.

    PubMed Central

    Arvan, P; Castle, D

    1998-01-01

    Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained. PMID:9620860

  17. Secretory Carcinoma of the Skin Harboring ETV6 Gene Fusions: A Cutaneous Analogue to Secretory Carcinomas of the Breast and Salivary Glands.

    PubMed

    Bishop, Justin A; Taube, Janis M; Su, Albert; Binder, Scott W; Kazakov, Dmitry V; Michal, Michal; Westra, William H

    2017-01-01

    Mammary analogue secretory carcinoma is a low-grade salivary gland carcinoma that exhibits analogous features to secretory carcinoma of the breast including the presence of a t(12;15) translocation resulting in the ETV6-NTRK3 gene fusion. Rare cases of purported secretory carcinoma of the skin adnexa have been reported, but their relationship to true secretory carcinoma of the breast and salivary glands is unclear, as they generally do not harbor ETV6 rearrangements. Cases of cutaneous neoplasms with histologic features identical to secretory carcinoma of the breast and salivary glands were identified from the consultation files of 3 academic medical institutions. Immunohistochemistry was performed for S100 protein, mammaglobin and STAT5a. Break-apart fluorescence in situ hybridization was used evaluate for disruption of the ETV6 gene. Six cases of cutaneous secretory carcinoma were identified. The tumors arose in 4 women and 2 men, ranging from 24 to 71 years in age (mean, 47 y). The carcinomas presented in the skin of the axilla (n=4), ventral neck (n=1), and cheek (n=1). The tumors arose in the superficial dermis in association with adnexal structures. None of the patients had a prior or concurrent breast or salivary gland tumor. They were histologically characterized by well-circumscribed but unencapsulated proliferations of bland, eosinophilic cells arranged in microcysts and follicles with intraluminal secretions. Ectopic breast or salivary gland tissue was not identified. The cases were diffusely positive for S100 protein (6 of 6), mammaglobin (6 of 6), and STAT5a (5 of 5). All 6 cases harbored rearrangements of ETV6. All tumors were treated by simple excision alone. No recurrences or metastases developed in the 2 cases with follow-up. Secretory carcinoma of the skin represents a phenotypic, immunohistochemical, and genetic counterpart to secretory carcinoma of the breast and salivary glands. This tumor entity is less anatomically restricted than previously

  18. Secretory function in subplate neurons during cortical development

    PubMed Central

    Kondo, Shinichi; Al-Hasani, Hannah; Hoerder-Suabedissen, Anna; Wang, Wei Zhi; Molnár, Zoltán

    2015-01-01

    Subplate cells are among the first generated neurons in the mammalian cerebral cortex and have been implicated in the establishment of cortical wiring. In rodents some subplate neurons persist into adulthood. Here we would like to highlight several converging findings which suggest a novel secretory function of subplate neurons during cortical development. Throughout the postnatal period in rodents, subplate neurons have highly developed rough endoplasmic reticulum (ER) and are under an ER stress condition. By comparing gene expression between subplate and layer 6, we found that several genes encoding secreted proteins are highly expressed in subplate neurons. One of these secreted proteins, neuroserpin, encoded by the serpini1 gene, is localized to the ER in subplate cells. We propose that subplate might influence cortical circuit formation through a transient secretory function. PMID:25859180

  19. Evolution of apicomplexan secretory organelles

    PubMed Central

    Gubbels, Marc-Jan; Duraisingh, Manoj T.

    2013-01-01

    The alveolate superphylum includes many free-living and parasitic organisms, which are united by the presence of alveolar sacs lying proximal to the plasma membrane, providing cell structure. All species comprising the apicomplexan group of alveolates are parasites and have adapted to the unique requirements of the parasitic lifestyle. Here the evolution of apicomplexan secretory organelles that are involved in the critical process of egress from one cell and invasion of another is explored. The variations within the Apicomplexa and how these relate to species-specific biology will be discussed. In addition, recent studies have identified specific calcium-sensitive molecules that coordinate the various events and regulate the release of these secretory organelles within apicomplexan parasites. Some aspects of this machinery are conserved outside the Apicomplexa, and are beginning to elucidate the conserved nature of the machinery. Briefly, the relationship of this secretion machinery within the Apicomplexa will be discussed, compared with free-living and predatory alveolates, and how these might have evolved from a common ancestor. PMID:23068912

  20. Unremitting Cell Proliferation in the Secretory Phase of Eutopic Endometriosis

    PubMed Central

    Franco-Murillo, Yanira; Miranda-Rodríguez, José Antonio; Rendón-Huerta, Erika; Montaño, Luis F.; Cornejo, Gerardo Velázquez; Gómez, Lucila Poblano; Valdez-Morales, Francisco Javier; Gonzalez-Sanchez, Ignacio

    2014-01-01

    Objective: Endometriosis is linked to altered cell proliferation and stem cell markers c-kit/stem cell factor (SCF) in ectopic endometrium. Our aim was to investigate whether c-kit/SCF also plays a role in eutopic endometrium. Design: Eutopic endometrium obtained from 35 women with endometriosis and 25 fertile eumenorrheic women was analyzed for in situ expression of SCF/c-kit, Ki67, RAC-alpha serine/threonine-protein kinase (Akt), phosphorylated RAC-alpha serine/threonin-protein kinase (pAkt), Glycogen synthase kinase 3 beta (GSK3β), and phosphorylated glycogen synthase kinase 3 beta (pGSK3β), throughout the menstrual cycle. Results: Expression of Ki67 and SCF was higher in endometriosis than in control tissue (P < .05) and greater in secretory rather than proliferative (P < .01) endometrium in endometriosis. Expression of c-kit was also higher in endometriosis although similar in both phases. Expression of Akt and GSK3β was identical in all samples and cycle phases, whereas pAkt and pGSK3β, opposed to control tissue, remained overexpressed in the secretory phase in endometriosis. Conclusion: Unceasing cell proliferation in the secretory phase of eutopic endometriosis is linked to deregulation of c-kit/SCF-associated signaling pathways. PMID:25194152

  1. Drug-induced secretory diarrhea: A role for CFTR.

    PubMed

    Moon, Changsuk; Zhang, Weiqiang; Sundaram, Nambirajan; Yarlagadda, Sunitha; Reddy, Vadde Sudhakar; Arora, Kavisha; Helmrath, Michael A; Naren, Anjaparavanda P

    2015-12-01

    Many medications induce diarrhea as a side effect, which can be a major obstacle to therapeutic efficacy and also a life-threatening condition. Secretory diarrhea can be caused by excessive fluid secretion in the intestine under pathological conditions. The cAMP/cGMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) is the primary chloride channel at the apical membrane of intestinal epithelial cells and plays a major role in intestinal fluid secretion and homeostasis. CFTR forms macromolecular complexes at discreet microdomains at the plasma membrane, and its chloride channel function is regulated spatiotemporally through protein-protein interactions and cAMP/cGMP-mediated signaling. Drugs that perturb CFTR-containing macromolecular complexes in the intestinal epithelium and upregulate intracellular cAMP and/or cGMP levels can hyperactivate the CFTR channel, causing excessive fluid secretion and secretory diarrhea. Inhibition of CFTR chloride-channel activity may represent a novel approach to the management of drug-induced secretory diarrhea.

  2. BPIFB6 Regulates Secretory Pathway Trafficking and Enterovirus Replication

    PubMed Central

    Morosky, Stefanie; Lennemann, Nicholas J.

    2016-01-01

    ABSTRACT Bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) is an endoplasmic reticulum (ER)-localized host factor that negatively regulates coxsackievirus B (CVB) replication through its control of the autophagic pathway. Here, we show that another member of the BPIFB family, BPIFB6, functions as a positive regulator of CVB, and other enterovirus, replication by controlling secretory pathway trafficking and Golgi complex morphology. We show that similar to BPIFB3, BPIFB6 localizes exclusively to the ER, where it associates with other members of the BPIFB family. However, in contrast to our findings that RNA interference (RNAi)-mediated silencing of BPIFB3 greatly enhances CVB replication, we show that silencing of BPIFB6 expression dramatically suppresses enterovirus replication in a pan-viral manner. Mechanistically, we show that loss of BPIFB6 expression induces pronounced alterations in retrograde and anterograde trafficking, which correlate with dramatic fragmentation of the Golgi complex. Taken together, these data implicate BPIFB6 as a key regulator of secretory pathway trafficking and viral replication and suggest that members of the BPIFB family participate in diverse host cell functions to regulate virus infections. IMPORTANCE Enterovirus infections are associated with a number of severe pathologies, such as aseptic meningitis, dilated cardiomyopathy, type I diabetes, paralysis, and even death. These viruses, which include coxsackievirus B (CVB), poliovirus (PV), and enterovirus 71 (EV71), co-opt the host cell secretory pathway, which controls the transport of proteins from the endoplasmic reticulum to the Golgi complex, to facilitate their replication. Here we report on the identification of a novel regulator of the secretory pathway, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 6 (BPIFB6), whose expression is required for enterovirus replication. We show that loss of

  3. Electroimmunodiffusion Studies of alpha Chain, Secretory Piece and Secretory IgA.

    DTIC Science & Technology

    1977-03-15

    riflIy b, bloeti nw~.b.r) V • V V VCommercially available antisera to secretory piece, colostrum , and a chain ‘ V were evaluated for use in...secretorj IgA Is laborious and expensive.. Problems in using currently available V commercial antisera to colostrum and free secretory piece arise because...V Coninercially available antisera to secretory piece, colostrum , V and ~t chain were evaluated for use in electroimmunodiffusj on of

  4. Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

    PubMed

    Kienzle, Christine; Basnet, Nirakar; Crevenna, Alvaro H; Beck, Gisela; Habermann, Bianca; Mizuno, Naoko; von Blume, Julia

    2014-09-01

    The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca(2+) entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca(2+) import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.

  5. RNAi knockdown of parafusin inhibits the secretory pathway.

    PubMed

    Liu, Li; Wyroba, Elzbieta; Satir, Birgit H

    2011-10-01

    Several glycolytic enzymes and their isoforms have been found to be important in cell signaling unrelated to glycolysis. The involvement of parafusin (PFUS), a member of the phosphoglucomutase (PGM) superfamily with no phosphoglucomutase activity, in Ca(2+)-dependent exocytosis has been controversial. This protein was first described in Paramecium tetraurelia, but is widely found. Earlier work showed that parafusin is a secretory vesicle scaffold component with unusual post-translational modifications (cyclic phosphorylation and phosphoglucosylation) coupled to stages in the exocytic process. Using RNAi, we demonstrate that parafusin synthesis can be reversibly blocked, with minor or no effect on other PGM isoforms. PFUS knockdown produces an inhibition of dense core secretory vesicle (DCSV) synthesis leading to an exo(-) phenotype. Although cell growth is unaffected, vesicle content is not packaged properly and no new DCSVs are formed. We conclude that PFUS and its orthologs are necessary for proper scaffold maturation. Because of this association, parafusin is an important signaling component for regulatory control of the secretory pathway.

  6. Reversible condensation of mast cell secretory products in vitro.

    PubMed Central

    Fernandez, J M; Villalón, M; Verdugo, P

    1991-01-01

    We have investigated the mechanisms responsible for the condensation and decondensation of secretory products that occur in mast cell secretion. We show here that the hydrated matrix of an exocytosed secretory granule can be recondensed to its original volume by exposure to acidic solutions containing histamine at concentrations that mimic those found in vivo. Recondensation by acidic histamine began in the range of 1-10 mM with a dose response curve that was accurately predicted by a Hill type equation with four highly cooperative binding sites and a half maximum concentration of [Hi++] = 3.9 mM. Recondensation by histamine showed a sigmoidal dependency on pH (critical range pH 5.5-6.5) and was fully reversible. These experiments suggest that histamine, possibly by binding to anionic sites in the protein-heparin complex of the granule matrix, triggers a change in the polymeric structures of the granule matrix from an extended coil to a collapsed globular state. This may be a useful model for understanding the condensation of secretory products into dense core granules and their subsequent decondensation upon exocytosis. Images FIGURE 1 FIGURE 4 PMID:1868152

  7. [Overweight and secretory male infertility].

    PubMed

    Oshakbaev, K P; Abylaĭuly, Zh; Dukenbaeva, B A

    2009-01-01

    We have performed a trial with participation of 60 males aged 23-52. Of them, 30 had secretory male iufertility (SMI) and obesity. The control 30 patients were healthy volunteers. The protocol was performed by two stages. Stage 1 included: investigation of a clinico-laboratory status, of correlation between a sorption function of erythrocytes, endogenic metabolic intoxication (EMI) and spermogram parameters, concentration of serum testosterone in SMI patients. Stage 2 consisted in treatment of the intoxication by reducing body mass. All the infertile men were obese; 30% of them had low glucose tolerance, 46.7% had stage 2 hypertension, 23.3%--seasonal allergic symptoms. The level of organic substances on the surface of erythrocytes in infertile men was higher than in the controls (p < 0.01). A negative correlation was seen between spermogram parameters and organic substances content on erythrocytic surface (p < 0/05), concentration of serum testosterone and the above substances (p < 0.01). The loss of fat tissue by 7-14 kg by infertile men resulted in a positive trend in spermogram parameters and the level of serum testosterone (p < 0.01).

  8. Secretory granule biogenesis and neuropeptide sorting to the regulated secretory pathway in neuroendocrine cells.

    PubMed

    Loh, Y Peng; Kim, Taeyoon; Rodriguez, Yazmin M; Cawley, Niamh X

    2004-01-01

    Neuropeptide precursors synthesized at the rough endoplasmic reticulum are transported and sorted at the trans-Golgi network (TGN) to the granules of the regulated secretory pathway (RSP) of neuroendocrine cells. They are then processed into active peptides and stored in large dense-core granules (LDCGs) until secreted upon stimulation. We have studied the regulation of biogenesis of the LDCGs and the mechanism by which neuropeptide precursors, such as pro-opiomelanocortin (POMC), are sorted into these LDCGs of the RSP in neuroendocrine and endocrine cells. We provide evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, plays an important role in the regulation of LDCG biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of secretory granule formation. Exogenously expressed POMC was neither stored nor secreted in a regulated manner in these CgA-deficient PC12 cells. Overexpression of CgA in a CgA- and LDCG-deficient endocrine cell line, 6T3, restored regulated secretion of transfected POMC and the presence of immunoreactive CgA at the tips of the processes of these cells. Unlike CgA, CgB, another granin protein, could not substitute for the role of CgA in regulating LDCG biogenesis. Thus, we conclude that CgA is a key player in the regulation of the biogenesis of LDCGs in neuroendocrine cells. To examine the mechanism of sorting POMC to the LDCGs, we carried out site-directed mutagenesis, transfected the POMC mutants into PC12 cells, and assayed for regulated secretion. Our previous molecular modeling studies predicted a three-dimensional sorting motif in POMC that can bind to a sorting receptor, membrane carboxypeptidase E (CPE). The sorting signal consists of four conserved residues at the N-terminal loop structure of POMC: two acidic residues and two hydrophobic residues. The two acidic residues were predicted to bind to a

  9. Immunomodulatory potential of particular Trichinella spiralis muscle larvae excretory-secretory components.

    PubMed

    Cvetkovic, J; Sofronic-Milosavljevic, Lj; Ilic, N; Gnjatovic, M; Nagano, I; Gruden-Movsesijan, A

    2016-12-01

    Excretory-secretory antigens of Trichinella spiralis muscle larvae can induce the semi-matured status of rat dendritic cells. This may at least partly be the consequence of transient activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Here we investigated the potential of several components of excretory-secretory antigens (native fraction containing 45, 49 and 53kDa proteins and recombinant Tsp53, representing one of the constituents of this fraction) to demonstrate previously observed effects of excretory-secretory antigens on dendritic cells in vitro, characterised by establishment of a particular phenotype (very low MHC II expression, moderate CD86 expression and significant ICAM-1 expression) and functional properties (low production of pro-inflammatory cytokine IL-12p70, and high production of IL-10 and TGF-β). Dendritic cells activated by these components were able to provoke proliferation of naïve T cells and their polarisation towards Th2 and anti-inflammatory responses. The investigated antigens had almost the same capacity to induce IL-4 and IL-10 production from T cells as excretory-secretory antigens, but failed to induce significant TGF-β synthesis. It could be concluded that the investigated excretory-secretory antigens components can largely reproduce the immunomodulatory effects of the complete excretory-secretory antigens and therefore may be considered as molecules important for creation of the anti-inflammatory milieu achieved by the parasite.

  10. HID-1 is required for homotypic fusion of immature secretory granules during maturation

    PubMed Central

    Du, Wen; Zhou, Maoge; Zhao, Wei; Cheng, Dongwan; Wang, Lifen; Lu, Jingze; Song, Eli; Feng, Wei; Xue, Yanhong; Xu, Pingyong; Xu, Tao

    2016-01-01

    Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules. DOI: http://dx.doi.org/10.7554/eLife.18134.001 PMID:27751232

  11. Estradiol increases cAMP in the oviductal secretory cells through a nongenomic mechanism.

    PubMed

    Oróstica, María L; Lopez, John; Rojas, Israel; Rocco, Jocelyn; Díaz, Patricia; Reuquén, Patricia; Cardenas, Hugo; Parada-Bustamante, Alexis; Orihuela, Pedro A

    2014-09-01

    In the rat oviduct, estradiol (E2) accelerates egg transport by a nongenomic action that requires previous conversion of E2 to methoxyestrogens via catechol-O-methyltranferase (COMT) and activation of estrogen receptor (ER) with subsequent production of cAMP and inositol triphosphate (IP3). However, the role of the different oviductal cellular phenotypes on this E2 nongenomic pathway remains undetermined. The aim of this study was to investigate the effect of E2 on the levels of cAMP and IP3 in primary cultures of secretory and smooth muscle cells from rat oviducts and determine the mechanism by which E2 increases cAMP in the secretory cells. In the secretory cells, E2 increased cAMP but not IP3, while in the smooth muscle cells E2 decreased cAMP and increased IP3. Suppression of protein synthesis by actinomycin D did not prevent the E2-induced cAMP increase, but this was blocked by the ER antagonist ICI 182 780 and the inhibitors of COMT OR 486, G protein-α inhibitory (Gαi) protein pertussis toxin and adenylyl cyclase (AC) SQ 22536. Expression of the mRNA for the enzymes that metabolizes estrogens, Comt, Cyp1a1, and Cyp1b1 was found in the secretory cells, but this was not affected by E2. Finally, confocal immunofluorescence analysis showed that E2 induced colocalization between ESR1 (ERα) and Gαi in extranuclear regions of the secretory cells. We conclude that E2 differentially regulates cAMP and IP3 in the secretory and smooth muscle cells of the rat oviduct. In the secretory cells, E2 increases cAMP via a nongenomic action that requires activation of COMT and ER, coupling between ESR1 and Gαi, and stimulation of AC.

  12. Porosome: The Universal Secretory Portal in Cells

    NASA Astrophysics Data System (ADS)

    Jena, Bhanu

    2012-10-01

    In the past 50 years it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the ``all or none'' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess such `all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. Therefore, in 1993 in a News and Views article in Nature, E. Neher wrote ``It seems terribly wasteful that, during the release of hormones and neurotransmitters from a cell, the membrane of a vesicle should merge with the plasma membrane to be retrieved for recycling only seconds or minutes later.'' This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the ``Porosome,'' the universal secretory machinery in cells. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nm resolution and in real time, and its functional reconstitution into artificial lipid membrane, have all been elucidated. Since porosomes in exocrine and neuroendocrine cells measure 100-180 nm

  13. Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

    PubMed Central

    Upadhyay, Srijana; Xu, Xinping

    2016-01-01

    ABSTRACT Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism. PMID:27879337

  14. Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

    PubMed Central

    Klemm, Robin W.; Ejsing, Christer S.; Surma, Michal A.; Kaiser, Hermann-Josef; Gerl, Mathias J.; Sampaio, Julio L.; de Robillard, Quentin; Ferguson, Charles; Proszynski, Tomasz J.; Shevchenko, Andrej

    2009-01-01

    The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery. PMID:19433450

  15. Cab45 is required for Ca2+-dependent secretory cargo sorting at the trans-Golgi network

    PubMed Central

    Alleaume, Anne-Marie; Kienzle, Christine; Carreras-Sureda, Amado; Valverde, Miguel

    2012-01-01

    Ca2+ import into the lumen of the trans-Golgi network (TGN) by the secretory pathway calcium ATPase1 (SPCA1) is required for the sorting of secretory cargo. How is Ca2+ retained in the lumen of the Golgi, and what is its role in cargo sorting? We show here that a soluble, lumenal Golgi resident protein, Cab45, is required for SPCA1-dependent Ca2+ import into the TGN; it binds secretory cargo in a Ca2+-dependent reaction and is required for its sorting at the TGN. PMID:23266954

  16. Characterization of a Secretory Annexin in Echinococcus granulosus

    PubMed Central

    Song, Xingju; Hu, Dandan; Zhong, Xiuqin; Wang, Ning; Gu, Xiaobin; Wang, Tao; Peng, Xuerong; Yang, Guangyou

    2016-01-01

    Cystic echinococcosis, caused by Echinococcus granulosus, is a widespread parasitic zoonosis causing economic loss and public health problems. Annexins are important proteins usually present in the plasma membrane, but previous studies have shown that an annexin B33 protein of E. granulosus (Eg-ANX) could be detected in the excretory/secretory products and cyst fluid. In this study, we cloned and characterized Eg-ANX. In silico analysis showed that the amino acid sequence of Eg-ANX was conserved and lacked any signal peptides. The phospholipid-binding activity of recombinant Eg-ANX (rEg-ANX) was tested; liposomes could bind to rEg-ANX only in the presence of Ca2+. In addition, we performed western blotting and immunohistochemical analyses to further validate the secretory properties of Eg-ANX. The protein could be detected in the cyst fluid of E. granulosus and was also present in the intermediate host tissues, which suggested that Eg-ANX might play an important role in parasite–host interaction. PMID:26787154

  17. Glycosylphosphatidylinositol-dependent secretory transport in Trypanosoma brucei.

    PubMed Central

    McDowell, M A; Ransom, D M; Bangs, J D

    1998-01-01

    We have investigated the role of glycosylphosphatidylinositol (GPI) anchors in forward secretory trafficking using African trypanosomes as a model system. Soluble GPI-minus forms of variant surface glycoprotein (VSG), in which the C-terminal GPI-addition peptide signal is deleted, are secreted from transformed procyclic trypanosomes with 5-fold reduced kinetics, relative to matched GPI-anchored constructs. Cell fractionation and immunofluorescence localization studies indicate that the GPI-minus VSG reporters accumulate in the endoplasmic reticulum (ER). This transport defect is specific, since overexpression of GPI-minus VSG has no effect on the rate of transport of a second soluble secretory reporter (BiPN) when co-expressed in the same cells. Two results suggest that delayed forward transport cannot be accounted for by failure to fold/assemble in the absence of a GPI anchor, thereby leading to prolonged association with ER quality-control machinery. First, no evidence was found for elevated association of GPI-minus VSG with the ER molecular chaperone, BiP. Secondly, newly synthesized GPI-minus VSG is dimerized efficiently, as judged by velocity-sedimentation analysis. GPI-dependent transport is not confined to the VSG reporters, because a similar dependence is found with another trypanosomal GPI-anchored protein, trans-sialidase. These findings suggest that GPI structures act in a positive manner to mediate efficient forward transport of some, and perhaps all, GPI-anchored proteins in the early secretory pathway of trypanosomes. Possible mechanisms for GPI-dependent transport are discussed with respect to current models of vesicular trafficking. PMID:9794811

  18. [Secretory immunoglobulin A in amniotic fluid].

    PubMed

    Briese, V; Straube, W; Brock, J; Lorenz, U

    1983-01-01

    Secretory immunoglobulin A (S-IgA) was estimated in amniotic fluid samples by means of the single radial immunodiffusion according to Mancini. A monospecific antiserum against human secretory component was used. 163 amniotic fluid samples from normal pregnancies and risk pregnancies respectively were investigated. Within the 3rd trimenon the S-IgA content in amniotic fluid increased significantly. With respect to literature and examinations performed previously a connection between S-IgA content in amniotic fluid and fetal lung maturity seems to be possible.

  19. Human Toxoplasma gondii-specific secretory immunoglobulin A reduces T. gondii infection of enterocytes in vitro.

    PubMed Central

    Mack, D G; McLeod, R

    1992-01-01

    Whey from 17 women (four acutely infected with Toxoplasma gondii, eight chronically infected, and five uninfected) was studied. T. gondii-specific secretory IgA antibodies were demonstrated by ELISA in whey from acutely infected and one of eight chronically infected women. Such antibodies to tachyzoite proteins of < or = 14, 22, 26-28, 30, 46, 60, 70-80, and > 100 kD (eliminated by protease but not periodate or neuraminidase treatment) were demonstrated in whey from acutely infected subjects when Western blots were probed with their whey and antibodies to human secretory IgA or IgA or secretory piece. Secretory IgA from four of eight chronically infected women recognized the 46- and 69-kD epitopes. Other whey samples were negative. Incubation of T. gondii tachyzoites with whey or purified secretory IgA from acutely infected (but not seronegative) women caused 50-75% reduction in infection of enterocytes in vitro. Whey reactive with the 46-kD epitope from three of six chronically infected women caused less (> or = 40%) inhibition. Whey and purified secretory IgA from two of three acutely infected women agglutinated tachyzoites. Whey did not result in complement-dependent lysis of T. gondii. These results indicate that it may be possible to produce human secretory IgA to T. gondii capable of reducing initial infection of enterocytes, as such IgA is present during natural infection. They also demonstrate candidate epitopes for such protection. Images PMID:1469104

  20. Stress modulates intestinal secretory immunoglobulin A

    PubMed Central

    Campos-Rodríguez, Rafael; Godínez-Victoria, Marycarmen; Abarca-Rojano, Edgar; Pacheco-Yépez, Judith; Reyna-Garfias, Humberto; Barbosa-Cabrera, Reyna Elizabeth; Drago-Serrano, Maria Elisa

    2013-01-01

    Stress is a response of the central nervous system to environmental stimuli perceived as a threat to homeostasis. The stress response triggers the generation of neurotransmitters and hormones from the hypothalamus pituitary adrenal axis, sympathetic axis and brain gut axis, and in this way modulates the intestinal immune system. The effects of psychological stress on intestinal immunity have been investigated mostly with the restraint/immobilization rodent model, resulting in an up or down modulation of SIgA levels depending on the intensity and time of exposure to stress. SIgA is a protein complex formed by dimeric (dIgA) or polymeric IgA (pIgA) and the secretory component (SC), a peptide derived from the polymeric immunoglobulin receptor (pIgR). The latter receptor is a transmembrane protein expressed on the basolateral side of gut epithelial cells, where it uptakes dIgA or pIgA released by plasma cells in the lamina propria. As a result, the IgA-pIgR complex is formed and transported by vesicles to the apical side of epithelial cells. pIgR is then cleaved to release SIgA into the luminal secretions of gut. Down modulation of SIgA associated with stress can have negative repercussions on intestinal function and integrity. This can take the form of increased adhesion of pathogenic agents to the intestinal epithelium and/or an altered balance of inflammation leading to greater intestinal permeability. Most studies on the molecular and biochemical mechanisms involved in the stress response have focused on systemic immunity. The present review analyzes the impact of stress (mostly by restraint/immobilization, but also with mention of other models) on the generation of SIgA, pIgR and other humoral and cellular components involved in the intestinal immune response. Insights into these mechanisms could lead to better therapies for protecting against pathogenic agents and avoiding epithelial tissue damage by modulating intestinal inflammation. PMID:24348350

  1. Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode.

    PubMed

    Rikihisa, Y; Lin, Y C; Fukaya, T

    1985-06-01

    In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.

  2. Secretory immunity with special reference to the oral cavity

    PubMed Central

    Brandtzaeg, Per

    2013-01-01

    The two principal antibody classes present in saliva are secretory IgA (SIgA) and IgG; the former is produced as dimeric IgA by local plasma cells (PCs) in the stroma of salivary glands and is transported through secretory epithelia by the polymeric Ig receptor (pIgR), also named membrane secretory component (SC). Most IgG in saliva is derived from the blood circulation by passive leakage mainly via gingival crevicular epithelium, although some may be locally produced in the gingiva or salivary glands. Gut-associated lymphoid tissue (GALT) and nasopharynx-associated lymphoid tissue (NALT) do not contribute equally to the pool of memory/effector B cells differentiating to mucosal PCs throughout the body. Thus, enteric immunostimulation may not be the best way to activate the production of salivary IgA antibodies although the level of specific SIgA in saliva may still reflect an intestinal immune response after enteric immunization. It remains unknown whether the IgA response in submandibular/sublingual glands is better related to B-cell induction in GALT than the parotid response. Such disparity is suggested by the levels of IgA in submandibular secretions of AIDS patients, paralleling their highly upregulated intestinal IgA system, while the parotid IgA level is decreased. Parotid SIgA could more consistently be linked to immune induction in palatine tonsils/adenoids (human NALT) and cervical lymph nodes, as supported by the homing molecule profile observed after immune induction at these sites. Several other variables influence the levels of antibodies in salivary secretions. These include difficulties with reproducibility and standardization of immunoassays, the impact of flow rate, acute or chronic stress, protein loss during sample handling, and uncontrolled admixture of serum-derived IgG and monomeric IgA. Despite these problems, saliva is an easily accessible biological fluid with interesting scientific and clinical potentials. PMID:23487566

  3. Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network

    PubMed Central

    Blank, Birgit; Maiser, Andreas; Emin, Derya; Prescher, Jens; Beck, Gisela; Kienzle, Christine; Bartnik, Kira; Habermann, Bianca; Pakdel, Mehrshad; Leonhardt, Heinrich; Lamb, Don C.

    2016-01-01

    Sorting and export of transmembrane cargoes and lysosomal hydrolases at the trans-Golgi network (TGN) are well understood. However, elucidation of the mechanism by which secretory cargoes are segregated for their release into the extracellular space remains a challenge. We have previously demonstrated that, in a reaction that requires Ca2+, the soluble TGN-resident protein Cab45 is necessary for the sorting of secretory cargoes at the TGN. Here, we report that Cab45 reversibly assembles into oligomers in the presence of Ca2+. These Cab45 oligomers specifically bind secretory proteins, such as COMP and LyzC, in a Ca2+-dependent manner in vitro. In intact cells, mutation of the Ca2+-binding sites in Cab45 impairs oligomerization, as well as COMP and LyzC sorting. Superresolution microscopy revealed that Cab45 colocalizes with secretory proteins and the TGN Ca2+ pump (SPCA1) in specific TGN microdomains. These findings reveal that Ca2+-dependent changes in Cab45 mediate sorting of specific cargo molecules at the TGN. PMID:27138253

  4. Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii.

    PubMed

    Stewart, Rebecca J; Ferguson, David J P; Whitehead, Lachlan; Bradin, Clare H; Wu, Hong J; Tonkin, Christopher J

    2016-02-01

    Upon infection, apicomplexan parasites quickly invade host cells and begin a replicative cycle rapidly increasing in number over a short period of time, leading to tissue lysis and disease. The secretory pathway of these highly polarized protozoan parasites tightly controls, in time and space, the biogenesis of specialized structures and organelles required for invasion and intracellular survival. In other systems, regulation of protein trafficking can occur by phosphorylation of vesicle fusion machinery. Previously, we have shown that Toxoplasma gondii αSNAP - a protein that controls the disassembly of cis-SNARE complexes--is phosphorylated. Here, we show that this post-translational modification is required for the correct function of αSNAP in controlling secretory traffic. We demonstrate that during intracellular development conditional expression of a non-phosphorylatable form of αSNAP results in Golgi fragmentation and vesiculation of all downstream secretory organelles. In addition, we show that the vestigial plastid (termed apicoplast), although reported not to be reliant on Golgi trafficking for biogenesis, is also affected upon overexpression of αSNAP and is much more sensitive to the levels of this protein than targeting to other organelles. This work highlights the importance of αSNAP and its phosphorylation in Toxoplasma organelle biogenesis and exposes a hereto fore-unexplored mechanism of regulation of vesicle fusion during secretory pathway trafficking in apicomplexan parasites.

  5. Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

    PubMed Central

    Urbé, Sylvie; Page, Lesley J.; Tooze, Sharon A.

    1998-01-01

    The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [35S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process. PMID:9864358

  6. PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

    PubMed Central

    Jansen, Anna M.; Jin, Chunyu; Rickhag, Mattias; Lund, Viktor K.; Jensen, Morten; Bhatia, Vikram; Sørensen, Gunnar; Madsen, Andreas N.; Xue, Zhichao; Møller, Siri K.; Woldbye, David; Qvortrup, Klaus; Huganir, Richard; Stamou, Dimitrios; Kjærulff, Ole; Gether, Ulrik

    2013-01-01

    Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine

  7. Morphological docking of secretory vesicles

    PubMed Central

    2010-01-01

    Calcium-dependent secretion of neurotransmitters and hormones is essential for brain function and neuroendocrine-signaling. Prior to exocytosis, neurotransmitter-containing vesicles dock to the target membrane. In electron micrographs of neurons and neuroendocrine cells, like chromaffin cells many synaptic vesicles (SVs) and large dense-core vesicles (LDCVs) are docked. For many years the molecular identity of the morphologically docked state was unknown. Recently, we resolved the minimal docking machinery in adrenal medullary chromaffin cells using embryonic mouse model systems together with electron-microscopic analyses and also found that docking is controlled by the sub-membrane filamentous (F-)actin. Currently it is unclear if the same docking machinery operates in synapses. Here, I will review our docking assay that led to the identification of the LDCV docking machinery in chromaffin cells and also discuss whether identical docking proteins are required for SV docking in synapses. PMID:20577884

  8. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    PubMed Central

    Jethwaney, Deepa; Islam, Md Rafiqul; Leidal, Kevin G; de Bernabe, Daniel Beltran-Valero; Campbell, Kevin P; Nauseef, William M; Gibson, Bradford W

    2007-01-01

    Background Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli. PMID:17692124

  9. The complete general secretory pathway in gram-negative bacteria.

    PubMed Central

    Pugsley, A P

    1993-01-01

    The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins. PMID:8096622

  10. An Alternative Terminal Step of the General Secretory Pathway in Staphylococcus aureus

    PubMed Central

    Craney, Arryn; Dix, Melissa M.; Adhikary, Ramkrishna; Cravatt, Benjamin F.

    2015-01-01

    ABSTRACT Type I signal peptidase (SPase) is essential for viability in wild-type bacteria because the terminal step of the bacterial general secretory pathway requires its proteolytic activity to release proteins from their membrane-bound N-terminal leader sequences after translocation across the cytoplasmic membrane. Here, we identify the Staphylococcus aureus operon ayrRABC (SA0337 to SA0340) and show that once released from repression by AyrR, the protein products AyrABC together confer resistance to the SPase inhibitor arylomycin M131 by providing an alternate and novel method of releasing translocated proteins. Thus, the derepression of ayrRABC allows cells to bypass the essentiality of SPase. We demonstrate that AyrABC functionally complements SPase by mediating the processing of the normally secreted proteins, albeit in some cases with reduced efficiency and either without cleavage or via cleavage at a site N-terminal to the canonical SPase cleavage site. Thus, ayrRABC encodes a secretion stress-inducible alternate terminal step of the general secretory pathway. Importance  Addressing proteins for proper localization within or outside a cell in both eukaryotes and prokaryotes is often accomplished with intrinsic signals which mediate membrane translocation and which ultimately must be removed. The canonical enzyme responsible for the removal of translocation signals is bacterial type I signal peptidase (SPase), which functions at the terminal step of the general secretory pathway and is thus essential in wild-type bacteria. Here, we identify a four-gene operon in S. aureus that encodes an alternate terminal step of the general secretory pathway and thus makes SPase nonessential. The results have important implications for protein secretion in bacteria and potentially for protein trafficking in prokaryotes and eukaryotes in general. PMID:26286693

  11. Non-secretory multiple myeloma: from biology to clinical management

    PubMed Central

    Dupuis, Megan Murray; Tuchman, Sascha A

    2016-01-01

    Multiple myeloma (MM) is the second most common hematologic malignancy in the US. It is typically characterized by production of large amounts of defective immunoglobulin (Ig). Diagnosing MM and monitoring treatment response, including eventual relapse, are largely based on sequential measurements of Ig. However, a small subset of MM called non-secretory multiple myeloma (NSMM) produces no detectable Ig. This subset of true NSMM has become even smaller over time, as the advent of the serum free light chain assay has resulted in the majority of NSMM patients being recategorized as light-chain MM – that is, MM cells that produce only the light-chain component of Ig. True forms of NSMM, meaning MM that secretes no monoclonal proteins whatsoever, constitute a distinct entity that is reviewed; definition of NSMM using current detection methods, discuss the biology underpinning NSMM development, and share recommendations for how NSMM should be managed clinically with respect to detection, treatment, and monitoring. PMID:28008276

  12. Accumulation of Major Histocompatibility Complex Class II Molecules in Mast Cell Secretory Granules and Their Release upon Degranulation

    PubMed Central

    Raposo, Graça; Tenza, Danielle; Mecheri, Salahedine; Peronet, Roger; Bonnerot, Christian; Desaymard, Catherine

    1997-01-01

    To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60–80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles. PMID:9398681

  13. Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation.

    PubMed

    Raposo, G; Tenza, D; Mecheri, S; Peronet, R; Bonnerot, C; Desaymard, C

    1997-12-01

    To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

  14. Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo

    PubMed Central

    Lwin, Kyaw Myo; Li, Donghao; Bretscher, Anthony

    2016-01-01

    The mechanisms by which molecular motors associate with specific cargo is a central problem in cell organization. The kinesin-like protein Smy1 of budding yeast was originally identified by the ability of elevated levels to suppress a conditional myosin-V mutation (myo2-66), but its function with Myo2 remained mysterious. Subsequently, Myo2 was found to provide an essential role in delivery of secretory vesicles for polarized growth and in the transport of mitochondria for segregation. By isolating and characterizing myo2 smy1 conditional mutants, we uncover the molecular function of Smy1 as a factor that enhances the association of Myo2 with its receptor, the Rab Sec4, on secretory vesicles. The tail of Smy1—which binds Myo2—its central dimerization domain, and its kinesin-like head domain are all necessary for this function. Consistent with this model, overexpression of full-length Smy1 enhances the number of Sec4 receptors and Myo2 motors per transporting secretory vesicle. Rab proteins Sec4 and Ypt11, receptors for essential transport of secretory vesicles and mitochondria, respectively, bind the same region on Myo2, yet Smy1 functions selectively in the transport of secretory vesicles. Thus a kinesin-related protein can function intimately with a myosin-V and its receptor in the transport of a specific cargo. PMID:27307583

  15. Excretory/secretory proteome of 14-day schistosomula, Schistosoma japonicum.

    PubMed

    Cao, Xiaodan; Fu, Zhiqiang; Zhang, Min; Han, Yanhui; Han, Qian; Lu, Ke; Li, Hao; Zhu, Chuangang; Hong, Yang; Lin, Jiaojiao

    2016-01-01

    Schistosomiasis remains a serious public health problem, with 200 million people infected and 779 million people at risk worldwide. The schistosomulum is the early stage of the complex lifecycle of Schistosoma japonicum in their vertebrate hosts, and is the main target of vaccine-induced protective immunity. Excretory/secretory (ES) proteins play a major role in host-parasite interactions and ES protein compositions of schistosomula of S. japonicum have not been characterized to date. In the present study, the proteome of ES proteins from 14 day schistosomula of S. japonicum was analyzed by liquid chromatography/tandem mass spectrometry and 713 unique proteins were finally identified. Gene ontology and pathway analysis revealed that identified proteins were mainly involved in carbohydrate metabolism, degradation, response to stimulus, oxidation-reduction, biological regulation and binding. Flow cytometry analysis demonstrated that thioredoxin peroxidase identified in this study had the effect on inhibiting MHCII and CD86 expression on LPS-activated macrophages. The present study provides insight into the growth and development of the schistosome in the final host and valuable information for screening vaccine candidates for schistosomiasis.

  16. Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

    PubMed

    Davila, Juanmahel; Laws, Mary J; Kannan, Athilakshmi; Li, Quanxi; Taylor, Robert N; Bagchi, Milan K; Bagchi, Indrani C

    2015-08-01

    During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal

  17. Rac1 Regulates Endometrial Secretory Function to Control Placental Development

    PubMed Central

    Davila, Juanmahel; Laws, Mary J.; Kannan, Athilakshmi; Li, Quanxi; Taylor, Robert N.; Bagchi, Milan K.; Bagchi, Indrani C.

    2015-01-01

    During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal

  18. Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes, is essential for the biogenesis of secretory organelles.

    PubMed

    Morlon-Guyot, Juliette; Pastore, Sandra; Berry, Laurence; Lebrun, Maryse; Daher, Wassim

    2015-08-01

    Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome-like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin-like receptor and syntaxin-6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab-GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps-C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal-related compartments, the vacuole and immature apical secretory organelles. Conditional knock-down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock-down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite-specific functions in the biogenesis of their unique secretory organelles.

  19. Influence of experimental hypokinesia on gastric secretory function

    NASA Technical Reports Server (NTRS)

    Markova, O. O.; Vavryshchuk, V. I.; Rozvodovskyy, V. I.; Proshcheruk, V. A.

    1980-01-01

    The gastric secretory function of rats was studied in 4, 8, 16 and 30 day hypokinesia. Inhibition of both the gastric juice secretory and acid producing functions was found. The greatest inhibition was observed on day 8 of limited mobility. By days 16 and 30 of the experiment, a tendency of the gastric secretory activity to return to normal was observed, although it remained reduced.

  20. Calcium mediation of the pig jejunal secretory response.

    PubMed Central

    Forsyth, G W; Wong, P H; Maenz, D D

    1985-01-01

    The involvement of Ca++ ions as secretory mediators in pig jejunal epithelia has been investigated with an in vitro system. Omission of Ca++ from the Ringer-HCO3 bathing media on both sides of the tissue had minor effects on the basal electrical activity of pig jejunal mucosa. There were only slight decreases in transepithelial potential difference and increases in conductance with Ca++ free media. Low EGTA concentrations which reversibly blocked potential difference responses to secretory agents also had minimal effects on basal electrical activity. The in vitro secretory responses to A23187, to theophylline, and to Escherichia coli heat-stable enterotoxin were all eliminated by Ca++ depletion and restored by replacing normal Ca++ concentrations in the bathing media. Dantrolene prevented the secretory response but not the potential difference increases caused by heat-stable enterotoxin and A23187, suggesting that intracellular Ca++ stores may be reservoirs of secretory signal agent. Verapamil only blocked the secretory response to heat-stable enterotoxin. Chlorpromazine had negligible effects on basal conditions, but totally blocked both the secretory response and the Ca++-dependent effects of A23187 and heat-stable enterotoxin on potential difference. The response to theophylline was only partially inhibited by chlorpromazine, implying some involvement of both cAMP and Ca++ as secretory signals for theophylline. Cytoplasmic Ca++ concentrations appear to be at least as important as cyclic nucleotides in regulating the secretory effects of pig jejunum. PMID:2410089

  1. A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes

    PubMed Central

    Tie, Hieng Chiong; Mahajan, Divyanshu; Chen, Bing; Cheng, Li; VanDongen, Antonius M. J.; Lu, Lei

    2016-01-01

    Cellular functions of the Golgi are determined by the unique distribution of its resident proteins. Currently, electron microscopy is required for the localization of a Golgi protein at the sub-Golgi level. We developed a quantitative sub-Golgi localization method based on centers of fluorescence masses of nocodazole-induced Golgi ministacks under conventional optical microscopy. Our method is rapid, convenient, and quantitative, and it yields a practical localization resolution of ∼30 nm. The method was validated by the previous electron microscopy data. We quantitatively studied the intra-Golgi trafficking of synchronized secretory membrane cargoes and directly demonstrated the cisternal progression of cargoes from the cis- to the trans-Golgi. Our data suggest that the constitutive efflux of secretory cargoes could be restricted at the Golgi stack, and the entry of the trans-Golgi network in secretory pathway could be signal dependent. PMID:26764092

  2. Serous cutaneous glands in anurans: Fourier transform analysis of the repeating secretory granule substructure

    NASA Astrophysics Data System (ADS)

    Nosi, D.; Delfino, G.; Quercioli, F.

    2013-03-01

    A combined transmission electron microscopy (TEM) and Fourier transform analysis has been performed on the secretory granules storing active peptides/proteins in serous cutaneous glands of n = 12 anuran species. Previous TEM investigation showed that the granules are provided with remarkable repeating substructures based on discrete subunits, arranged into a consistent framework. Furthermore, TEM findings revealed that this recurrent arrangement is acquired during a prolonged post-Golgian (or maturational) processing that affects the secretory product. Maturation leads to a variety of patterns depending on the degree of subunit clustering. This variety of recurrent patterns has been plotted into a range of frequency spectra. Through this quantitative approach, we found that the varying granule substructure can be reduced to a single mechanism of peptide/protein aggregation.

  3. Degradation of immunoglobulins, protease inhibitors, and interleukin-1 by a secretory proteinase of Acanthamoeba castellanii

    PubMed Central

    Na, Byoung-Kuk; Cho, Jong-Hwa; Song, Chul-Yong; Kim, Tong-Soo

    2002-01-01

    The effect of a secretory proteinase from the pathogenic amoebae Acanthamoeba castellanii on host's defense-oriented or regulatory proteins such as immunoglobulins, interleukin-1, and protease inhibitors was investigated. The enzyme was found to degrade secretory immunoglobulin A (sIgA), IgG, and IgM. It also degraded interleukin-1α (IL-1α) and IL-1β. Its activity was not inhibited by endogenous protease inhibitors, such as α2-macroglobulin, α1-trypsin inhibitor, and α2-antiplasmin. Furthermore, the enzyme rapidly degraded those endogenous protease inhibitors as well. The degradation of host's defense-oriented or regulatory proteins by the Acanthamoeba proteinase suggested that the enzyme might be an important virulence factor in the pathogenesis of Acanthamoeba infection. PMID:12073735

  4. Developmental genetics of secretory vesicle acidification during Caenorhabditis elegans spermatogenesis.

    PubMed

    Gleason, Elizabeth J; Hartley, Paul D; Henderson, Melissa; Hill-Harfe, Katherine L; Price, Paul W; Weimer, Robby M; Kroft, Tim L; Zhu, Guang-Dan; Cordovado, Suzanne; L'Hernault, Steven W

    2012-06-01

    Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis.

  5. Spiperone: evidence for uptake into secretory granules.

    PubMed Central

    Dannies, P S; Rudnick, M S; Fishkes, H; Rudnick, G

    1984-01-01

    Spiperone, a dopamine antagonist widely used as a specific ligand for dopamine and serotonin receptors, is actively accumulated into the F4C1 strain of rat pituitary tumor cells. The accumulation of 10 nM [3H]spiperone was linear for 3 min and reached a steady state after 10 min. Spiperone accumulation was reduced 50% by preincubation with 5 microM reserpine, an inhibitor of biogenic amine transport into secretory granules, and was also blocked by monensin and ammonium chloride, both of which increase the pH of intracellular storage organelles. Uptake was not affected by replacing sodium in the buffer with lithium at equimolar concentrations. Spiperone at 1 microM inhibited by over 50% serotonin transport into membrane vesicles isolated from platelet dense granules; this concentration inhibited the Na+-dependent plasma membrane transport system less than 10%. The data indicate spiperone specifically interacts with the secretory granule amine transport system and suggest that this transport system is found in the F4C1 pituitary cell strain as well as in platelets and neurons. The data also suggest that experiments utilizing spiperone to measure dopamine and serotonin receptors be interpreted with caution. PMID:6584920

  6. Brown adipose tissue as a secretory organ.

    PubMed

    Villarroya, Francesc; Cereijo, Rubén; Villarroya, Joan; Giralt, Marta

    2017-01-01

    Brown adipose tissue (BAT) is the main site of adaptive thermogenesis and experimental studies have associated BAT activity with protection against obesity and metabolic diseases, such as type 2 diabetes mellitus and dyslipidaemia. Active BAT is present in adult humans and its activity is impaired in patients with obesity. The ability of BAT to protect against chronic metabolic disease has traditionally been attributed to its capacity to utilize glucose and lipids for thermogenesis. However, BAT might also have a secretory role, which could contribute to the systemic consequences of BAT activity. Several BAT-derived molecules that act in a paracrine or autocrine manner have been identified. Most of these factors promote hypertrophy and hyperplasia of BAT, vascularization, innervation and blood flow, processes that are all associated with BAT recruitment when thermogenic activity is enhanced. Additionally, BAT can release regulatory molecules that act on other tissues and organs. This secretory capacity of BAT is thought to be involved in the beneficial effects of BAT transplantation in rodents. Fibroblast growth factor 21, IL-6 and neuregulin 4 are among the first BAT-derived endocrine factors to be identified. In this Review, we discuss the current understanding of the regulatory molecules (the so-called brown adipokines or batokines) that are released by BAT that influence systemic metabolism and convey the beneficial metabolic effects of BAT activation. The identification of such adipokines might also direct drug discovery approaches for managing obesity and its associated chronic metabolic diseases.

  7. ATP: The crucial component of secretory vesicles.

    PubMed

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  8. ATP: The crucial component of secretory vesicles

    PubMed Central

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R.; González-Santana, Ayoze; Westhead, Edward W.; Borges, Ricardo; Machado, José David

    2016-01-01

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission. PMID:27342860

  9. Secretory antibody following oral influenza immunization.

    PubMed

    Waldman, R H; Stone, J; Bergmann, K C; Khakoo, R; Lazzell, V; Jacknowitz, A; Waldman, E R; Howard, S

    1986-12-01

    Secretory IgA antibody may be important in protection against respiratory viral infections, and the concept of a common mucosal immune system offers the theoretical basis for the convenient stimulation of this antibody. Therefore, the oral route was compared with intramuscular injection in a double-blind, placebo-controlled study in young healthy volunteers. A killed influenza vaccine, given in enteric-coated capsules (total of 98 ug hemagglutinin of A/Bangkok) led to significant salivary and nasal IgA antibody rises in a 4-week period. The preimmunization titers in secretions were inversely correlated with the antibody rise after immunization. The orally administered vaccine was associated with no more side effects than placebo, in contradistinction to reactions following the intramuscular route. The latter route also was without significant effect in regard to a stimulation of secretory antibodies. The observed simultaneous induction of antibodies in saliva and nasal secretions following oral administration of killed vaccine gives further evidence of a common mucosal immune system and its possible clinical use.

  10. Novel N-Benzoyl-2-Hydroxybenzamide Disrupts Unique Parasite Secretory Pathway

    PubMed Central

    Fomovska, Alina; Huang, Qingqing; El Bissati, Kamal; Mui, Ernest J.; Witola, William H.; Cheng, Gang; Zhou, Ying; Sommerville, Caroline; Roberts, Craig W.; Bettis, Sam; Prigge, Sean T.; Afanador, Gustavo A.; Hickman, Mark R.; Lee, Patty J.; Leed, Susan E.; Auschwitz, Jennifer M.; Pieroni, Marco; Stec, Jozef; Muench, Stephen P.; Rice, David W.; Kozikowski, Alan P.

    2012-01-01

    Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3β, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites. PMID:22354304

  11. A genome-wide RNA interference screen identifies two novel components of the metazoan secretory pathway

    PubMed Central

    Wendler, Franz; Gillingham, Alison K; Sinka, Rita; Rosa-Ferreira, Cláudia; Gordon, David E; Franch-Marro, Xavier; Peden, Andrew A; Vincent, Jean-Paul; Munro, Sean

    2010-01-01

    Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts. PMID:19942856

  12. Novel N-benzoyl-2-hydroxybenzamide disrupts unique parasite secretory pathway.

    PubMed

    Fomovska, Alina; Huang, Qingqing; El Bissati, Kamal; Mui, Ernest J; Witola, William H; Cheng, Gang; Zhou, Ying; Sommerville, Caroline; Roberts, Craig W; Bettis, Sam; Prigge, Sean T; Afanador, Gustavo A; Hickman, Mark R; Lee, Patty J; Leed, Susan E; Auschwitz, Jennifer M; Pieroni, Marco; Stec, Jozef; Muench, Stephen P; Rice, David W; Kozikowski, Alan P; McLeod, Rima

    2012-05-01

    Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3β, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.

  13. The plant secretory pathway seen through the lens of the cell wall.

    PubMed

    van de Meene, A M L; Doblin, M S; Bacic, Antony

    2017-01-01

    Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes.

  14. Are there salvage routes within the general secretory pathway in yeast?

    PubMed

    Gozalbo, D; Martínez, J P; Sentandreu, R

    1992-04-01

    It is generally accepted that both extracellular protein secretion and plasma membrane expansion in yeast occur basically as in higher eukaryotic cells. In addition to the constitutive (default) secretory pathway, some specialized mammalian cells possess a regulated route which at present has not been detected in yeast. However, there is a body of experimental results suggesting that under certain circumstances export of integral plasma membrane and exocellular proteins may take place through alternative (salvage) pathways. The existence of these latter routes would enable the yeast cell to adapt more efficiently to distinct or adverse conditions requiring the secretion of discrete amounts of specific sets of proteins.

  15. Cysteine Cathepsins in the Secretory Vesicle Produce Active Peptides: Cathepsin L Generates Peptide Neurotransmitters and Cathepsin B Produces Beta-Amyloid of Alzheimer’s Disease

    PubMed Central

    Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory

    2011-01-01

    Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles has been demonstrated as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β-amyloid (Aβ) peptides that accumulate in Alzheimer’s disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrasts with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin function. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. PMID:21925292

  16. 21 CFR 866.5380 - Free secretory component immuno-logical test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... immunochemical techniques free secretory component (normally a portion of the secretory IgA antibody molecule) in... repetitive lung infections and other hypogammaglobulinemic conditions (low antibody levels)....

  17. 21 CFR 866.5380 - Free secretory component immuno-logical test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunochemical techniques free secretory component (normally a portion of the secretory IgA antibody molecule) in... repetitive lung infections and other hypogammaglobulinemic conditions (low antibody levels)....

  18. 21 CFR 866.5380 - Free secretory component immuno-logical test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... immunochemical techniques free secretory component (normally a portion of the secretory IgA antibody molecule) in... repetitive lung infections and other hypogammaglobulinemic conditions (low antibody levels)....

  19. The Actomyosin Ring Recruits Early Secretory Compartments to the Division Site in Fission Yeast

    PubMed Central

    Vjestica, Aleksandar; Tang, Xin-Zi

    2008-01-01

    The ultimate goal of cytokinesis is to establish a membrane barrier between daughter cells. The fission yeast Schizosaccharomyces pombe utilizes an actomyosin-based division ring that is thought to provide physical force for the plasma membrane invagination. Ring constriction occurs concomitantly with the assembly of a division septum that is eventually cleaved. Membrane trafficking events such as targeting of secretory vesicles to the division site require a functional actomyosin ring suggesting that it serves as a spatial landmark. However, the extent of polarization of the secretion apparatus to the division site is presently unknown. We performed a survey of dynamics of several fluorophore-tagged proteins that served as markers for various compartments of the secretory pathway. These included markers for the endoplasmic reticulum, the COPII sites, and the early and late Golgi. The secretion machinery exhibited a marked polarization to the division site. Specifically, we observed an enrichment of the transitional endoplasmic reticulum (tER) accompanied by Golgi cisternae biogenesis. These processes required actomyosin ring assembly and the function of the EFC-domain protein Cdc15p. Cdc15p overexpression was sufficient to induce tER polarization in interphase. Thus, fission yeast polarizes its entire secretory machinery to the cell division site by utilizing molecular cues provided by the actomyosin ring. PMID:18184749

  20. Saturated fatty acids alter the late secretory pathway by modulating membrane properties.

    PubMed

    Payet, Laurie-Anne; Pineau, Ludovic; Snyder, Ellen C R; Colas, Jenny; Moussa, Ahmed; Vannier, Brigitte; Bigay, Joelle; Clarhaut, Jonathan; Becq, Frédéric; Berjeaud, Jean-Marc; Vandebrouck, Clarisse; Ferreira, Thierry

    2013-12-01

    Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic β-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.

  1. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells.

    PubMed

    Cesaratto, Francesca; López-Requena, Alejandro; Burrone, Oscar R; Petris, Gianluca

    2015-10-20

    Tobacco etch virus protease (TEVp) is a unique endopeptidase with stringent substrate specificity. TEVp has been widely used as a purified protein for in vitro applications, but also as a biological tool directly expressing it in living cells. To adapt the protease to diverse applications, several TEVp mutants with different stability and enzymatic properties have been reported. Herein we describe the development of a novel engineered TEVp mutant designed to be active in the secretory pathway. While wild type TEVp targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalytically inactive enzyme, a TEVp mutant with selected mutations at two verified N-glycosylation sites and at an exposed cysteine was highly efficient. This mutant was very active in the endoplasmic reticulum (ER) of living cells and can be used as a biotechnological tool to cleave proteins within the secretory pathway. As an immediate practical application we report the expression of a complete functional monoclonal antibody expressed from a single polypeptide, which was cleaved by our TEVp mutant into the two antibody chains and secreted as an assembled and functional molecule. In addition, we show active TEVp mutants lacking auto-cleavage activity.

  2. Natural polyreactive secretory immunoglobulin A autoantibodies as a possible barrier to infection in humans.

    PubMed Central

    Quan, C P; Berneman, A; Pires, R; Avrameas, S; Bouvet, J P

    1997-01-01

    Secretory immunoglobulin A (S-IgA) was investigated in human secretions for the presence of natural antibodies (Abs) acting as the first "immune barrier" to infection before induction or boosting of specific responses. These molecules could be the secretory counterpart of the natural Abs in serum that were previously shown by our laboratory to be polyreactive to autoantigens. Significant levels of S-IgA Abs to human actin, myosin, tubulin, and spectrin were detected in 10 saliva and 8 colostrum samples from normal subjects. Computer-assisted analysis of immunoblots of extracts from human muscles showed these Abs to react with a large number of autoantigens. Their polyreactivity was confirmed by cross-inhibition and by immunoblotting studies of affinity-purified natural Abs, assayed against a large variety of surface or secreted antigens from Streptococcus pyogenes. The thiocyanate elution method showed that functional affinities of some natural Abs can be of the same order of magnitude as those of tetanus vaccine antitoxins. Moreover, nonimmune binding of these natural Abs to the gut protein Fv (Fv-fragment binding protein) can enhance their effector functions. This demonstrates that human secretions contain polyreactive auto-Abs which can also react with pathogens. These secretory Abs of "skeleton key" specificities are possibly produced by a primordial B-1-cell-associated immune system and can be involved in a plurispecific mucosal protection against pathogens, irrespective of the conventional immune response. PMID:9316998

  3. Pancreatic endoproteases and pancreatic secretory trypsin inhibitor immunoreactivity in human Paneth cells.

    PubMed Central

    Bohe, M; Borgström, A; Lindström, C; Ohlsson, K

    1986-01-01

    Normal and metaplastic gastrointestinal mucosa obtained at surgical resection were studied by light microscopy, using the unlabelled antibody enzyme method for immunohistochemical staining of lysozyme, pancreatic endoproteases, and pancreatic secretory trypsin inhibitor (PSTI). Paneth cells in the mucosa of normal small intestine, gastric mucosa with intestinal metaplasia, and colonic metaplastic mucosa were found to contain anionic trypsin, cationic trypsin, lysozyme, and PSTI immunoreactivity, but not chymotrypsin and elastase immunoreactivity. Normal gastric and colonic mucosa and some goblet cells in the small intestine showed positive PSTI immunoreactivity but no endoprotease immunoreactivity. The presence of immunoreactive trypsin and immunoreactive PSTI in the Paneth cells, which are of secretory type, probably indicates an important extrapancreatic source of these proteins rather than a storage of endocytosed material. Images PMID:3525612

  4. Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

    PubMed

    Love, Kerry Routenberg; Politano, Timothy J; Panagiotou, Vasiliki; Jiang, Bo; Stadheim, Terrance A; Love, J Christopher

    2012-01-01

    Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

  5. Development and validation of a UHPLC-UV method for the determination of a prostate secretory protein 94-derived synthetic peptide (PCK3145) in human plasma and assessment of its stability in human plasma.

    PubMed

    El Mubarak, Mohamed A; Leontari, Iliana; Danika, Charikleia; Katsila, Theodora; Sivolapenko, Gregory

    2016-09-01

    PCK3145 is a synthetic peptide, derived from the Prostate Secreted Protein 94 (PSP94), with promising in vitro and animal in vivo results in prostate cancer. The aim of the present study was to develop and validate a fast and robust ultra-high-performance liquid chromatography with ultraviolet detection for the determination of PCK3145 in human plasma which would be suitable for the assessment of PCK3145 stability to proteolytic degradation. Following protein precipitation, chromatographic separation was carried out on an Aeris Peptide C18 column with mobile phase consisting of acetonitrile-water at a flow-rate of 0.50 mL/min. The calibration curve was linear over the range 0.50-20.00 μg/mL. Intra- and inter-day percentage relative standard deviation and relative error were ≤10%. The limit of detection and the lower limit of quantification were 0.15 and 0.50 μg/mL, respectively. Recovery of PCK3145 from human plasma was ≥96%. The peptide presented high stability in whole blood and in human plasma (>98% intact peptide after 24 h incubation at 37°C in human plasma), which represents a distinctive advantage in the therapeutic use of the compound. This is the first validated UHPLC method for the determination of PCK3145 reported, and it was successfully applied in the study of the proteolytic stability of PCK3145 in human plasma ex vivo. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Ca-dependent Nonsecretory Vesicle Fusion in a Secretory Cell

    PubMed Central

    Wang, Tzu-Ming; Hilgemann, Donald W.

    2008-01-01

    We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 μM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P2, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII−/− mouse embryonic fibroblasts (MEFs), as well as in PLCδ1, PLC δ1/δ4, and PLCγ1−/− MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (KD ∼71 μM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP

  7. Scanning electron microscopy of the endometrium during the secretory phase.

    PubMed Central

    Motta, P M; Andrews, P M

    1976-01-01

    Scanning electron microscopy was used to study the surface morphology of the rabbit endometrium during the secretory phase of the oestrous cycle. The free surfaces of ciliated and of inactive active secretory cells are described. Changes in secretory cell surface morphology resulting from accumulation and secretion of material involve the apparent retraction of microvilli and the formation of one or more bulbous protrusions of the cell's apical surface. These protrusions may be relatively smooth surfaced or exhibit long slender micro-extensions. The protrusions grow in size and are eventually pinched off. Loss of the bulbous protrusions often leaves behind crater-like invaginations of the cell's surface. Secretory cells adjacent to the endometrial glands are the first to exhibit signs of mucin accumulation and secretion. The single cilium of a secretory cell is not apparently affected by the secretory process. Signs of ciliated and secretory cell degeneration, and possible sloughing, are also described. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:1033932

  8. Ultrastructural features of the early secretory pathway in Trichoderma reesei.

    PubMed

    Nykänen, Marko; Birch, Debra; Peterson, Robyn; Yu, Hong; Kautto, Liisa; Gryshyna, Anna; Te'o, Junior; Nevalainen, Helena

    2016-05-01

    We have systematically analysed the ultrastructure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase-overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24 h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24 h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls; also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFP fusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.

  9. Identification of CTLA2A, DEFB29, WFDC15B, SERPINA1F and MUP19 as Novel Tissue-Specific Secretory Factors in Mouse

    PubMed Central

    Zhang, Jibin; Ahn, Jinsoo; Suh, Yeunsu; Hwang, Seongsoo; Davis, Michael E.; Lee, Kichoon

    2015-01-01

    Secretory factors in animals play an important role in communication between different cells, tissues and organs. Especially, the secretory factors with specific expression in one tissue may reflect important functions and unique status of that tissue in an organism. In this study, we identified potential tissue-specific secretory factors in the fat, muscle, heart, lung, kidney and liver in the mouse by analyzing microarray data from NCBI’s Gene Expression Omnibus (GEO) public repository and searching and predicting their subcellular location in GeneCards and WoLF PSORT, and then confirmed tissue-specific expression of the genes using semi-quantitative PCR reactions. With this approach, we confirmed 11 lung, 7 liver, 2 heart, 1 heart and muscle, 7 kidney and 2 adipose and liver-specific secretory factors. Among these genes, 1 lung-specific gene - CTLA2A (cytotoxic T lymphocyte-associated protein 2 alpha), 3 kidney-specific genes - SERPINA1F (serpin peptidase inhibitor, Clade A, member 1F), WFDC15B (WAP four-disulfide core domain 15B) and DEFB29 (defensin beta 29) and 1 liver-specific gene - MUP19 (major urinary protein 19) have not been reported as secretory factors. These genes were tagged with hemagglutinin at the 3’end and then transiently transfected to HEK293 cells. Through protein detection in cell lysate and media using Western blotting, we verified secretion of the 5 genes and predicted the potential pathways in which they may participate in the specific tissue through data analysis of GEO profiles. In addition, alternative splicing was detected in transcripts of CTLA2A and SERPINA1F and the corresponding proteins were found not to be secreted in cell culture media. Identification of novel secretory factors through the current study provides a new platform to explore novel secretory factors and a general direction for further study of these genes in the future. PMID:25946105

  10. [Clinicopathologic features of mammary analogue secretory carcinoma of salivary glands].

    PubMed

    Zhang, X P; Ni, H; Wang, X; Chen, H; Shi, S S; Yu, B; Zhou, X J; Rao, Q

    2017-01-08

    Objective: To investigate the clinicopathological features of mammary analogue secretory carcinoma (MASC) of salivary glands, and its diagnosis, differential diagnosis, immunohistochemistry and molecular pathology. Methods: Seventeen cases of MASC were enrolled, with 9 cases of salivary acinar cell carcinoma and 18 cases of adenoid cystic carcinoma as control groups from Nanjing General Hospital from 1997 to 2014 were included in this retrospective study, combined with immunohistochemistry and molecular detection of ETV6-NTRK3 gene fusion. All cases were histologically reviewed with immunohistochemical staining (EnVision) for S-100 protein, SOX10, GATA3, CD117 expression in each group. Fluorescence in situ hybridization (FISH) was used to detect the ETV6-NTRK3 gene fusion. Results: The age of MASC patients ranged from 27 to 74 years with mean age of 47 and ratio of male and female was 4∶3. All cases showed infiltrative growth and diverse cytology and histology, including lobular (8 cases), cystic papillary (3 cases), cribriform mixed with papillary and glandular structures (6 cases) at various proportions. Some tumors of MASC also exhibited solid growth areas with occasional microcystic honeycombed pattern composed of small cysts merged into larger cysts resembling thyroid follicles. S-100 protein and SOX10 were strongly positive in all MASC cases (17/17). In addition, there was insignificant positivity for GATA3 (3/17) and CD117 (4/17). ETV6 gene fusion detection was informative in 12 MASC cases by FISH with 10 positive cases and 2 negative cases. Conclusions: Combined immunohistochemical positivity of S-100 protein, CD117 and SOX10 are useful in the diagnosis and differential diagnosis of MASC. FISH detection of ETV6-NTRK3 fusion offers an additional molecular diagnostic marker for the diagnosis.

  11. Hepatitis C Virus Is Released via a Noncanonical Secretory Route.

    PubMed

    Bayer, Karen; Banning, Carina; Bruss, Volker; Wiltzer-Bach, Linda; Schindler, Michael

    2016-12-01

    We analyzed hepatitis C virus (HCV) morphogenesis using viral genomes encoding a mCherry-tagged E1 glycoprotein. HCV-E1-mCherry polyprotein expression, intracellular localization, and replication kinetics were comparable to those of untagged HCV, and E1-mCherry-tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and nonstructural NS5A followed a temporospatial pattern with a succinct decrease in the number of replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core, and E2 increased at E1-mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating an absence of viral glycoprotein processing by the Golgi apparatus. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggest that assembled HCV particles are released via a noncanonical secretory route involving the endosomal compartment.

  12. Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA

    PubMed Central

    Nakanishi, Katsuhiro; Morikane, Shota; Ichikawa, Shiori; Kurohane, Kohta; Niwa, Yasuo; Akimoto, Yoshihiro; Matsubara, Sachie; Kawakami, Hayato; Kobayashi, Hirokazu; Imai, Yasuyuki

    2017-01-01

    Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection. PMID:28368034

  13. Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA.

    PubMed

    Nakanishi, Katsuhiro; Morikane, Shota; Ichikawa, Shiori; Kurohane, Kohta; Niwa, Yasuo; Akimoto, Yoshihiro; Matsubara, Sachie; Kawakami, Hayato; Kobayashi, Hirokazu; Imai, Yasuyuki

    2017-04-03

    Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection.

  14. Augmentation of arginase Ⅱ expression in the human endometrial epithelium in the secretory phase.

    PubMed

    Tajima, Makiko; Harada, Tatsuya; Ishikawa, Tomonori; Iwahara, Yuki; Kubota, Toshiro

    2012-12-03

    L-arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts L-arginine to urea and L-ornithine. L-Ornithine is the principal precursor for the production of polyamines and L-proline, which are required for cell proliferation and collagen synthesis. Endothelial NOS is expressed in the human endometrial glandular epithelium, but the expression and physiological roles of arginase in the human endometrium are not clear. The objective of this study was to investigate the expression and distribution patterns of arginases Ⅰ (A-Ⅰ) and Ⅱ (A-Ⅱ) in the human endometrium by using immunohistochemistry, reverse transcription-polymerase chain reaction (RTPCR), and western blotting. A-Ⅰ and A-Ⅱ were detected by immunohistochemistry in human endometrial epithelial cells during the proliferative and secretory phases of the menstrual cycle. RT-PCR showed that A-Ⅰ and A-Ⅱ mRNA were expressed in human endometrial tissue. Western blotting analysis results showed the expression of A-Ⅱ protein. Immunohistochemistry and western blotting results showed that expression levels of A-Ⅱ were significantly higher in the secretory phase than in the proliferative phase. Increased A-Ⅱ levels in the secretory phase may be responsible for endometrial growth by increasing polyamines and proline products.

  15. A pediatric case of mammary analogue secretory carcinoma within the parotid.

    PubMed

    Quattlebaum, S Craig; Roby, Brianne; Dishop, Megan K; Said, M Sherif; Chan, Kenny

    2015-01-01

    Mammary analogue secretory carcinoma (MASC) is a recently described entity in the differential diagnosis of salivary gland tumors. It is notable for a characteristic t(12;15)(p13;q25) translocation that results in a unique fusion protein, ETV6-NTRK3. While several studies have retrospectively identified this translocation in cases previously diagnosed as a different salivary malignancy, there have been relatively few cases where this translocation was identified on initial pathology results, and fewer still in a pediatric population. We present a case of a 15 year old female with a slowly enlarging, painless, left facial mass. MRI demonstrated a cystic mass extending into the deep lobe of the parotid, and she underwent parotidectomy. The tumor cells stained positive for S100 and CK19. ETV6 translocation was present, confirming the diagnosis. Mammary analogue secretory carcinoma is a recently described tumor of the salivary glands, which often masquerades as more common primary salivary gland tumors and cysts. More research is needed to characterize the typical behavior of this neoplasm and the optimal treatment regimen. With identification of its characteristic translocation, mammary analogue secretory carcinoma can be easily differentiated from its more prevalent counterparts, and should therefore remain within the differential of the pathologist and head and neck surgeon.

  16. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis.

    PubMed

    Dettmer, Jan; Hong-Hermesdorf, Anne; Stierhof, York-Dieter; Schumacher, Karin

    2006-03-01

    In eukaryotic cells, compartments of the highly dynamic endomembrane system are acidified to varying degrees by the activity of vacuolar H(+)-ATPases (V-ATPases). In the Arabidopsis thaliana genome, most V-ATPase subunits are encoded by small gene families, thus offering potential for a multitude of enzyme complexes with different kinetic properties and localizations. We have determined the subcellular localization of the three Arabidopsis isoforms of the membrane-integral V-ATPase subunit VHA-a. Colocalization experiments as well as immunogold labeling showed that VHA-a1 is preferentially found in the trans-Golgi network (TGN), the main sorting compartment of the secretory pathway. Uptake experiments with the endocytic tracer FM4-64 revealed rapid colocalization with VHA-a1, indicating that the TGN may act as an early endosomal compartment. Concanamycin A, a specific V-ATPase inhibitor, blocks the endocytic transport of FM4-64 to the tonoplast, causes the accumulation of FM4-64 together with newly synthesized plasma membrane proteins, and interferes with the formation of brefeldin A compartments. Furthermore, nascent cell plates are rapidly stained by FM4-64, indicating that endocytosed material is redirected into the secretory flow after reaching the TGN. Together, our results suggest the convergence of the early endocytic and secretory trafficking pathways in the TGN.

  17. [Serum and secretory immunoglobulins in allergic diseases].

    PubMed

    Atovmian, O I; German, G P; Chernokhvostova, E V

    1985-07-01

    A total of 158 patients with pollinosis, bronchial asthma, urticaria and Quincke's edema were examined. The immunoglobulin and C3 levels in sera and the immunoglobulin and albumin levels in saliva were determined by the method of single radial immunodiffusion with the corresponding monospecific antisera. In all the groups of patients subjected to examination the presence of polyclonal hypergammaglobulinemia was detected, which was manifested by a rise in the levels of IgG, IgA and especially IgM; the level of IgD was low. A decrease in the level of C3 was detected in pollinosis patients in the absence of the exacerbation of the disease. No circulating immune complexes were detected. An essential increase in the level of IgG in saliva was revealed, which was due to the local synthesis of this immunoglobulin. In winter the level of salivary IgA in pollinosis patients was found to be essentially below normal, but at the period of exacerbation it increased twofold, probably in response to local stimulation with antigen-allergen. Patients with bronchial asthma and pollinosis were found to have a high level of free secretory component (SC); in pollinosis the level of free SC sharply increased during the stage of exacerbation, which was due to the increase of its synthesis and secretion by the epithelial cells of the mucous membranes. The importance of these data for the pathogenesis of allergic diseases are discussed.

  18. Mammary analogue secretory carcinoma mimicking salivary adenoma.

    PubMed

    Williams, Lindsay; Chiosea, Simion I

    2013-12-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor characterized by ETV6 translocation. It appears that prior studies have identified MASC by reviewing salivary gland carcinomas, such as acinic cell carcinoma and adenocarcinoma, not otherwise specified. To address the possibility of MASC mimicking benign salivary neoplasms we reviewed 12 salivary gland (cyst)adenomas diagnosed prior to the discovery of MASC. One encapsulated (cyst)adenoma of the parotid gland demonstrated features of MASC. The diagnosis was confirmed by fluorescence in situ hybridization with an ETV6 break-apart probe. An unusual complex pattern of ETV6 rearrangement with duplication of the telomeric/distal ETV6 probe was identified. This case illustrates that MASC may mimic salivary (cyst)adenomas. To more accurately assess true clinical and morphologic spectrum of MASC, future studies may have to include review of salivary (cyst)adenomas. The differential diagnosis of MASC may have to be expanded to include cases resembling salivary (cyst)adenomas.

  19. Identification of the Moving Junction Complex of Toxoplasma gondii: A Collaboration between Distinct Secretory Organelles

    PubMed Central

    2005-01-01

    Apicomplexan parasites, including Toxoplasma gondii and Plasmodium sp., are obligate intracellular protozoa. They enter into a host cell by attaching to and then creating an invagination in the host cell plasma membrane. Contact between parasite and host plasma membranes occurs in the form of a ring-shaped moving junction that begins at the anterior end of the parasite and then migrates posteriorly. The resulting invagination of host plasma membrane creates a parasitophorous vacuole that completely envelops the now intracellular parasite. At the start of this process, apical membrane antigen 1 (AMA1) is released onto the parasite surface from specialized secretory organelles called micronemes. The T. gondii version of this protein, TgAMA1, has been shown to be essential for invasion but its exact role has not previously been determined. We identify here a trio of proteins that associate with TgAMA1, at least one of which associates with TgAMA1 at the moving junction. Surprisingly, these new proteins derive not from micronemes, but from the anterior secretory organelles known as rhoptries and specifically, for at least two, from the neck portion of these club-shaped structures. Homologues for these AMA1-associated proteins are found throughout the Apicomplexa strongly suggesting that this moving junction apparatus is a conserved feature of this important class of parasites. Differences between the contributing proteins in different species may, in part, be the result of selective pressure from the different niches occupied by these parasites. PMID:16244709

  20. Identification of a Chromogranin A Domain That Mediates Binding to Secretogranin III and Targeting to Secretory Granules in Pituitary Cells and Pancreatic β-Cells

    PubMed Central

    Hosaka, Masahiro; Watanabe, Tsuyoshi; Sakai, Yuko; Uchiyama, Yasuo; Takeuchi, Toshiyuki

    2002-01-01

    Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca2+-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48–111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214–373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells. PMID:12388744

  1. Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production.

    PubMed

    Chaiyadet, Sujittra; Smout, Michael; Johnson, Michael; Whitchurch, Cynthia; Turnbull, Lynne; Kaewkes, Sasithorn; Sotillo, Javier; Loukas, Alex; Sripa, Banchob

    2015-10-01

    Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south

  2. Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

    PubMed Central

    Chiang, Lilian; Karvar, Serhan; Hamm-Alvarez, Sarah F.

    2012-01-01

    This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the “nascent vesicle site,” from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules. PMID:22363735

  3. CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Robichaud, Patrick; Voorhees, John J; Fisher, Gary J

    2011-08-01

    Dermal connective tissue collagen is the major structural protein in skin. Fibroblasts within the dermis are largely responsible for collagen production and turnover. We have previously reported that dermal fibroblasts, in aged human skin in vivo, express elevated levels of CCN1, and that CCN1 negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation (Quan et al. Am J Pathol 169:482-90, 2006, J Invest Dermatol 130:1697-706, 2010). In further investigations of CCN1 actions, we find that CCN1 alters collagen homeostasis by promoting expression of specific secreted proteins, which include matrix metalloproteinases and proinflammatory cytokines. We also find that CCN1-induced secretory proteins are elevated in aged human skin in vivo. We propose that CCN1 induces an "Age-Associated Secretory Phenotype", in dermal fibroblasts, which mediates collagen reduction and fragmentation in aged human skin.

  4. Fetal Exposure of Rhesus Macaques to Bisphenol A Alters Cellular Development of the Conducting Airway by Changing Epithelial Secretory Product Expression

    PubMed Central

    Murphy, Shannon R.; Boetticher, Miriam V.; VandeVoort, Catherine A.

    2013-01-01

    Background: Bisphenol A (BPA) exposure early in life results in organizational changes in reproductive organs, but the effect of BPA on conducting airway cellular maturation has not been studied. Late gestation is characterized by active differentiation of secretory cells in the lung epithelium. Objective: We evaluated the hypothesis that BPA exposure disrupts epithelial secretory cell development in the fetal conducting airway of the rhesus macaque. Methods: We exposed animals to BPA during either the second (early term) or the third (late term) trimester. There were four treatment groups: a) sham control early term, b) sham control late term, c) BPA early term (BPA-early), and d) BPA late term (BPA-late). Because cellular maturation occurs nonuniformly in the lung, we defined mRNA and protein expression by airway level using microdissection. Results: BPA exposure of the dam during late term significantly accelerated secretory cell maturation in the proximal airways of the fetus; both Clara cell secretory protein (CCSP) and MUC5AC/5B mRNA and protein expression increased. Conclusions: BPA exposure during late gestation accelerates secretory cell maturation in the proximal conducting airways. We identified a critical window of fetal susceptibility for BPA effects on lung epithelial cell maturation in the third trimester. This is of environmental health importance because increases in airway mucins are hallmarks of a number of childhood lung diseases that may be affected by BPA exposure. PMID:23757601

  5. A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes.

    PubMed

    Moreno, Alfredo; SantoDomingo, Jaime; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Alvarez, Javier

    2010-12-01

    Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH(3) cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide (GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques.

  6. Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants.

    PubMed

    Avaro, Sandrine; Belgareh-Touzé, Naïma; Sibella-Argüelles, Carla; Volland, Christiane; Haguenauer-Tsapis, Rosine

    2002-03-15

    We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble alpha-factor, the periplasmic glycoprotein invertase, the plasma membrane GPI-anchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases.

  7. Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense.

    PubMed

    Crottet, P; Corthésy, B

    1998-11-15

    Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

  8. The Nonplanar Secretory IgA2 and Near Planar Secretory IgA1 Solution Structures Rationalize Their Different Mucosal Immune Responses*S⃞

    PubMed Central

    Bonner, Alexandra; Almogren, Adel; Furtado, Patricia B.; Kerr, Michael A.; Perkins, Stephen J.

    2009-01-01

    Secretory IgA (SIgA) is the most prevalent human antibody and is central to mucosal immunity. It exists as two subclasses, SIgA1 and SIgA2, where SIgA2 has a shorter hinge joining the Fab and Fc regions. Both forms of SIgA are predominantly dimeric and contain an additional protein called the secretory component (SC) that is attached during the secretory process and is believed to protect SIgA in harsh mucosal conditions. Here we locate the five SC domains relative to dimeric IgA2 within SIgA2 using constrained scattering modeling. The x-ray and sedimentation parameters showed that SIgA2 has an extended solution structure. The constrained modeling of SIgA2 was initiated using two IgA2 monomers that were positioned according to our best fit solution structure for dimeric IgA1. SC was best located along the convex edge of the Fc-Fc region. The best fit models showed that SIgA2 is significantly nonplanar in its structure, in distinction to our previous near planar SIgA1 structure. Both the shorter IgA2 hinges and the presence of SC appear to displace the four Fab regions out of the Fc plane in SIgA2. This may explain the noncovalent binding of SC in some SIgA2 molecules. This nonplanar structure is predicted to result in specific immune properties for SIgA2 and SIgA1. It may explain differences observed between the SIgA1 and SIgA2 subclasses in terms of their interactions with antigens, susceptibility to proteases, effects on receptors, and distribution in different tissues. The different structures account for the prevalence of both forms in mucosal secretions. PMID:19109255

  9. Axons provide the secretory machinery for trafficking of voltage-gated sodium channels in peripheral nerve

    PubMed Central

    González, Carolina; Cánovas, José; Fresno, Javiera; Couve, Eduardo; Court, Felipe A.; Couve, Andrés

    2016-01-01

    The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and are mostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodium channels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retention-release methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons. PMID:26839409

  10. Mammary analogue secretory carcinoma of salivary gland origin: an update and expanded morphologic and immunohistochemical spectrum of recently described entity.

    PubMed

    Skalova, Alena

    2013-07-01

    Mammary analogue secretory carcinoma of salivary gland origin (MASC) is a recently described tumor with ETV6 translocation. Akin to secretory breast cancer, MASC expresses S-100 protein, mammaglobin, vimentin, and harbors a t(12;15) (p13;q25) translocation which leads to ETV6-NTRK3 fusion product. Histologically, MASC displays a lobulated growth pattern and is often composed of microcystic, tubular, and solid structures with abundant eosinophilic homogeneous or bubbly secretions. Colloid-like secretory material stains positive for periodic acid-Schiff (PAS) with and without diastase and for Alcian blue. The cells of MASC are devoid of PAS-positive secretory zymogen granules. These features help to exclude the most important differential diagnostic considerations, namely acinic cell carcinoma, low-grade cribriform cystadenocarcinoma, cystadenocarcinoma (not otherwise specified), and low-grade mucoepidermoid carcinoma. To date the presence of the ETV6-NTRK3 fusion gene has not been demonstrated in any other salivary gland tumor than MASC. It is likely that MASC is more common than currently recognized and with further studies, the clinical need for molecular studies of the ETV6-NTRK3 fusion may diminish. However, molecular testing is recommended at this time to arrive at the diagnosis of MASC.

  11. Dynamic Regulation of Ero1α and Peroxiredoxin 4 Localization in the Secretory Pathway*

    PubMed Central

    Kakihana, Taichi; Araki, Kazutaka; Vavassori, Stefano; Iemura, Shun-ichiro; Cortini, Margherita; Fagioli, Claudio; Natsume, Tohru; Sitia, Roberto; Nagata, Kazuhiro

    2013-01-01

    In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis. PMID:23979138

  12. Dynamic regulation of Ero1α and peroxiredoxin 4 localization in the secretory pathway.

    PubMed

    Kakihana, Taichi; Araki, Kazutaka; Vavassori, Stefano; Iemura, Shun-ichiro; Cortini, Margherita; Fagioli, Claudio; Natsume, Tohru; Sitia, Roberto; Nagata, Kazuhiro

    2013-10-11

    In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.

  13. Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast.

    PubMed

    Gurunathan, Sangiliyandi; David, Doris; Gerst, Jeffrey E

    2002-02-15

    Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis.

  14. BACE2 is stored in secretory granules of mouse and rat pancreatic beta cells.

    PubMed

    Finzi, Giovanna; Franzi, Francesca; Placidi, Claudia; Acquati, Francesco; Palumbo, Elisa; Russo, Antonella; Taramelli, Roberto; Sessa, Fausto; La Rosa, Stefano

    2008-01-01

    BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of beta cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of beta cells suggests that it may play a role in diabetes-associated amyloidogenesis.

  15. Flow cytometry-assisted purification and proteomic analysis of the corticotropes dense-core secretory granules.

    PubMed

    Gauthier, Daniel J; Sobota, Jacqueline A; Ferraro, Francesco; Mains, Richard E; Lazure, Claude

    2008-09-01

    The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density-based gradient and/or immunoaffinity purification steps followed by extraction, 1-DE or 2-DE, gel staining, in-gel tryptic digestion, and protein identification by MS. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel-free procedure involves fluorescence-assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and MS. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through MS.

  16. Identification and Characterization of an Escorter for Two Secretory Adhesins in Toxoplasma gondii

    PubMed Central

    Reiss, Matthias; Viebig, Nicola; Brecht, Susan; Fourmaux, Marie-Noelle; Soete, Martine; Di Cristina, Manlio; Dubremetz, Jean François; Soldati, Dominique

    2001-01-01

    The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite. PMID:11157983

  17. Sphingomyelin is sorted at the trans Golgi network into a distinct class of secretory vesicle

    PubMed Central

    Deng, Yongqiang; Rivera-Molina, Felix E.; Toomre, Derek K.; Burd, Christopher G.

    2016-01-01

    One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers. PMID:27247384

  18. Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition

    PubMed Central

    Stoffel, Wilhelm; Hammels, Ina; Jenke, Bitta; Binczek, Erika; Schmidt-Soltau, Inga; Brodesser, Susanne; Schauss, Astrid; Etich, Julia; Heilig, Juliane; Zaucke, Frank

    2016-01-01

    Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus–pituitary–somatotropic hypoplasia. The unbiased smpd3−/− mouse mutant and derived smpd3−/− primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3−/− mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition. PMID:27882938

  19. Neuronal porosome - The secretory portal at the nerve terminal: Its structure-function, composition, and reconstitution

    NASA Astrophysics Data System (ADS)

    Jena, Bhanu P.

    2014-09-01

    Cup-shaped secretory portals at the cell plasma membrane called porosomes mediate secretion from cells. Membrane bound secretory vesicles transiently dock and fuse at the cytosolic compartment of the porosome base to expel intravesicular contents to the outside during cell secretion. In the past decade, the structure, isolation, composition, and functional reconstitution of the neuronal porosome complex has been accomplished providing a molecular understanding of its structure-function. Neuronal porosomes are 15 nm cup-shaped lipoprotein structures composed of nearly 40 proteins; compared to the 120 nm nuclear pore complex comprised of over 500 protein molecules composed of 30 different proteins. Being a membrane-associated supramolecular complex has precluded determination of the atomic structure of the porosome. However recent studies using small-angle X-ray solution scattering (SAXS), provide at sub-nanometer resolution, the native 3D structure of the neuronal porosome complex associated with docked synaptic vesicle at the nerve terminal. Additionally, results from the SAXS study and earlier studies using atomic force microscopy, provide the possible molecular mechanism involved in porosome-mediated neurotransmitter release at the nerve terminal.

  20. Short-term cholinergic desensitization of rat pancreatic secretory response

    SciTech Connect

    Asselin, J.; Larose, L.; Morisset, J.

    1987-03-01

    Dispersed pancreatic acini were first exposed to carbamylcholine (10/sup -7/-10/sup -4/ M) for 60 min, washed, and reexposed to this same agonist (10/sup -8/-10/sup -3/ M) for 15 min. During this second incubation, the functional secretory capacity of these acini was evaluated by measuring amylase release. Acini preexposed to concentrations of carbamylcholine of 10/sup -6/ M or greater showed shifts to the right in the subsequent carbamylcholine dose-response curves of amylase release. A 3-h recovery period (without carbamylcholine) did not restore the altered carbamylcholine dose-response curve. Ca/sup 2 +/ concentrations of 10/sup -7/ M or 2.5 x 10/sup -3/ M instead of 0.5 x 10/sup -3/ M during the 60-min preincubation did not affect the desensitization process. With use of N-(/sup 3/H)methylscopolamine to evaluate muscarinic receptors, the only changes observed after desensitization were a significant decrease in the high-affinity and an equivalent increase in that of the low-affinity receptors. After cholinergic exposure amylase release stimulated by caerulein was only slightly modified, whereas amylase release in response to a phorbol ester 12-O-tetradecanoylphorbol-13-acetate and to the ionophore A23187 was not altered. These data indicate that short-term desensitization with a cholinergic agent is relatively specific to muscarinic agonists, causes changes in the muscarinic receptor high-and low-affinity concentration but does not alter intracellular steps after calcium mobilization or protein kinase C activation known to be involved in the secretion process.

  1. Large-scale identification of endogenous secretory peptides using electron transfer dissociation mass spectrometry.

    PubMed

    Sasaki, Kazuki; Osaki, Tsukasa; Minamino, Naoto

    2013-03-01

    Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

  2. Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry*

    PubMed Central

    Sasaki, Kazuki; Osaki, Tsukasa; Minamino, Naoto

    2013-01-01

    Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554–577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics. PMID:23250050

  3. [Effect of plant hormones on the components of secretory pathway in human normal and tumor cells].

    PubMed

    Vil'danova, M S; Savitskaia, M A; Onishchenko, G E; Smirnova, E A

    2014-01-01

    Plant hormones play a key role in plant growth and differentiation. Many hormones are known as potential antitumor agents, yet others appear to affect the secretory activity and are produced by mammalian cells as pro-inflammatory cytokines. The goal of this research was to study the effect of abscisic and gibberellic acids on the secretory system of human cultured epidermoid carcinoma cells A431 and keratinocytes HaCat. Immunocytochemical and morphometric analysis demonstrated that subtoxic concentration of plant hormones induced the broadening of the ER network and increased the size of Golgi complex. Electron microscopy studies confirmed the hypertrophic changes of the Golgi apparatus, specifically, the swelling of cisternae in the trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartment of dictyosomes after exposure to abscisic acid, and swelling of cis- and trans-compartments of dictyosomes after exposure to gibberellic acid. Using of Click-iT technique allowed to detect the elevation of the total protein synthesis only in A431 cells exposed to abscisic acid. Cumulative data suggests that, under these conditions, the hypertrophy of Golgi apparatus may reflect the enhanced secretory activity of cells. In other experiments, the hypertrophy of Golgi is not related to increased protein synthesis and therefore may suggest the stress-related changes of ER and Golgi apparatus. Our results demonstrate that morphologically similar reaction of cellular organelles, such as hypertrophy of Golgi apparatus, is the result of different functional activities, and that molecular mechanisms underlying the changes induced in cells need further investigations.

  4. Ultrastructural patterns of secretory activity in poison cutaneous glands of larval and juvenile Dendrobates auratus (Amphibia, Anura).

    PubMed

    Angel, R; Delfino, G; Parra, G J

    2003-01-01

    A transmission electron-microscope study has been performed on larval and juvenile skin of the Central American arrow-frog Dendrobates auratus to investigate early secretory processes and maturational changes in the serous (poison) glands. Poison biosynthesis involves the endoplasmic reticulum (both smooth and rough types), as well as Golgi stacks which release early serous product as secretory vesicles (or pre-granules). These vesicles contain fine-grained material, along with single electron-opaque bodies, spheroidal in shape, that accompany the grained product throughout its post-Gogian, maturational change. The first steps of this process involve condensation and lead to the formation of secretory granules with a glomerular-like substructure, resulting from a thick, random aggregation of rods (secretory granule subunits). Advanced maturational activity causes the loss of peculiar granule substructure: the dense bodies split into fragments, whereas the thick glomerular arrangement becomes looser, until the secretory product changes into a dispersed material. This ultrastructural study revealed biosynthesis and maturation processes in close sequence, suggesting the poison of D. auratus contains proteins and/or peptides as well as lipophilic compounds. Molecules of both these classes are known to perform several roles relevant to survival strategies in extant anurans. Furthermore, the ephemeral granules with a glomerular-like substructure detected in tadpoles and froglets exhibit the complex patterns of mature poisons in adult specimens of other anurans: Hylidae and related families. This agrees with current trends in the taxonomy of these advanced frogs and underlines the pertinence of an ontogenetic approach in investigating anuran phylogenesis.

  5. Targeting vaccinia virus-expressed secretory beta subunit of human chorionic gonadotropin to the cell surface induces antibodies.

    PubMed Central

    Srinivasan, J; Singh, O; Chakrabarti, S; Talwar, G P

    1995-01-01

    We carried out experiments designed to study the effect of a protein's localization on its immunogenicity. A novel cell-surface protein was generated from a small, glycosylated secretory protein. The DNA sequence encoding the entire precursor of the human chorionic gonadotropin beta (beta hCG) subunit was fused in the correct reading frame to the DNA sequence encoding the transmembrane and cytoplasmic domains of vesicular stomatitis virus glycoprotein. This chimeric gene was introduced into the vaccinia virus genome to generate a recombinant virus. The recombinant virus, when used to infect animal cells, expressed a 135-amino-acid beta hCG subunit anchored in cellular membranes by the 48 carboxy-terminal amino acids of vesicular stomatitis virus glycoprotein. The immunogenicity of this recombinant virus with respect to its ability to generate anti-hCG antibodies was compared with that of a second recombinant vaccinia virus expressing the native secretory form of beta hCG. All animals immunized with the vaccinia virus expressing beta hCG on the cell surface elicited high titers of anti-hCG antibodies. Even after a single immunization with the recombinant vaccinia virus, the anti-hCG antibody titers persisted for a long period of time (more than 6 months). None of the animals immunized with vaccinia virus expressing the native secretory form of beta hCG showed any hCG-specific antibody response. PMID:7591154

  6. Characterization of excretory-secretory antigens of adult Toxocara canis by western blotting.

    PubMed

    Sudhakar, N R; Samanta, S; Sahu, Shivani; Raina, O K; Gupta, S C; Goswami, T K; Lokesh, K M; Kumar, Ashok

    2014-06-01

    Toxocara canis is one of the most common helminth worm of dogs which continues to stimulate both public health concern alongside the higher scientific interest. It may cause visceral and ocular damage in humans especially in children. The identification of specific antigens of T. canis is important so as to develop better diagnostic techniques. Excretory-secretory (ES) antigens were prepared by culturing the adult T. canis worms in RPMI 1640 medium without serum supplementation followed by ammonium sulphate precipitation. These antigens were separated using sodium dodecyl sulphate-electrophoresis (SDS-PAGE). Recovered proteins ranged from 30 to 384 kDa. The specific reactivity of the T. canis excretory-secretory (TC-ES) proteins was checked by western blotting. The immuno-reactivity of the naturally infected dog sera with the TC-ES antigens showed five bands at 43, 57,105, 139 and 175 kDa. The immuno-reactivity of the hyper immune serum raised in rabbits against TC-ES antigens was observed with ten polypeptides of 21, 25, 30, 37, 45, 50, 57, 69, 77 and 105 kDa. Common antigens band were observed at 57 and 105 KDa. These antigens merit further evaluation as candidate for use in diagnosis of toxocariasis in humans and adult dogs.

  7. Engineering Yarrowia lipolytica to express secretory invertase with strong FBA1IN promoter.

    PubMed

    Hong, Seung-Pyo; Seip, John; Walters-Pollak, Dana; Rupert, Ross; Jackson, Raymond; Xue, Zhixiong; Zhu, Quinn

    2012-02-01

    Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipid-derived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1IN promoter (FBA1 sequence of -826 to +169) containing an intron (+64 to +165) showed five-fold higher expression than the FBA1 promoter (-831 to -1). The transcriptional enhancement by the 5'-region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC+ phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro-region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC+ strains, our engineered stains secreted most of invertase into the medium.

  8. Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.

    PubMed

    de Wit, Joris; Toonen, Ruud F; Verhage, Matthijs

    2009-01-07

    Neurons secrete many diffusible signals from synaptic and other secretory vesicles. We characterized secretion of guidance cues, neuropeptides, neurotrophins, and proteases from single secretory vesicles using pHluorin-tagged cargo in cortical neurons. Stimulation triggered transient and persistent fusion events. Transient events represented full release followed by cargo diffusion or incomplete release followed by vesicle retrieval, as previously observed in neuroendocrine cells. Unexpectedly, we also observed that certain cargo, such as Semaphorin 3A (Sema3A), was delivered at the cell surface as stable deposits. Stable deposits and transient events were observed for single cargo and both were SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and calcium dependent. The ratio between stable and transient events did not depend on cargo size, subcellular localization (synaptic vs extrasynaptic secretion), or the presence of the extracellular matrix. Instead, the ratio is cargo specific and depends on an interaction with the vesicle matrix through a basic domain in the cargo protein. Inhibition of this interaction through deletion of the basic domain in Sema3A abolished stable deposits and rendered all events transient. Strikingly, cargo favoring transient release was stably deposited after corelease with cargo favoring stable deposit. These data argue against cargo diffusion after exocytosis as a general principle. Instead, the vesicle matrix retains secreted signals, probably for focal signaling at the cell surface.

  9. Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillus subtilis SCK6

    PubMed Central

    Bashir, Shaista; Sadaf, Saima; Ahmad, Sajjad; Akhtar, Muhammad Waheed

    2015-01-01

    This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer. PMID:26881203

  10. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    PubMed

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils.

  11. Secretory COPII coat component Sec23a is essential for craniofacial chondrocyte maturation.

    PubMed

    Lang, Michael R; Lapierre, Lynne A; Frotscher, Michael; Goldenring, James R; Knapik, Ela W

    2006-10-01

    An increasing number of human disorders have been linked to mutations in genes of the secretory pathway. The chemically induced zebrafish crusher variant results in malformed craniofacial skeleton, kinked pectoral fins and a short body length. By positional cloning, we identified a nonsense mutation converting leucine to a stop codon (L402X) in the sec23a gene, an integral component of the COPII complex, which is critical for anterograde protein trafficking between endoplasmic reticulum and Golgi apparatus. Zebrafish crusher mutants develop normally until the onset of craniofacial chondrogenesis. crusher chondrocytes accumulate proteins in a distended endoplasmic reticulum, resulting in severe reduction of cartilage extracellular matrix (ECM) deposits, including type II collagen. We demonstrate that the paralogous gene sec23b is also an essential component of the ECM secretory pathway in chondrocytes. In contrast, knockdown of the COPI complex does not hinder craniofacial morphogenesis. As SEC23A lesions cause the cranio-lenticulo-sutural dysplasia syndrome, crusher provides the first vertebrate model system that links the biology of endoplasmic reticulum to Golgi trafficking with a clinically relevant dysmorphology.

  12. Secretory vesicle priming by CAPS is independent of its SNARE-binding MUN domain.

    PubMed

    Nguyen Truong, Cuc Quynh; Nestvogel, Dennis; Ratai, Olga; Schirra, Claudia; Stevens, David R; Brose, Nils; Rhee, JeongSeop; Rettig, Jens

    2014-11-06

    Priming of secretory vesicles is a prerequisite for their Ca(2+)-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca(2+)-dependent activator protein for secretion (CAPS) also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

  13. Pasteurella multocida toxin: Targeting mast cell secretory granules during kiss-and-run secretion.

    PubMed

    Danielsen, Elisabeth M; Christiansen, Nina; Danielsen, E Michael

    2016-02-01

    Pasteurella multocida toxin (PMT), a virulence factor of the pathogenic Gram-negative bacterium P. multocida, is a 146 kDa protein belonging to the A-B class of toxins. Once inside a target cell, the A domain deamidates the α-subunit of heterotrimeric G-proteins, thereby activating downstream signaling cascades. However, little is known about how PMT selects and enters its cellular targets. We therefore studied PMT binding and uptake in porcine cultured intestinal mucosal explants to identify susceptible cells in the epithelium and underlying lamina propria. In comparison with Vibrio cholera B-subunit, a well-known enterotoxin taken up by receptor-mediated endocytosis, PMT binding to the epithelial brush border was scarce, and no uptake into enterocytes was detected by 2h, implying that none of the glycolipids in the brush border are a functional receptor for PMT. However, in the lamina propria, PMT distinctly accumulated in the secretory granules of mast cells. This also occurred at 4 °C, ruling out endocytosis, but suggestive of uptake via pores that connect the granules to the cell surface. Mast cell granules are known to secrete their contents by a "kiss-and-run" mechanism, and we propose that PMT may exploit this secretory mechanism to gain entry into this particular cell type.

  14. A Western Blot-based Investigation of the Yeast Secretory Pathway Designed for an Intermediate-Level Undergraduate Cell Biology Laboratory

    ERIC Educational Resources Information Center

    Hood-DeGrenier, Jennifer K.

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in…

  15. Characterisation of a secretory serine protease inhibitor (SjB6) from Schistosoma japonicum

    PubMed Central

    2014-01-01

    Background Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential physiological roles in many organisms. In pathogens, serpins are thought to have evolved specifically to limit host immune responses by interfering with the host immune-stimulatory signals. Serpins are less well characterised in parasitic helminths, although some are thought to be involved in mechanisms associated with host immune modulation. In this study, we cloned and partially characterised a secretory serpin from Schistosoma japonicum termed SjB6, these findings provide the basis for possible functional roles. Methods SjB6 gene was identified through database mining of our previously published microarray data, cloned and detailed sequence and structural analysis and comparative modelling carried out using various bioinformatics and proteomics tools. Gene transcriptional profiling was determined by real-time PCR and the expression of native protein determined by immunoblotting. An immunological profile of the recombinant protein produced in insect cells was determined by ELISA. Results SjB6 contains an open reading frame of 1160 base pairs that encodes a protein of 387 amino acid residues. Detailed sequence analysis, comparative modelling and structural-based alignment revealed that SjB6 contains the essential structural motifs and consensus secondary structures typical of inhibitory serpins. The presence of an N-terminal signal sequence indicated that SjB6 is a secretory protein. Real-time data indicated that SjB6 is expressed exclusively in the intra-mammalian stage of the parasite life cycle with its highest expression levels in the egg stage (p < 0.0001). The native protein is approximately 60 kDa in size and recombinant SjB6 (rSjB6) was recognised strongly by sera from rats experimentally infected with S. japonicum. Conclusions The significantly high expression of SjB6 in schistosome eggs, when compared to other life cycle stages, suggests a possible

  16. Ca(2+) and H+ homeostasis in fission yeast: a role of Ca(2+)/H+ exchange and distinct V-H+-ATPases of the secretory pathway organelles.

    PubMed

    Okorokov, L A; Silva, F E; Okorokova Façanha, A L

    2001-09-14

    We determined the H+ and Ca(2+) uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca(2+) sequestering is due to Ca(2+)/H+ antiporter(s) localized to secretory pathway organelles while Ca(2+)-ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V-H+-ATPases acidify the lumen of the secretory pathway organelles. The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca(2+) and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca(2+) signaling in a differential and local manner.

  17. Protein secretion in Bacillus species.

    PubMed Central

    Simonen, M; Palva, I

    1993-01-01

    Bacilli secrete numerous proteins into the environment. Many of the secretory proteins, their export signals, and their processing steps during secretion have been characterized in detail. In contrast, the molecular mechanisms of protein secretion have been relatively poorly characterized. However, several components of the protein secretion machinery have been identified and cloned recently, which is likely to lead to rapid expansion of the knowledge of the protein secretion mechanism in Bacillus species. Comparison of the presently known export components of Bacillus species with those of Escherichia coli suggests that the mechanism of protein translocation across the cytoplasmic membrane is conserved among gram-negative and gram-positive bacteria differences are found in steps preceding and following the translocation process. Many of the secretory proteins of bacilli are produced industrially, but several problems have been encountered in the production of Bacillus heterologous secretory proteins. In the final section we discuss these problems and point out some possibilities to overcome them. PMID:8464403

  18. Separation of rat pituitary secretory granules by continuous flow electrophoresis

    NASA Technical Reports Server (NTRS)

    Hayes, Daniel; Exton, Carrie; Salada, Thomas; Shellenberger, Kathy; Waddle, Jenny; Hymer, W. C.

    1990-01-01

    The separation of growth hormone-containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.

  19. The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals.

    PubMed

    McGirr, Rebecca; Guizzetti, Leonardo; Dhanvantari, Savita

    2013-05-01

    Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1-6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

  20. Fine structure of the Caenorhabditis elegans secretory-excretory system.

    PubMed

    Nelson, F K; Albert, P S; Riddle, D L

    1983-02-01

    The secretory-excretory system of C. elegans, reconstructed from serial-section electron micrographs of larvae, is composed of four cells, the nuclei of which are located on the ventral side of the pharynx and adjacent intestine. (1) The pore cell encloses the terminal one-third of the excretory duct which leads to an excretory pore at the ventral midline. (2) The duct cell surrounds the excretory duct with a lamellar membrane from the origin of the duct at the excretory sinus to the pore cell boundary. (3) A large H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The excretory sinus within the cell body joins the lumena of the canals with the origin of the duct. (4) A binucleate, A-shaped gland cell extends bilateral processes anteriorly from cell bodies located just behind the pharynx. These processes are fused at the anterior tip of the cell, where the cell enters the circumpharyngeal nerve ring. The processes are also joined at the anterior edge of the excretory cell body, where the excretory cell and gland are joined to the duct cell at the origin of the duct. Secretory granules may be concentrated in the gland near this secretory-excretory junction. Although the gland cells of all growing developmental stages stain positively with paraldehyde-fuchsin, the gland of the dauer larva stage (a developmentally arrested third-stage larva) does not stain, nor do glands of starved worms of other stages. Dauer larvae uniquely lack secretory granules, and the gland cytoplasm is displaced by a labyrinth of large, transparent spaces. Exit from the dauer stage results in the return of active secretory morphology in fourth-stage larvae.

  1. Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

    PubMed Central

    Son, Sung Min; Cha, Moon-Yong; Choi, Heesun; Kang, Seokjo; Choi, Hyunjung; Lee, Myung-Shik; Park, Sun Ah; Mook-Jung, Inhee

    2016-01-01

    ABSTRACT The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology. PMID:26963025

  2. The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway.

    PubMed

    Janßen, Linda; Ramnarayan, Venkat Raman; Aboelmagd, Mohamed; Iliopoulou, Maro; Hein, Zeynep; Majoul, Irina; Fritzsche, Susanne; Halenius, Anne; Springer, Sebastian

    2016-01-01

    In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.

  3. Quantification of pancreatic secretory trypsin inhibitor in colonic carcinoma and normal adjacent colonic mucosa.

    PubMed Central

    Bohe, H; Bohe, M; Jönsson, P; Lindström, C; Ohlsson, K

    1992-01-01

    AIMS: To measure the content of immunoreactive human pancreatic secretory trypsin inhibitor (irPSTI) in colonic carcinoma and adjacent normal colonic mucosa. METHODS: From a stable hybridoma cell line producing monoclonal antibodies specific for human PSTI, a specific enzyme linked immunosorbent assay (ELISA) for human PSTI was developed. In a precipitation assay system these antibodies bound human PSTI in a dose-dependent manner. The specimens were obtained from resectional surgery. RESULTS: The content of irPSTI was 19.9 micrograms/g protein (0.55 micrograms/g tissue wet weight) in colonic carcinoma. In adjacent normal colonic mucosa 43.6 micrograms/g protein (1.12 micrograms/g tissue wet weight) was shown. CONCLUSIONS: The enzymatic degradation of surrounding tissue necessary for tumour cell invasion could be facilitated by this relative deficit of the inhibitor in infiltrative carcinoma. PMID:1479031

  4. Subcellular localization and trafficking of phytolongins (non-SNARE longins) in the plant secretory pathway

    PubMed Central

    de Marcos Lousa, Carine; Soubeyrand, Eric; Bolognese, Paolo; Wattelet-Boyer, Valerie; Bouyssou, Guillaume; Marais, Claireline; Boutté, Yohann; Filippini, Francesco; Moreau, Patrick

    2016-01-01

    SNARE proteins are central elements of the machinery involved in membrane fusion of eukaryotic cells. In animals and plants, SNAREs have diversified to sustain a variety of specific functions. In animals, R-SNARE proteins called brevins have diversified; in contrast, in plants, the R-SNARE proteins named longins have diversified. Recently, a new subfamily of four longins named ‘phytolongins’ (Phyl) was discovered. One intriguing aspect of Phyl proteins is the lack of the typical SNARE motif, which is replaced by another domain termed the ‘Phyl domain’. Phytolongins have a rather ubiquitous tissue expression in Arabidopsis but still await intracellular characterization. In this study, we found that the four phytolongins are distributed along the secretory pathway. While Phyl2.1 and Phyl2.2 are strictly located at the endoplasmic reticulum network, Phyl1.2 associates with the Golgi bodies, and Phyl1.1 locates mainly at the plasma membrane and partially in the Golgi bodies and post-Golgi compartments. Our results show that export of Phyl1.1 from the endoplasmic reticulum depends on the GTPase Sar1, the Sar1 guanine nucleotide exchange factor Sec12, and the SNAREs Sec22 and Memb11. In addition, we have identified the Y48F49 motif as being critical for the exit of Phyl1.1 from the endoplasmic reticulum. Our results provide the first characterization of the subcellular localization of the phytolongins, and we discuss their potential role in regulating the secretory pathway. PMID:26962210

  5. Amyloid formation of growth hormone in presence of zinc: Relevance to its storage in secretory granules

    PubMed Central

    Jacob, Reeba S.; Das, Subhadeep; Ghosh, Saikat; Anoop, Arunagiri; Jha, Narendra Nath; Khan, Tuhin; Singru, Praful; Kumar, Ashutosh; Maji, Samir K.

    2016-01-01

    Amyloids are cross-β-sheet fibrillar aggregates, associated with various human diseases and native functions such as protein/peptide hormone storage inside secretory granules of neuroendocrine cells. In the current study, using amyloid detecting agents, we show that growth hormone (GH) could be stored as amyloid in the pituitary of rat. Moreover, to demonstrate the formation of GH amyloid in vitro, we studied various conditions (solvents, glycosaminoglycans, salts and metal ions) and found that in presence of zinc metal ions (Zn(II)), GH formed short curvy fibrils. The amyloidogenic nature of these fibrils was examined by Thioflavin T binding, Congo Red binding, transmission electron microscopy and X-ray diffraction. Our biophysical studies also suggest that Zn(II) initiates the early oligomerization of GH that eventually facilitates the fibrillation process. Furthermore, using immunofluorescence study of pituitary tissue, we show that GH in pituitary significantly co-localizes with Zn(II), suggesting the probable role of zinc in GH aggregation within secretory granules. We also found that GH amyloid formed in vitro is capable of releasing monomers. The study will help to understand the possible mechanism of GH storage, its regulation and monomer release from the somatotrophs of anterior pituitary. PMID:27004850

  6. Mammary analogue secretory carcinoma: the first submandibular case reported including findings on fine needle aspiration cytology.

    PubMed

    Petersson, Fredrik; Lian, Derrick; Chau, Yuk Ping; Yan, Benedict

    2012-03-01

    We present the first case (male, 35 years old) of a mammary analogue secretory carcinoma occurring in a submandibular gland and document findings on fine needle aspiration cytology. On histology, the tumor displayed characteristic features: circumscribed nodules composed of bland, pink to light red neoplastic cells with low proliferative/mitotic activity arranged in tubular, vaguely cribriform, and microcystic structures containing Periodic acid Schiff-positive, diastase-resistant secretory material. Immunohistochemistry showed strong and diffuse positivity for cytokeratin 7, S100 protein, and vimentin, as well as moderate to strong immunoreactivity for c-kit in the majority of tumor cells. A rearrangement of the ETV6 gene on fluorescence in situ hybridization was documented. The patient underwent an ipsilateral selective (levels I-IV) neck dissection which showed metastasis in 3 out of 36 lymph nodes (levels 1-3). Adjuvant radiotherapy was administered. No local recurrence or metastatic disease has been detected during a follow up period of 28 months.

  7. Multiple effects of the phenylhydrazone derivative FCCP on the secretory pathway in rat plasma cells.

    PubMed

    Antoine, J C; Jouanne, C

    1986-10-01

    We studied the sensitivity of the last steps of the secretory process of antibody-producing cells to carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and sodium azide (NaN3), agents which lower the cellular ATP content by inhibiting oxidative phosphorylation and mitochondrial electron transport, respectively. Popliteal lymph node cells or purified plasma cells from rats immunized against horseradish peroxidase were incubated with the drugs. The rate of secretion of anti-HRP antibodies was measured by an enzyme-linked immunoadsorbent assay or after biosynthetic labeling with L-[3H]fucose. FCCP as well as NaN3 were shown to rapidly inhibit (in less than 5 min) the secretion of immunoglobulins (Ig) and to partially block the release of fucosylated Ig. This indicates that the drugs inhibit the transport of Ig from the Golgi apparatus (GA) (where fucose is added to Ig) to the plasma membrane. However, the degree of inhibition reached 40 to 50% with NaN3 and 70 to 80% with FCCP, whereas both drugs similarly depleted ATP stores by 45 to 55%. These results are consistent with multiple effects of FCCP on the secretion pathway of Ig. As a tentative explanation, we suggest that FCCP, because of its protonophore properties, not only reduces cellular ATP levels but may also neutralize the Golgi or post-Golgi acidic compartments recently shown to be involved in the transport of plasma membrane and secretory proteins.

  8. Proteomics of the human endometrial glandular epithelium and stroma from the proliferative and secretory phases of the menstrual cycle.

    PubMed

    Hood, Brian L; Liu, Baoquan; Alkhas, Addie; Shoji, Yutaka; Challa, Rusheeswar; Wang, Guisong; Ferguson, Susan; Oliver, Julie; Mitchell, Dave; Bateman, Nicholas W; Zahn, Christopher M; Hamilton, Chad A; Payson, Mark; Lessey, Bruce; Fazleabas, Asgerally T; Maxwell, G Larry; Conrads, Thomas P; Risinger, John I

    2015-04-01

    Despite its importance in reproductive biology and women's health, a detailed molecular-level understanding of the human endometrium is lacking. Indeed, no comprehensive studies have been undertaken to elucidate the important protein expression differences between the endometrial glandular epithelium and surrounding stroma during the proliferative and midsecretory phases of the menstrual cycle. We utilized laser microdissection to harvest epithelial cells and stromal compartments from proliferative and secretory premenopausal endometrial tissue and performed a global, quantitative mass spectrometry-based proteomics analysis. This analysis identified 1224 total proteins from epithelial cells, among which 318 were differentially abundant between the proliferative and secretory phases (q < 0.05), and 1005 proteins from the stromal compartments, 19 of which were differentially abundant between the phases (q < 0.05). Several proteins were chosen for validation by immunohistochemistry in an independent set of uterine tissues, including carboxypeptidase M, tenascin C, neprilysin, and ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3). ENPP3, which was elevated in epithelial glandular cells in the secretory phase, was confirmed to be elevated in midsecretory-phase baboon uterine lavage samples and also observed to have an N-linked glycosylated form that was not observed in the proliferative phase. This study provides a detailed view into the global proteomic alterations of the epithelial cells and stromal compartments of the cycling premenopausal endometrium. These proteomic alterations during endometrial remodeling provide a basis for numerous follow-up investigations on the function of these differentially regulated proteins and their role in reproductive biology and endometrial pathologies.

  9. Antigen Detection in Enteropathogenic Escherichia coli Using Secretory Immunoglobulin A Antibodies Isolated from Human Breast Milk

    PubMed Central

    Manjarrez-Hernandez, H. A.; Gavilanes-Parra, S.; Chavez-Berrocal, E.; Navarro-Ocaña, A.; Cravioto, A.

    2000-01-01

    Enteropathogenic Escherichia coli (EPEC) produces a characteristic attaching and effacing (A/E) lesion in the small intestines of infected children. The immune response to EPEC infection remains poorly characterized. The molecular targets that elicit protective immunity against EPEC disease are unknown. In this study protein antigens from EPEC were identified using secretory immunoglobulin A (sIgA) antibodies isolated from milk from Mexican women by Western blot analysis. Purified sIgA antibodies, which inhibit the adherence of EPEC to cells, reacted to many EPEC proteins, the most prominent of which were intimin (a 94-kDa outer membrane protein) and two unknown proteins with apparent molecular masses of 80 and 70 kDa. A culture supernatant protein of 110 kDa also reacted strongly with the sIgA antibodies. The molecular size of this protein and its reactivity with specific anti-EspC antiserum suggest that it is EPEC-secreted protein C (EspC). These EPEC surface protein antigens were consistently recognized by all the different sIgA samples obtained from 15 women. Screening of clinical isolates of various O serogroups from cases of severe infantile diarrhea revealed that all EPEC strains able to produce the A/E lesion showed expression of intimin and the 80- and 70-kDa proteins. Such proteins reacted strongly with the purified sIgA pool. Moreover, nonvirulent E. coli strains were unable to generate a sIgA response. The immunogenic capacities of the 80- and 70-kDa proteins as virulence antigens have not been previously reported. The strong sIgA response to intimin and the 80- and 70-kDa proteins obtained in this study indicates that such antigens stimulate intestinal immune responses and may elicit protective immunity against EPEC disease. PMID:10948121

  10. Free radicals enzymatically triggered by Clonorchis sinensis excretory-secretory products cause NF-κB-mediated inflammation in human cholangiocarcinoma cells.

    PubMed

    Nam, Joo-Hyun; Moon, Ju Hyun; Kim, In Ki; Lee, Myoung-Ro; Hong, Sung-Jong; Ahn, Joong Ho; Chung, Jong Woo; Pak, Jhang Ho

    2012-01-01

    Chronic clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis worms and their excretory-secretory products, is associated with hepatobiliary damage, inflammation, periductal fibrosis and even development of cholangiocarcinoma. Our previous report revealed that intracellular reactive oxygen species were generated in C. sinensis excretory-secretory product-treated human cholangiocarcinoma cells; however, their endogenous sources and pathophysiological roles in host cells were not determined. In the present study, we found that treatment of human cholangiocarcinoma cells with excretory-secretory products triggered increases in free radicals via a time-dependent activation of NADPH oxidase, xanthine oxidase and inducible nitric oxide synthase. This increase in free radicals substantially promoted the degradation of cytosolic IκB-α, nuclear translocation of nuclear factor-κB subunits (RelA and p50), and increased κB consensus DNA-binding activity. Excretory-secretory product-induced nuclear factor-κB activation was markedly attenuated by preincubation with specific inhibitors of each free radical-producing enzyme or the antioxidant, N-acetylcysteine. Moreover, excretory-secretory products induced an increase in the mRNA and protein expression of the proinflammatory cytokines, IL-1β and IL-6, in an nuclear factor-κB-dependent manner, indicating that enzymatic production of free radicals in ESP-treated cells participates in nuclear factor-κB-mediated inflammation. These findings provide new insights into the pathophysiological role of C. sinensis excretory-secretory products in host chronic inflammatory processes, which are initial events in hepatobiliary diseases.

  11. Relationships between levels of lysozyme, lactoferrin, salivary peroxidase, and secretory immunoglobulin A in stimulated parotid saliva.

    PubMed Central

    Rudney, J D; Smith, Q T

    1985-01-01

    Recent studies suggest that salivary lysozyme (Lz), lactoferrin (Lf), peroxidase (Spx), and secretory immunoglobulin A (sIgA) may interact in a common antimicrobial system. A multiple protein approach therefore may be needed to determine the role of this system in oral health and ecology. In the present study we investigate the relationships between levels of Lz, Lf, Spx, and sIgA (adjusted for flow rate and total protein) in stimulated parotid saliva from 44 dental students. Principal components analysis was used to determine major patterns of intercorrelation between variables; cluster analysis was used to identify groups of subjects with similar salivary profiles for Lz, Lf, Spx, and sIgA. Spx tended to vary independently of Lz and Lf, which, in turn, tended to vary together. sIgA showed a weak negative relationship with Spx and a weak positive relationship with Lz and Lf. Six major clusters of subjects with similar antimicrobial protein profiles were found. These were significantly different at P less than 0.0001. Spx was the most important determinant of cluster membership followed (in order of importance) by Lz, Lf, and sIgA. Cluster profiles were Spx-, sIgAmu, Lf-, Lz-; Spx-, sIgA+, Lfmu, Lz+; Spxmu, sIgAmu, Lfmu, Lzmu; Spx+, sIgA-, Lf-, Lz-; Spx+, sIgAmu, Lf+, Lz-; and Spx+, sIgAmu, Lf+, Lz+ (-, mu, and + refer to the position of the cluster mean each variable relative to the overall mean for that variable). Results suggest that clusters may be a product of independent variation in the secretory activity of acinar and intercalated duct cells. PMID:4030086

  12. [Mammary analog secretory carcinoma of the parotid gland].

    PubMed

    Guérin, Maxime; Diedhiou, Abdoulaye; Nallet, Emmanuel; Duflo, Suzy; Laé, Marick; Wassef, Michel

    2014-10-01

    Mammary analog secretory carcinoma (MASC) of the parotid gland is a rare and recently described lesion. We report the case of a 46-year-old man with a tumor of the parotid gland which was carried to the diagnosis of MASC. Diagnostic was confirmed by highlighting the ETV6-NTRK3 gene translocation. However, some morphologic and immunohistochemical features are suggestive of this entity. This carcinoma should be distinguished from its main differential diagnoses: acinic cell carcinoma and low grade cribriform cystadenocarcinoma.

  13. Secretory pattern and regulatory mechanism of growth hormone in cattle

    PubMed Central

    2016-01-01

    Abstract The ultradian rhythm of growth hormone (GH) secretion has been known in several animal species for years and has recently been observed in cattle. Although the physiological significance of the rhythm is not yet fully understood, it appears essential for normal growth. In this review, previous studies concerning the GH secretory pattern in cattle, including its ultradian rhythm, are introduced and the regulatory mechanism is discussed on the basis of recent findings. PMID:26260675

  14. Isolation of intact sub-dermal secretory cavities from Eucalyptus

    PubMed Central

    2010-01-01

    Background The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils. Results Leaves were digested using a variety of commercially available enzymes. A pectinase from Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories. Conclusions The protocol described herein is likely to be adaptable to a range of Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain. PMID:20807444

  15. Altered secretory immunoglobulin A on skin surface after intensive exercise.

    PubMed

    Eda, Nobuhiko; Shimizu, Kazuhiro; Suzuki, Satomi; Tanabe, Yoko; Lee, Eunjae; Akama, Takao

    2013-09-01

    The aim of this study was to determine the effects of high-intensity endurance exercise on skin immunity by estimating secretory immunoglobulin A (SIgA) and staphylococci on skin surface. Seven healthy adult men (age, 22.3 ± 2.0 years) performed bicycle exercise at 75% HRmax for 60 minutes from 2030 to 2130 hours. Secretory immunoglobulin A was obtained from 1 ml extraction liquids stirred with the microtube homogenizer in the open end of a polypropylene tube for 60 seconds. Secretory immunoglobulin A concentrations were measured using enzyme-linked immunosorbent assay. Staphylococci were harvested by pressed agar-based media against skin surface. Skin surface samples were collected from the chest and the forearm on the first day at 2030 hours (before rest, A1), 2130 hours (after rest, A2), and 2230 hours (after showering, A3); the next morning at 0700 hours (A4); on the second day at 2030 hours (before exercise, B1), 2130 hours (after exercise, B2), and 2230 hours (after showering, B3); and the next morning at 0700 hours (B4). Secretory immunoglobulin A concentration on the forearm was significantly lower at B2 (p < 0.05) and B3 (p < 0.05) than that at B1 and that on the chest at B1 tended to be higher compared with B2 (p = 0.084) and B3 (p = 0.075). The number of staphylococci was significantly higher at B2 than that at B1 (p < 0.01) and B4 (p < 0.01) on the forearm. We conclude that high-intensity endurance exercise might depress immune function and enhance infectious risk on skin surface. Coaches should encourage their athletes to take a shower and change into clean clothes immediately after sports activities and athletes should maintain a clean skin surface to decrease the infectious risk on skin surface.

  16. Secretory pattern and regulatory mechanism of growth hormone in cattle.

    PubMed

    Kasuya, Etsuko

    2016-02-01

    The ultradian rhythm of growth hormone (GH) secretion has been known in several animal species for years and has recently been observed in cattle. Although the physiological significance of the rhythm is not yet fully understood, it appears essential for normal growth. In this review, previous studies concerning the GH secretory pattern in cattle, including its ultradian rhythm, are introduced and the regulatory mechanism is discussed on the basis of recent findings.

  17. Targeted ablation of the chromogranin a (Chga) gene: normal neuroendocrine dense-core secretory granules and increased expression of other granins.

    PubMed

    Hendy, Geoffrey N; Li, Tong; Girard, Martine; Feldstein, Richard C; Mulay, Shree; Desjardins, Roxane; Day, Robert; Karaplis, Andrew C; Tremblay, Michel L; Canaff, Lucie

    2006-08-01

    Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.

  18. Observations of Calcium Dynamics in Cortical Secretory Vesicles

    PubMed Central

    Raveh, Adi; Valitsky, Michael; Shani, Liora; Coorssen, Jens R.; Blank, Paul S.; Zimmerberg, Joshua; Rahamimoff, Rami

    2012-01-01

    SUMMARY Calcium (Ca2+) dynamics were evaluated in fluorescently labeled sea urchin secretory vesicles using confocal microscopy. 71% of the vesicles examined exhibited one or more transient increases in the fluorescence signal that was damped in time. The detection of transient increases in signal was dependent upon the affinity of the fluorescence indicator; the free Ca2+ concentration in the secretory vesicles was estimated to be in the range of ~10 – 100 μM. Non-linear stochastic analysis revealed the presence of extra variance in the Ca2+ dependent fluorescence signal. This noise process increased linearly with the amplitude of the Ca2+ signal. Both the magnitude and spatial properties of this noise process were dependent upon the activity of vesicle p-type (Cav2.1) Ca2+ channels. Blocking the p-type Ca2+ channels with ω-agatoxin decreased signal variance, and altered the spatial noise pattern within the vesicle. These fluorescence signal properties are consistent with vesicle Ca2+ dynamics and not simply due to obvious physical properties such as gross movement artifacts or pH driven changes in Ca2+ indicator fluorescence. The results suggest that the free Ca2+ content of cortical secretory vesicles is dynamic; this property may modulate the exocytotic fusion process. PMID:22831912

  19. Souffle/Spastizin controls secretory vesicle maturation during zebrafish oogenesis.

    PubMed

    Kanagaraj, Palsamy; Gautier-Stein, Amandine; Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-06-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.

  20. Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis

    PubMed Central

    Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-01-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research. PMID:24967841

  1. THE OPPORTUNISTIC PATHOGEN TOXOPLASMA GONDII DEPLOYS A DIVERSE LEGION OF INVASION AND SURVIVAL PROTEINS

    PubMed Central

    Zhou, Xing W.; Kafsack, Björn F. C.; Cole, Robert N.; Beckett, Phil; Shen, Rong F.; Carruthers, Vern B.

    2006-01-01

    Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins using two complementary methodologies, 2-DE/MS and LC/ESI-MS-MS (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by 2-DE revealed ∼100 spots, most of which were successfully identified by protein microsequencing or MALDI-MS analysis. Many proteins were present in multiple species suggesting they are subjected to substantial posttranslational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. The findings provide a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection. PMID:16002397

  2. Nutrients related to GLP1 secretory responses.

    PubMed

    Mansour, Asieh; Hosseini, Saeed; Larijani, Bagher; Pajouhi, Mohamad; Mohajeri-Tehrani, Mohammad Reza

    2013-06-01

    The hormone glucagon-like peptide (GLP-1) is secreted from gut endocrine L cells in response to ingested nutrients. The activities of GLP-1 include stimulating insulin gene expression and biosynthesis, improving β-cell proliferation, exogenesis, and survival. Additionally, it prevents β-cell apoptosis induced by a variety of cytotoxic agents. In extrapancreatic tissues, GLP-1 suppresses hunger, delays gastric emptying, acts as an ileal brake, and increases glucose uptake. The pleiotropic actions of GLP-1, especially its glucose-lowering effect, gave rise to the suggestion that it is a novel approach to insulin resistance treatment. Hormones secreted from the gut including GLP-1, which are involved in the regulation of insulin sensitivity and secretions, have been found to be affected by nutrient intake. In recent years, there has been a growing interest in the effect nutrients may have on GLP-1 secretion; some frequently studied dietary constituents include monounsaturated fatty acids, fructooligosaccharides, and glutamine. This review focuses on the influence that the carbohydrate, fat, and protein components of a meal may have on the GLP-1 postprandial responses.

  3. Two dipolar α-helices within hormone-encoding regions of proglucagon are sorting signals to the regulated secretory pathway.

    PubMed

    Guizzetti, Leonardo; McGirr, Rebecca; Dhanvantari, Savita

    2014-05-23

    Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229-240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting "signature."

  4. Increased expression of Candida albicans secretory proteinase, a putative virulence factor, in isolates from human immunodeficiency virus-positive patients.

    PubMed Central

    Ollert, M W; Wende, C; Görlich, M; McMullan-Vogel, C G; Borg-von Zepelin, M; Vogel, C W; Korting, H C

    1995-01-01

    The increased prevalence and the severity of oropharyngeal candidiasis in human immunodeficiency virus (HIV)-positive patients are attributed exclusively to the virus-induced immune deficiency of the host. The present study was aimed at answering the question of whether Candida albicans secretory proteinase, a putative virulence factor of the opportunistic C. albicans yeast, has any potential influence on the clinical manifestation of oropharyngeal candidiasis in HIV-positive patients. We measured the secretory proteinase activities of clinical C. albicans isolates from the oropharynges of either HIV-positive individuals (n = 100) or a control group (n = 122). The mean secretory proteinase activity of C. albicans isolates from the HIV-positive group (4,255 +/- 2,372 U/liter) was significantly higher compared with that of isolates from the control group (2,324 +/- 1,487 U/liter) (P < 0.05). The higher level of secretory proteinase activity in the culture supernatants of individual C. albicans isolates correlated with the increased level of proteinase expression on the cell surface, as revealed by cytofluorometry, and with higher levels of secretion of the immunodetectable protein, as shown by Western blotting (immunoblotting). Proteinase activity within the population of C. albicans isolates from HIV-positive individuals was independent of the patient's clinical disease stage and the CD4+/CD8+ cell numbers. Furthermore, no correlation of the proteinase activities with the C. albicans serotype was found, although C. albicans serotype B was significantly more frequent in the HIV-positive group (40%) compared with that in the control group (12%). However, a positive correlation of proteinase activity to antifungal susceptibility was evident.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8567880

  5. The secretory pathway calcium ATPase PMR-1/SPCA1 has essential roles in cell migration during Caenorhabditis elegans embryonic development.

    PubMed

    Praitis, Vida; Simske, Jeffrey; Kniss, Sarah; Mandt, Rebecca; Imlay, Leah; Feddersen, Charlotte; Miller, Michael B; Mushi, Juliet; Liszewski, Walter; Weinstein, Rachel; Chakravorty, Adityarup; Ha, Dae-Gon; Schacht Farrell, Angela; Sullivan-Wilson, Alexander; Stock, Tyson

    2013-05-01

    Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA) is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600), a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family.

  6. The Secretory Pathway Calcium ATPase PMR-1/SPCA1 Has Essential Roles in Cell Migration during Caenorhabditis elegans Embryonic Development

    PubMed Central

    Praitis, Vida; Simske, Jeffrey; Kniss, Sarah; Mandt, Rebecca; Imlay, Leah; Feddersen, Charlotte; Miller, Michael B.; Mushi, Juliet; Liszewski, Walter; Weinstein, Rachel; Chakravorty, Adityarup; Ha, Dae-Gon; Schacht Farrell, Angela; Sullivan-Wilson, Alexander; Stock, Tyson

    2013-01-01

    Maintaining levels of calcium in the cytosol is important for many cellular events, including cell migration, where localized regions of high calcium are required to regulate cytoskeletal dynamics, contractility, and adhesion. Studies show inositol-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), which release calcium into the cytosol, are important regulators of cell migration. Similarly, proteins that return calcium to secretory stores are likely to be important for cell migration. The secretory protein calcium ATPase (SPCA) is a Golgi-localized protein that transports calcium from the cytosol into secretory stores. SPCA has established roles in protein processing, metal homeostasis, and inositol-trisphosphate signaling. Defects in the human SPCA1/ATP2C1 gene cause Hailey-Hailey disease (MIM# 169600), a genodermatosis characterized by cutaneous blisters and fissures as well as keratinocyte cell adhesion defects. We have determined that PMR-1, the Caenorhabditis elegans ortholog of SPCA1, plays an essential role in embryogenesis. Pmr-1 strains isolated from genetic screens show terminal phenotypes, such as ventral and anterior enclosure failures, body morphogenesis defects, and an unattached pharynx, which are caused by earlier defects during gastrulation. In Pmr-1 embryos, migration rates are significantly reduced for cells moving along the embryo surface, such as ventral neuroblasts, C-derived, and anterior-most blastomeres. Gene interaction experiments show changing the activity of itr-1/IP3R and unc-68/RyR modulates levels of embryonic lethality in Pmr-1 strains, indicating pmr-1 acts with these calcium channels to regulate cell migration. This analysis reveals novel genes involved in C. elegans cell migration, as well as a new role in cell migration for the highly conserved SPCA gene family. PMID:23696750

  7. Analysis of Arf1 GTPase-dependent membrane binding and remodeling using the exomer secretory vesicle cargo adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2016-01-01

    Summary Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest. PMID:27632000

  8. Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae.

    PubMed

    Petrovic, Uros; Sribar, Jernej; Matis, Maja; Anderluh, Gregor; Peter-Katalinić, Jasna; Krizaj, Igor; Gubensek, Franc

    2005-10-15

    Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to g and e isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.

  9. A senescence secretory switch mediated by PI3K/AKT/mTOR activation controls chemoprotective endothelial secretory responses

    PubMed Central

    Bent, Eric H.; Gilbert, Luke A.; Hemann, Michael T.

    2016-01-01

    Cancer therapy targets malignant cells that are surrounded by a diverse complement of nonmalignant stromal cells. Therapy-induced damage of normal cells can alter the tumor microenvironment, causing cellular senescence and activating cancer-promoting inflammation. However, how these damage responses are regulated (both induced and resolved) to preserve tissue homeostasis and prevent chronic inflammation is poorly understood. Here, we detail an acute chemotherapy-induced secretory response that is self-limiting in vitro and in vivo despite the induction of cellular senescence. We used tissue-specific knockout mice to demonstrate that endothelial production of the proinflammatory cytokine IL-6 promotes chemoresistance and show that the chemotherapeutic doxorubicin induces acute IL-6 release through reactive oxygen species-mediated p38 activation in vitro. Doxorubicin causes endothelial senescence but, surprisingly, without a typical senescence secretory response. We found that endothelial cells repress senescence-associated inflammation through the down-regulation of PI3K/AKT/mTOR signaling and that reactivation of this pathway restores senescence-associated inflammation. Thus, we describe a mechanism by which damage-associated paracrine secretory responses are restrained to preserve tissue homeostasis and prevent chronic inflammation. PMID:27566778

  10. Calcineurin is universally involved in vesicle endocytosis at neuronal and nonneuronal secretory cells.

    PubMed

    Wu, Xin-Sheng; Zhang, Zhen; Zhao, Wei-Dong; Wang, Dongsheng; Luo, Fujun; Wu, Ling-Gang

    2014-05-22

    Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), we found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. We therefore suggest that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis.

  11. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

    PubMed

    Barg, S; Knowles, M K; Chen, X; Midorikawa, M; Almers, Wolfhard

    2010-11-30

    Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

  12. Mitochondrial calcium in the life and death of exocrine secretory cells.

    PubMed

    Voronina, Svetlana; Tepikin, Alexei

    2012-07-01

    The remarkable recent discoveries of the proteins mediating mitochondrial Ca(2+) transport (reviewed in this issue) provide an exciting opportunity to utilise this new knowledge to improve our fundamental understanding of relationships between Ca(2+) signalling and bioenergetics and, importantly, to improve the understanding of diseases in which Ca(2+) toxicity and mitochondrial malfunction play a crucial role. Ca(2+) is an important activator of exocrine secretion, a regulator of the bioenergetics of exocrine cells and a contributor to exocrine cell damage. Exocrine secretory cells, exocrine tissues and diseases affecting exocrine glands (like Sjögren's syndrome and acute pancreatitis) will, therefore, provide worthy research areas for the application of this new knowledge of the Ca(2+) transport mechanisms in mitochondria.

  13. Quantification of secretory leukocyte protease inhibitor (SLPI) in oral gargle specimens collected using mouthwash

    PubMed Central

    Pierce Campbell, Christine M.; Guan, Wei; Sprung, Robert; Koomen, John M.; O’Keefe, Michael T.; Ingles, Donna J.; Abrahamsen, Martha; Giuliano, Anna R.

    2014-01-01

    Background Secretory leukocyte protease inhibitor (SLPI) is an innate immunity-associated protein known to inhibit HIV transmission, and is thought to inhibit a variety of infectious agents, including human papillomaviruses (HPVs). We aimed to optimize an established ELISA-based SLPI quantification assay for use with oral gargle specimens collected using mouthwash, and to assess preliminary associations with age, smoking status, and alcohol intake. Methods Oral gargle supernatants from 50 individuals were used to optimize the Human SLPI Quantikine ELISA Kit. Sample suitability was assessed and quality control analyses were conducted. Results Salivary SLPI was successfully recovered from oral gargles with low intra-assay and high inter-individual variability. Initial measurements showed that salivary SLPI varied considerably across individuals, and that SLPI was inversely associated with age. Conclusions This optimized assay can be used to examine the role of SLPI in the acquisition of oral HPV and other infections. PMID:24140751

  14. Excavating the surface-associated and secretory proteome of Mycobacterium leprae for identifying vaccines and diagnostic markers relevant immunodominant epitopes.

    PubMed

    Rana, Aarti; Thakur, Shweta; Bhardwaj, Nupur; Kumar, Devender; Akhter, Yusuf

    2016-12-01

    For centuries, Mycobacterium leprae, etiological agent of leprosy, has been afflicting mankind regardless of extensive use of live-attenuated vaccines and antibiotics. Surface-associated and secretory proteins (SASPs) are attractive targets against bacteria. We have integrated biological knowledge with computational approaches and present a proteome-wide identification of SASPs. We also performed computational assignment of immunodominant epitopes as coordinates of prospective antigenic candidates in most important class of SASPs, the outer membrane proteins (OMPs). Exploiting the known protein sequence and structural characteristics shared by the SASPs from bacteria, 17 lipoproteins, 11 secretory and 19 novel OMPs (including 4 essential proteins) were identified in M. leprae As OMPs represent the most exposed antigens on the cell surface, their immunoinformatics analysis showed that the identified 19 OMPs harbor T-cell MHC class I epitopes and class II epitopes against HLA-DR alleles (54), while 15 OMPs present potential T-cell class II epitopes against HLA-DQ alleles (6) and 7 OMPs possess T-cell class II epitopes against HLA-DP alleles (5) of humans. Additionally, 11 M. leprae OMPs were found to have B-cell epitopes and these may be considered as prime candidates for the development of new immunotherapeutics against M. leprae.

  15. Characterisation and expression of secretory phospholipase A2 group IB during ontogeny of Atlantic cod ( Gadus morhua).

    PubMed

    Sæle, Øystein; Nordgreen, Andreas; Olsvik, Pål A; Hamre, Kristin

    2011-01-01

    The pancreatic enzyme secretory phospholipase A2 group IB (sPLA2 IB) hydrolyses phospholipids at the sn-2 position, resulting in a NEFA and a lyso-phospholipid, which are then absorbed by the enterocytes. The sPLA2 IB is a member of a family of nineteen enzymes sharing the same catalytic ability, of which nine are cytosolic and ten are secretory. Presently, there are no pharmacological tools to separate between the different secretory enzymes when measuring the enzymatic activity. Thus, it is important to support activity data with more precise techniques when isolation of intestinal content is not possible for analysis, as in the case of small teleost larvae, where the whole animal is sometimes analysed. In the present study, we characterise the sPLA2 IB gene in Atlantic cod (Gadus morhua) and describe its ontogeny at the genetic and protein level and compare this to the total sPLA2 activity level. A positive correlation was found between the expression of sPLA2 IB mRNA and protein. Both remained stable and low during the larval stage followed by an increase from day 62 posthatch, coinciding with the development of the pyloric ceaca. Meanwhile, total sPLA2 enzyme activity in cod was stable and relatively high during the early stages when larvae were fed live prey, followed by a decrease in activity when the fish were weaned to a formulated diet. Thus, the expression of sPLA2 IB mRNA and protein did not correlate with total sPLA2 activity.

  16. Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae.

    PubMed Central

    Salama, S R; Cleves, A E; Malehorn, D E; Whitters, E A; Bankaitis, V A

    1990-01-01

    The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for secretory protein movement from the Golgi complex. That some conservation of SEC14p function may exist was initially suggested by experiments that revealed immunoreactive polypeptides in cell extracts of the divergent yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. We have cloned and characterized the K. lactis SEC14 gene (SEC14KL). Immunoprecipitation experiments indicated that SEC14KL encoded the K. lactis structural homolog of SEC14p. In agreement with those results, nucleotide sequence analysis of SEC14KL revealed a gene product of 301 residues (Mr, 34,615) and 77% identity to SEC14p. Moreover, a single ectopic copy of SEC14KL was sufficient to render S. cerevisiae sec14-1(Ts) mutants, or otherwise inviable sec14-129::HIS3 mutant strains, completely proficient for secretory pathway function by the criteria of growth, invertase secretion, and kinetics of vacuolar protein localization. This efficient complementation of sec14-129::HIS3 was observed to occur when the rates of SEC14pKL and SEC14p synthesis were reduced by a factor of 7 to 10 with respect to the wild-type rate of SEC14p synthesis. Taken together, these data provide evidence that the high level of structural conservation between SEC14p and SEC14pKL reflects a functional identity between these polypeptides as well. On the basis of the SEC14p and SEC14pKL primary sequence homology to the human retinaldehyde-binding protein, we suggest that the general function of these SEC14p species may be to regulate the delivery of a hydrophobic ligand to Golgi membranes so that biosynthetic secretory traffic can be supported. Images PMID:2198263

  17. Effect of the Secretory Small GTPase Rab27B on Breast Cancer Growth, Invasion, and Metastasis

    PubMed Central

    Hendrix, An; Maynard, Dawn; Pauwels, Patrick; Braems, Geert; Denys, Hannelore; Van den Broecke, Rudy; Lambert, Jo; Van Belle, Simon; Cocquyt, Veronique; Gespach, Christian; Bracke, Marc; Seabra, Miguel C.; Gahl, William A.

    2010-01-01

    Background Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. Methods Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)–positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. Results Increased expression of Rab27B promoted G1 to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B–injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90α as key proinvasive growth regulator. Heat-shock protein 90α secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but

  18. Secretory IgA: Designed for Anti-Microbial Defense.

    PubMed

    Brandtzaeg, Per

    2013-01-01

    Prevention of infections by vaccination remains a compelling goal to improve public health. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion - a term coined for non-inflammatory antibody shielding of internal body surfaces, mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA), produced by local plasma cells (PCs) stimulated by antigens that target the mucose. SIgA was early shown to be complexed with an epithelial glycoprotein - the secretory component (SC). A common SC-dependent transport mechanism for pIgA and pentameric IgM was then proposed, implying that membrane SC acts as a receptor, now usually called the polymeric Ig receptor (pIgR). From the basolateral surface, pIg-pIgR complexes are taken up by endocytosis and then extruded into the lumen after apical cleavage of the receptor - bound SC having stabilizing and innate functions in the secretory antibodies. Mice deficient for pIgR show that this is the only receptor responsible for epithelial export of IgA and IgM. These knockout mice show a variety of defects in their mucosal defense and changes in their intestinal microbiota. In the gut, induction of B-cells occurs in gut-associated lymphoid tissue, particularly the Peyer's patches and isolated lymphoid follicles, but also in mesenteric lymph nodes. PC differentiation is accomplished in the lamina propria to which the activated memory/effector B-cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue but by different homing receptors. This compartmentalization is a challenge for mucosal vaccination, as are the mechanisms used by the mucosal immune system to discriminate between commensal symbionts (mutualism), pathobionts, and overt pathogens (elimination).

  19. Stage- and Gender-Specific Proteomic Analysis of Brugia malayi Excretory-Secretory Products

    PubMed Central

    Moreno, Yovany; Geary, Timothy G.

    2008-01-01

    Introduction While we lack a complete understanding of the molecular mechanisms by which parasites establish and achieve protection from host immune responses, it is accepted that many of these processes are mediated by products, primarily proteins, released from the parasite. Parasitic nematodes occur in different life stages and anatomical compartments within the host. Little is known about the composition and variability of products released at different developmental stages and their contribution to parasite survival and progression of the infection. Methodology/Principal Findings To gain a deeper understanding on these aspects, we collected and analyzed through 1D-SDS PAGE and LC-MS/MS the Excretory-Secretory Products (ESP) of adult female, adult male and microfilariae of the filarial nematode Brugia malayi, one of the etiological agents of human lymphatic filariasis. This proteomic analysis led to the identification of 228 proteins. The list includes 76 proteins with unknown function as well as also proteins with potential immunoregulatory properties, such as protease inhibitors, cytokine homologues and carbohydrate-binding proteins. Larval and adult ESP differed in composition. Only 32 proteins were shared between all three stages/genders. Consistent with this observation, different gene ontology profiles were associated with the different ESP. Conclusions/Significance A comparative analysis of the proteins released in vitro by different forms of a parasitic nematode dwelling in the same host is presented. The catalog of secreted proteins reflects different stage- and gender-specific related processes and different strategies of immune evasion, providing valuable insights on the contribution of each form of the parasite for establishing the host–parasite interaction. PMID:18958170

  20. Cutis laxa: intersection of elastic fiber biogenesis, TGFβ signaling, the secretory pathway and metabolism.

    PubMed

    Urban, Zsolt; Davis, Elaine C

    2014-01-01

    Cutis laxa (CL), a disease characterized by redundant and inelastic skin, displays extensive locus heterogeneity. Together with geroderma osteodysplasticum and arterial tortuosity syndrome, which show phenotypic overlap with CL, eleven CL-related genes have been identified to date, which encode proteins within 3 groups. Elastin, fibulin-4, fibulin-5 and latent transforming growth factor-β-binding protein 4 are secreted proteins which form elastic fibers and are involved in the sequestration and subsequent activation of transforming growth factor-β (TGFβ). Proteins within the second group, localized to the secretory pathway, perform transport and membrane trafficking functions necessary for the modification and secretion of elastic fiber components. Key proteins include a subunit of the vacuolar-type proton pump, which ensures the efficient secretion of tropoelastin, the precursor or elastin. A copper transporter is required for the activity of lysyl oxidases, which crosslink collagen and elastin. A Rab6-interacting goglin recruits kinesin motors to Golgi-vesicles facilitating the transport from the Golgi to the plasma membrane. The Rab and Ras interactor 2 regulates the activity of Rab5, a small guanosine triphosphatase essential for the endocytosis of various cell surface receptors, including integrins. Proteins of the third group related to CL perform metabolic functions within the mitochondria, inhibiting the accumulation of reactive oxygen species. Two of these proteins catalyze subsequent steps in the conversion of glutamate to proline. The third transports dehydroascorbate into mitochondria. Recent studies on CL-related proteins highlight the intricate connections among membrane trafficking, metabolism, extracellular matrix assembly, and TGFβ signaling.

  1. [Secretory immunoglobulin of the stomach in patients with chronic gastritis].

    PubMed

    Ostrovskiĭ, A B; Nikolaeva, O V; Isakova, V N

    1988-01-01

    The determination of the level of secretory IgA by a method of radial immunodiffusion after Mancini in the gastric juice of 48 patients with chronic gastritis in correlation with the status of the gastric mucosa, the level of acidification and the phase of exacerbation has shown diagnostic potentialities of the method. The highest IgA level was detected in patients with "rearrangement" gastritis and in patients with sharply suppressed gastric secretion. In marked atrophy of the gastric mucosa IgA secretion was significantly lowered. The period of remission was attended by a decrease in IgA secretion as compared with the phase of exacerbation.

  2. von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

    PubMed Central

    Lopes da Silva, Mafalda

    2016-01-01

    The von Willebrand factor (VWF) synthesized and secreted by endothelial cells is central to hemostasis and thrombosis, providing a multifunctional adhesive platform that brings together components needed for these processes. VWF secretion can occur from both apical and basolateral sides of endothelial cells, and from constitutive, basal, and regulated secretory pathways, the latter two via Weibel-Palade bodies (WPB). Although the amount and structure of VWF is crucial to its function, the extent of VWF release, multimerization, and polarity of the 3 secretory pathways have only been addressed separately, and with conflicting results. We set out to clarify these relationships using polarized human umbilical vein endothelial cells (HUVECs) grown on Transwell membranes. We found that regulated secretion of ultra–large (UL)-molecular-weight VWF predominantly occurred apically, consistent with a role in localized platelet capture in the vessel lumen. We found that constitutive secretion of low-molecular-weight (LMW) VWF is targeted basolaterally, toward the subendothelial matrix, using the adaptor protein complex 1 (AP-1), where it may provide the bulk of collagen-bound subendothelial VWF. We also found that basally-secreted VWF is composed of UL-VWF, released continuously from WPBs in the absence of stimuli, and occurs predominantly apically, suggesting this could be the main source of circulating plasma VWF. Together, we provide a unified dataset reporting the amount and multimeric state of VWF secreted from the constitutive, basal, and regulated pathways in polarized HUVECs, and have established a new role for AP-1 in the basolateral constitutive secretion of VWF. PMID:27106123

  3. Unprecedented multiplicity of Ig transmembrane and secretory mRNA forms in the cartilaginous fish.

    PubMed

    Rumfelt, Lynn L; Diaz, Marilyn; Lohr, Rebecca L; Mochon, Evonne; Flajnik, Martin F

    2004-07-15

    In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (C(H)) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgW(short)) having an unusual Sec tail, which is orthologous to skate IgX(short) cDNA. The IgW and IgW(short) Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface.

  4. Distribution of secretory component in hepatocytes and its mode of transfer into bile

    PubMed Central

    Mullock, Barbara M.; Hinton, Richard H.; Dobrota, Miloslav; Peppard, Jane; Orlans, Eva

    1980-01-01

    Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound 125I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [14C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile. ImagesPLATE 1Fig. 5. PMID:7470082

  5. A light-triggered protein secretion system.

    PubMed

    Chen, Daniel; Gibson, Emily S; Kennedy, Matthew J

    2013-05-13

    Optical control of protein interactions has emerged as a powerful experimental paradigm for manipulating and studying various cellular processes. Tools are now available for controlling a number of cellular functions, but some fundamental processes, such as protein secretion, have been difficult to engineer using current optical tools. Here we use UVR8, a plant photoreceptor protein that forms photolabile homodimers, to engineer the first light-triggered protein secretion system. UVR8 fusion proteins were conditionally sequestered in the endoplasmic reticulum, and a brief pulse of light triggered robust forward trafficking through the secretory pathway to the plasma membrane. UVR8 was not responsive to excitation light used to image cyan, green, or red fluorescent protein variants, allowing multicolor visualization of cellular markers and secreted protein cargo as it traverses the cellular secretory pathway. We implemented this novel tool in neurons to demonstrate restricted, local trafficking of secretory cargo near dendritic branch points.

  6. The Senescence-Associated Secretory Phenotype Promotes Benign Prostatic Hyperplasia

    PubMed Central

    Vital, Paz; Castro, Patricia; Tsang, Susan; Ittmann, Michael

    2015-01-01

    Benign prostatic hyperplasia (BPH) is characterized by increased tissue mass in the transition zone of the prostate, which leads to obstruction of urine outflow and considerable morbidity in a majority of older men. Senescent cells accumulate in human tissues, including the prostate, with increasing age. Expression of proinflammatory cytokines is increased in these senescent cells, a manifestation of the senescence-associated secretory phenotype. Multiplex analysis revealed that multiple cytokines are increased in BPH, including GM-CSF, IL-1α, and IL-4, and that these are also increased in senescent prostatic epithelial cells in vitro. Tissue levels of these cytokines were correlated with a marker of senescence (cathepsin D), which was also strongly correlated with prostate weight. IHC analysis revealed the multifocal epithelial expression of cathepsin D and coexpression with IL-1α in BPH tissues. In tissue recombination studies in nude mice with immortalized prostatic epithelial cells expressing IL-1α and prostatic stromal cells, both epithelial and stromal cells exhibited increased growth. Expression of IL-1α in prostatic epithelial cells in a transgenic mouse model resulted in increased prostate size and bladder obstruction. In summary, both correlative and functional evidence support the hypothesis that the senescence-associated secretory phenotype can promote the development of BPH, which is the single most common age-related pathology in older men. PMID:24434012

  7. Assessing the biosynthetic capabilities of secretory glands in Citrus peel.

    PubMed

    Voo, Siau Sie; Grimes, Howard D; Lange, B Markus

    2012-05-01

    Epithelial cells (ECs) lining the secretory cavities of Citrus peel have been hypothesized to be responsible for the synthesis of essential oil, but direct evidence for such a role is currently sparse. We used laser-capture microdissection and pressure catapulting to isolate ECs and parenchyma cells (as controls not synthesizing oil) from the peel of young grapefruit (Citrus × paradisi 'Duncan'), isolated RNA, and evaluated transcript patterns based on oligonucleotide microarrays. A Gene Ontology analysis of these data sets indicated an enrichment of genes involved in the biosynthesis of volatile terpenoids and nonvolatile phenylpropanoids in ECs (when compared with parenchyma cells), thus indicating a significant metabolic specialization in this cell type. The gene expression patterns in ECs were consistent with the accumulation of the major essential oil constituents (monoterpenes, prenylated coumarins, and polymethoxylated flavonoids). Morphometric analyses demonstrated that secretory cavities are formed early during fruit development, whereas the expansion of cavities, and thus oil accumulation, correlates with later stages of fruit expansion. Our studies have laid the methodological and experimental groundwork for a vastly improved knowledge of the as yet poorly understood processes controlling essential oil biosynthesis in Citrus peel.

  8. Widespread occurrence of expressed fungal secretory peroxidases in forest soils.

    PubMed

    Kellner, Harald; Luis, Patricia; Pecyna, Marek J; Barbi, Florian; Kapturska, Danuta; Krüger, Dirk; Zak, Donald R; Marmeisse, Roland; Vandenbol, Micheline; Hofrichter, Martin

    2014-01-01

    Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.

  9. Salivary Secretory Disorders, Inducing Drugs, and Clinical Management

    PubMed Central

    Miranda-Rius, Jaume; Brunet-Llobet, Lluís; Lahor-Soler, Eduard; Farré, Magí

    2015-01-01

    Background: Salivary secretory disorders can be the result of a wide range of factors. Their prevalence and negative effects on the patient's quality of life oblige the clinician to confront the issue. Aim: To review the salivary secretory disorders, inducing drugs and their clinical management. Methods: In this article, a literature search of these dysfunctions was conducted with the assistance of a research librarian in the MEDLINE/PubMed Database. Results: Xerostomia, or dry mouth syndrome, can be caused by medication, systemic diseases such as Sjögren's Syndrome, glandular pathologies, and radiotherapy of the head and neck. Treatment of dry mouth is aimed at both minimizing its symptoms and preventing oral complications with the employment of sialogogues and topical acting substances. Sialorrhea and drooling, are mainly due to medication or neurological systemic disease. There are various therapeutic, pharmacologic, and surgical alternatives for its management. The pharmacology of most of the substances employed for the treatment of salivary disorders is well-known. Nevertheless, in some cases a significant improvement in salivary function has not been observed after their administration. Conclusion: At present, there are numerous frequently prescribed drugs whose unwanted effects include some kind of salivary disorder. In addition, the differing pathologic mechanisms, and the great variety of existing treatments hinder the clinical management of these patients. The authors have designed an algorithm to facilitate the decision making process when physicians, oral surgeons, or dentists face these salivary dysfunctions. PMID:26516310

  10. Pathogen-induced secretory diarrhea and its prevention.

    PubMed

    Anand, S; Mandal, S; Patil, P; Tomar, S K

    2016-11-01

    Secretory diarrhea is a historically known serious health implication around the world which primarily originates through pathogenic microorganisms rather than immunological or genetical disorders. This review highlights infective mechanisms of non-inflammatory secretory diarrhea causing pathogens, known therapeutics and their efficacy against them. These non-inflammatory diarrheal pathogens breach cell barriers, induce inflammation, disrupt fluid secretion across the epithelium by alteration in ion transport by faulting cystic fibrosis transmembrane conductance regulator (CFTR), calcium activated chloride channels and ion exchanger functions. Currently, a variety of prevention strategies have been used to treat these symptoms like use of antibacterial drugs, vaccines, fluid and nutritional therapy, probiotics and prebiotics as adjuncts. In progression of the need for a therapy having quick physiological effects, withdrawing the symptoms with a wide and safe therapeutic index, newer antisecretory agents like potent inhibitors, agonists and herbal remedies are some of the interventions which have come into light through greater understanding of the mechanisms and molecular targets involved in intestinal fluid secretion. Although these therapies have their own pros and cons inside the host, the quest for new antisecretory agents has been a successful elucidation to reduce burden of diarrheal disease.

  11. Cell-specific analysis of tracheobronchial secretory cells and secretions

    SciTech Connect

    Finkbeiner, W.E.

    1989-01-01

    In these studies, two methods (cell culture and monoclonal antibody production) that allowed cell-specific analysis of tracheobronchial secretion were used. Bovine tracheal submucosal gland cells were isolated, placed into culture and serially propagated. In culture, the cells maintained features of serous cells. The cells incorporated {sup 35}S into high molecular weight molecules. {beta}-adrenergic agonists stimulated release of radiolabeled molecules and elevations in intracellular cAMP levels, responses that could be blocked by the {beta}-adrenergic antagonist propranolol. Cyclic AMP appeared to be involved in the stimulus-secretion coupling events in serous cells since the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine potentiated the effects of isoproterenol on the secretory response and the elevation of intracellular cAMP levels. Furthermore, cAMP analogues elicited a secretory response in the absence of cAMP. The phosphorylation state of several cytosolic and particulate phosphoproteins was altered by cAMP-activated kinase activity. Monoclonal antibodies were produced against human airway secretions.

  12. From acute to chronic pancreatitis: the role of mutations in the pancreatic secretory trypsin inhibitor gene.

    PubMed

    Hirota, Masahiko; Kuwata, Kinuko; Ohmuraya, Masaki; Ogawa, Michio

    2003-03-01

    Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor of trypsin. We proposed the hypothesis that, if the function of the PSTI is impaired by its genetic mutation, trypsin may easily promote autodigestion causing pancreatitis and we performed a mutational analysis of the PSTI gene in patients with pancreatitis. Two exonic mutations (N34S and R67C) were thought to be associated with a predisposition to pancreatitis. The N34S mutation was co-segregated with two intronic mutations, IVS1-37T>C and IVS3-69insTTTT. Although we analyzed the function of the recombinant N34S protein, we could not demonstrate the loss of function of this protein. Intronic mutations, rather than N34S itself (IVS1-37T>C + N34S + IVS3-69insTTTT complex), may be associated with the decreased function of the PSTI. Alternatively, increased digestion of N34S in vivo may be applicable. As for R67C, the conformational alteration of the protein by forming intra-molecular or inter-molecular disulfide bonds with 67Cys was strongly suggested. These results, along with the brand-new findings in PSTI knockout mice, suggest that the genetic mutation of the PSTI is one of the important mechanisms for predisposition to pancreatitis by lowering the trypsin inhibitory function.

  13. Regulation and activity of secretory leukoprotease inhibitor (SLPI) is altered in smokers.

    PubMed

    Meyer, Megan; Bauer, Rebecca N; Letang, Blanche D; Brighton, Luisa; Thompson, Elizabeth; Simmen, Rosalia C M; Bonner, James; Jaspers, Ilona

    2014-02-01

    A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1(-/-) mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection.

  14. Xbp1-based engineering of secretory capacity enhances the productivity of Chinese hamster ovary cells.

    PubMed

    Tigges, Marcel; Fussenegger, Martin

    2006-05-01

    A variety of successful transcription and translation engineering strategies implemented during the past decade have driven the specific productivity of mammalian cells to an apparent limit. Restricted post-translation competence has since been considered the major bottleneck preventing mammalian cells from fully exploiting their physiologic production capacity in a biopharmaceutical manufacturing scenario. Through ectopic expression of the human transcription factor Xbp1 (X-box-binding-protein 1), evolved to manage plasma cell differentiation and coordinate the unfolded protein response, we have specifically expanded the endoplasmic reticulum and the Golgi of transgenic Chinese hamster ovary (CHO-K1)-derived cell lines with a resulting increase in overall production capacity. Xbp-1-based engineering of secretory bottlenecks was compatible with a variety of different promoter–product gene configurations suggesting that Xbp-1 induces generic production increases in CHO-K1 cell derivatives. Secretion engineering, illustrated here by Xbp1-based reprogramming of the post-translational processing machinery, provides a first insight into mastering a major system bottleneck which impacts biopharmaceutical manufacturing of secreted protein therapeutics.

  15. A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

    PubMed

    Hood-Degrenier, Jennifer K

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

  16. Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism

    USGS Publications Warehouse

    Ramirez-Gomez, F.; Greene, W.; Rego, K.; Hansen, J.D.; Costa, G.; Kataria, P.; Bromage, E.S.

    2012-01-01

    The gene encoding IgH δ has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated δ-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory δ transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory δ transcript resulted in two δ-H chains, which incorporated Cμ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ mRNA and IgD+ plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.

  17. Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism.

    PubMed

    Ramirez-Gomez, Francisco; Greene, Whitney; Rego, Katherine; Hansen, John D; Costa, Greg; Kataria, Priti; Bromage, Erin S

    2012-02-01

    The gene encoding IgH δ has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated δ-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory δ transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory δ transcript resulted in two δ-H chains, which incorporated Cμ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ mRNA and IgD(+) plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.

  18. Mammary analogue secretory carcinoma (MASC) of the salivary gland: A new tumor entity

    PubMed Central

    Damjanov, Ivan; Skenderi, Faruk; Vranic, Semir

    2016-01-01

    Mammary analogue secretory carcinoma (MASC) is a recently described low-grade malignant tumor of the salivary glands, biologically and morphologically equivalent to secretory breast carcinoma. We give a brief overview of this new entity, including morphological, immunohistochemical, molecular-genetic, clinical, epidemiologic features, differential diagnosis, and outcome results. PMID:27131022

  19. Mammary Analogue Secretory Carcinoma (MASC) of the salivary gland: A new tumor entity.

    PubMed

    Damjanov, Ivan; Skenderi, Faruk; Vranic, Semir

    2016-08-02

    Mammary analogue secretory carcinoma (MASC) is a recently described low-grade malignant tumor of the salivary glands, biologically and morphologically equivalent to secretory breast carcinoma. We give a brief overview of this new entity, including morphological, immunohistochemical, molecular-genetic, clinical, epidemiologic features, differential diagnosis, and outcome results.

  20. [Secretory immunoglobulin A in the amniotic fluid of healthy pregnant females].

    PubMed

    Briese, V; Straube, W; Brock, J; Stark, K H; Lorenz, U

    1983-01-01

    Amniotic fluid levels of secretory immunoglobulin A (S-AgA) were measured by simple radial immunodiffusion according to the method of Mancini using a monospecific antiserum against the human secretory component. 256 samples from healthy pregnant women were examined. Amniotic fluid S-IgA concentration increases significantly during normal pregnancy and shows a loose correlation to the phospholipid level.

  1. Dark matter in archaeal genomes: a rich source of novel mobile elements, defense systems and secretory complexes.

    PubMed

    Makarova, Kira S; Wolf, Yuri I; Forterre, Patrick; Prangishvili, David; Krupovic, Mart; Koonin, Eugene V

    2014-09-01

    Microbial genomes encompass a sizable fraction of poorly characterized, narrowly spread fast-evolving genes. Using sensitive methods for sequences comparison and protein structure prediction, we performed a detailed comparative analysis of clusters of such genes, which we denote "dark matter islands", in archaeal genomes. The dark matter islands comprise up to 20% of archaeal genomes and show remarkable heterogeneity and diversity. Nevertheless, three classes of entities are common in these genomic loci: (a) integrated viral genomes and other mobile elements; (b) defense systems, and (c) secretory and other membrane-associated systems. The dark matter islands in the genome of thermophiles and mesophiles show similar general trends of gene content, but thermophiles are substantially enriched in predicted membrane proteins whereas mesophiles have a greater proportion of recognizable mobile elements. Based on this analysis, we predict the existence of several novel groups of viruses and mobile elements, previously unnoticed variants of CRISPR-Cas immune systems, and new secretory systems that might be involved in stress response, intermicrobial conflicts and biogenesis of novel, uncharacterized membrane structures.

  2. Development and Essential Oil Content of Secretory Glands of Sage (Salvia officinalis) 1

    PubMed Central

    Venkatachalam, K. V.; Kjonaas, Robert; Croteau, Rodney

    1984-01-01

    Scanning electron microscopy of sage (Salvia officinalis L.) leaves confirmed the presence of two basic types of glandular trichomes consisting of a capitate stalked form containing a multicellular stalk and surmounted by a unicellular secretory head, and a capitate sessile form containing a unicellular stalk and unicellular, or multicellular, secretory head. In the latter type, secretory activity and filling of the subcuticular cavity may begin at virtually any stage of the division cycle affording fully developed glands containing from one to twelve cells in the secretory head. Gas liquid chromatographic analysis of the oil content of the most numerous gland species (capitate stalked, capitate sessile with one and with eight secretory cells) indicated only minor quantitative differences in essential oil composition. Thus, each gland type is capable of producing the four major monoterpene families (p-menthanes, pinanes, bornanes and thujanes) characteristic of sage. Images Fig. 1 PMID:16663786

  3. Development and essential oil content of secretory glands of sage (Salvia officinalis)

    SciTech Connect

    Venkatachalam, K.V.; Kjonaas, R.; Croteau, R.

    1984-09-01

    Scanning electron microscopy of sage (Salvia officinalis L.) leave confirmed the presence of two basic types of glandular trichomes consisting of a capitate stalked form containing a multicellular stalk and surmounted by a unicellular secretory head, and a capitate sessile form containing a unicellular stalk and unicellular, or multicellular, secretory head. In the latter type, secretory activity and filling of the subcuticular cavity may begin at virtually any stage of the division cycle affording fully developed glands containing from one to twelve cells in the secretory head. Gas liquid chromatographic analysis of the oil content of the most numerous gland species (capitate stalked, capitate sessile with one and with eight secretory cells) indicated only minor quantitative differences in essential oil composition. Thus, each gland type is capable of producing the four major monoterpene families (p-menthanes, pinanes, bornanes and thujanes) characteristic of sage. 21 references, 2 figures.

  4. Protein export by the mycobacterial SecA2 system is determined by the preprotein mature domain.

    PubMed

    Feltcher, Meghan E; Gibbons, Henry S; Ligon, Lauren S; Braunstein, Miriam

    2013-02-01

    At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles "housekeeping" export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.

  5. Fallopian tube secretory cell expansion: a sensitive biomarker for ovarian serous carcinogenesis.

    PubMed

    Wang, Yiying; Li, Li; Wang, Yue; Tang, Sarah Ngocvi; Zheng, Wenxin

    2015-01-01

    Recent advances suggest that precancerous lesions of pelvic serous carcinoma originate from tubal secretory cells. The purpose of our study was to determine if an increased number of secretory cells varies with age or location in the fallopian tube and to examine its association with serous neoplasia. Three groups (benign control, high-risk, and pelvic serous carcinoma) of age-matched patients were studied. The age data were stratified into 10-year intervals ranging from 20-29 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data were analyzed by standard contingency table and Poisson distribution methods after age justification. We found that the absolute number of tubal secretory cells increased significantly with age in all three groups. But a more dramatic increase of secretory cells was observed in high-risk and pelvic serous carcinoma patients. Secretory cell expansion is more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and is significantly associated with serous neoplasia (P < 0.001). Furthermore, age remained a significant risk factor for serous neoplasia after age adjustment. These findings suggest that secretory cell expansion could serve as a potential sensitive biomarker for early serous carcinogenesis within the fallopian tube. The study also supports a relationship between serous neoplasia and increased secretory to ciliated cell ratios, and the relationship between frequency of secretory cell expansion within the fallopian tube and increasing age and-more significantly-presence of high-risk factors or co-existing serous cancers.

  6. Fallopian tube secretory cell expansion: a sensitive biomarker for ovarian serous carcinogenesis.

    PubMed

    Wang, Yiying; Li, Li; Wang, Yue; Tang, Sarah Ngocvi; Zheng, Wenxin

    2016-01-01

    Recent advances suggest that precancerous lesions of pelvic serous carcinoma originate from tubal secretory cells. The purpose of our study was to determine if an increased number of secretory cells vary with age or location in the fallopian tube and to examine its association with serous neoplasia. Three groups (benign control, high-risk, and pelvic serous carcinoma) of age-matched patients were studied. The age data were stratified into 10-year intervals ranging from 20-29 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data were analyzed by standard contingency table and Poisson distribution methods after age justification. We found that the absolute number of tubal secretory cells increased significantly with age in all three groups. But a more dramatic increase of secretory cells was observed in high-risk and pelvic serous carcinoma patients. Secretory cell expansion is more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and is significantly associated with serous neoplasia (p < 0.001). Furthermore, age remained a significant risk factor for serous neoplasia after age adjustment. These findings suggest that secretory cell expansion could serve as a potential sensitive biomarker for early serous carcinogenesis within the fallopian tube. The study also supports a relationship between serous neoplasia and increased secretory to ciliated cell ratios, and the relationship between frequency of secretory cell expansion within the fallopian tube and increasing age and-more significantly-presence of high-risk factors or co-existing serous cancers.

  7. The secretory granule matrix: a fast-acting smart polymer.

    PubMed

    Nanavati, C; Fernandez, J M

    1993-02-12

    The secretory granule matrix is a miniature biopolymer that consists of a charged polymer network that traps peptides and transmitters when it condenses and releases them on exocytotic decondensation. Models of exocytotic fusion have treated this matrix as a short circuit and have neglected its electrical contributions. This matrix responded to negative voltages by swelling, which was accompanied by a large increase in conductance, and to positive voltages by condensing. Thus, the matrix resembled a diode. The swollen matrix exerted large pressures on the order of 12 bar. The responses took place within milliseconds of the application of the electric field. These findings suggest that matrix decondensation, and therefore product release, is controlled by potential gradients.

  8. Mammary analog secretory carcinoma of thyroid: A case report.

    PubMed

    Rupp, Aaron P; Bocklage, Thèrése J

    2017-01-01

    Mammary analog secretory carcinoma (MASC) is a recently described rare neoplasm that was first reported in the salivary gland with an associated ETV6-NTRK3 fusion. We present a case of MASC involving and presumably arising in the thyroid, which was originally diagnosed as papillary thyroid carcinoma on fine needle aspiration and surgical resection. The later correct diagnosis of MASC was confirmed by immunohistochemistry and molecular studies. The cytopathological features of MASC in the salivary gland are previously described; however, we present the first cytopathological description of MASC arising in the thyroid with the unique feature of prominent nuclear grooves. Differentiating MASC from overlapping features of cytopathologic mimics such as papillary thyroid carcinoma may carry crucial therapeutic implications. Diagn. Cytopathol. 2017;45:45-50. © 2016 Wiley Periodicals, Inc.

  9. Secretory expression of a heterologous nattokinase in Lactococcus lactis.

    PubMed

    Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

    2007-05-01

    Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

  10. Novel distribution of the secretory leucocyte proteinase inhibitor in kidney.

    PubMed Central

    Ohlsson, S; Ljungkrantz, I; Ohlsson, K; Segelmark, M; Wieslander, J

    2001-01-01

    The secretory leucocyte proteinase inhibitor (SLPI) is a low molecular weight, tissue-specific inhibitor of, for example, elastase and cathepsin G, which also have antimicrobial capacity. SLPI has been localised to the respiratory, gastrointestinal and genital tracts, but so far not to the kidney. The presence of SLPI in renal tubuli cells was demonstrated using immunohistochemistry and, by means of in situ hybridisation on human renal biopsies, we were able to demonstrate SLPI production. In various inflammatory conditions in the kidneys, the protease-antiprotease balance is disturbed. For this reason, as well as the possible role in the defence against ascending urinary tract infections, it is interesting to establish a source of SLPI in renal tubuli cells. PMID:11817677

  11. Characterization of mast cell secretory granules and their cell biology.

    PubMed

    Azouz, Nurit Pereg; Hammel, Ilan; Sagi-Eisenberg, Ronit

    2014-10-01

    Exocytosis and secretion of secretory granule (SG) contained inflammatory mediators is the primary mechanism by which mast cells exert their protective immune responses in host defense, as well as their pathological functions in allergic reactions and anaphylaxis. Despite their central role in mast cell function, the molecular mechanisms underlying the biogenesis and secretion of mast cell SGs remain largely unresolved. Early studies have established the lysosomal nature of the mast cell SGs and implicated SG homotypic fusion as an important step occurring during both their biogenesis and compound secretion. However, the molecular mechanisms that account for key features of this process largely remain to be defined. A novel high-resolution imaging based methodology allowed us to screen Rab GTPases for their phenotypic and functional impact and identify Rab networks that regulate mast cell secretion. This screen has identified Rab5 as a novel regulator of homotypic fusion of the mast cell SGs that thereby regulates their size and cargo composition.

  12. Silent reading and secretory otitis media in school children.

    PubMed

    Lous, J

    1993-01-01

    In an unselected cohort of 366 8-year-old children, the relationship between secretory otitis media and reading achievement was investigated. The children underwent 10 impedance audiometries and 5 pure tone audiometries during their first year at school. At the beginning of the second grade they all had a Silent Reading Word Test (OS-400). The background parameters were recorded by an interview with one of the parents. There was a significant but small correlation between type B tympanograms in the first grade and silent word reading. No association between silent reading score and otological history or pure tone screening was found. In a stepwise multiple regression model, 37% of the variance could be 'explained' by the included variables. The 'classroom factor' could 'explain' about 17% of the variance, followed by phonology at the start of school (6%), gender (5%), social group of the mother (4%), type B tympanogram (2%), absence from school (2%) and allergy (1%).

  13. Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2

    PubMed Central

    Cavigiolio, Giorgio; Jayaraman, Shobini

    2014-01-01

    In the acute phase of the inflammatory response, secretory phospholipase A2 (sPLA2) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA2 in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA2-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA2 reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA2 reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA2 by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA2 with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA2 with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA2 was Ca2+-independent, implicating a different mechanism from the Ca2+-dependent PLA2-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA2 proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA2 that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis. PMID:24523407

  14. Prolactin and growth hormone aggregates in secretory granules: the need to understand the structure of the aggregate.

    PubMed

    Dannies, Priscilla S

    2012-04-01

    Prolactin and GH form reversible aggregates in the trans-Golgi lumen that become the dense cores of secretory granules. Aggregation is an economical means of sorting, because self-association removes the hormones from other possible pathways. Secretory granules containing different aggregates show different behavior, such as the reduction in stimulated release of granules containing R183H-GH compared with release of those containing wild-type hormone. Aggregates may facilitate localization of membrane proteins necessary for transport and exocytosis of secretory granules, and therefore understanding their properties is important. Three types of self-association have been characterized: dimers of human GH that form with Zn(2+), low-affinity self-association of human prolactin caused by acidic pH and Zn(2+) with macromolecular crowding, and amyloid fibers of prolactin. The best candidate for the form in most granules may be low-affinity self-association because it occurs rapidly at Zn(2+) concentrations that are likely to be in granules and reverses rapidly in neutral pH. Amyloid may form in older granules. Determining differences between aggregates of wild type and those of R183H-GH should help to understand why granules containing the mutant behave differently from those containing wild-type hormone. If reversible aggregation of other hormones, including those that are proteolytically processed, is the crucial act in forming granules, rather than use of a sorting signal, then prohormones should form reversible aggregates in solution in conditions that resemble those of the trans-Golgi lumen, including macromolecular crowding.

  15. Thyroid hormone status regulates the expression of secretory phospholipases.

    PubMed

    Sharma, Pragya; Levesque, Tania; Boilard, Eric; Park, Edwards A

    2014-01-31

    Thyroid hormone (T3) stimulates various metabolic pathways and the hepatic actions of T3 are mediated primarily through the thyroid hormone receptor beta (TRβ). Hypothyroidism has been linked with low grade inflammation, elevated risk of hepatic steatosis and atherosclerosis. Secretory phospholipases (sPLA2) are associated with inflammation, hyperlipidemia and atherosclerosis. Due to potential linkage between thyroid hormone and sPLA2, we investigated the effect of thyroid hormone status on the regulation of secretory phospholipases in mice, rats and human liver. T3 suppressed the expression of the sPLA2 group IIa (PLA2g2a) gene in the liver of BALB/c mice and C57BL/6 transgenic mice expressing the human PLA2g2a. PLA2g2a was elevated with hypothyroidism and high fat diets which may contribute to the low grade inflammation associated with hypothyroidism and diet induced obesity. We also examined the effects of the TRβ agonist eprotirome on hepatic gene regulation. We observed that eprotirome inhibited the expression of selected sPLA2 genes and furthermore the cytokine mediated induction PLA2g2a was suppressed. In addition, eprotirome induced genes involved in fatty acid oxidation and cholesterol clearance while inhibiting lipogenic genes. Our results indicate that in vivo thyroid hormone status regulates the abundance of sPLA2 and the inhibition of PLA2g2a by T3 is conserved across species. By regulating sPLA2 genes, T3 may impact processes associated with atherosclerosis and inflammation and TRβ agonists may ameliorate inflammation and hyperlipidemia.

  16. Thyroid hormone status regulates the expression of secretory phospholipases

    PubMed Central

    Sharma, Pragya; Levesque, Tania; Boilard, Eric; Park, Edwards A.

    2014-01-01

    Thyroid hormone (T3) stimulates various metabolic pathways and the hepatic actions of T3 are mediated primarily through the thyroid hormone receptor beta (TRβ). Hypothyroidism has been linked with low grade inflammation, elevated risk of hepatic steatosis and atherosclerosis. Secretory phospholipases (sPLA2) are associated with inflammation, hyperlipidemia and atherosclerosis. Due to potential linkage between thyroid hormone and sPLA2, we investigated the effect of thyroid hormone status on the regulation of secretory phospholipases in mice, rats and human liver. T3 suppressed the expression of the sPLA2 group IIa (PLA2g2a) gene in the liver of BALB/c mice and C57BL/6 transgenic mice expressing the human PLA2g2a. PLA2g2a was elevated with hypothyroidism and high fat diets which may contribute to the low grade inflammation associated with hypothyroidism and diet induced obesity. We also examined the effects of the TRβ agonist eprotirome on hepatic gene regulation. We observed that eprotirome inhibited the expression of selected sPLA2 genes and furthermore the cytokine mediated induction PLA2g2a was suppressed. In addition, eprotirome induced genes involved in fatty acid oxidation and cholesterol clearance while inhibiting lipogenic genes. Our results indicate that in vivo thyroid hormone status regulates the abundance of sPLA2 and the inhibition of PLA2g2a by T3 is conserved across species. By regulating sPLA2 genes, T3 may impact processes associated with atherosclerosis and inflammation and TRβ agonists may ameliorate inflammation and hyperlipidemia. PMID:24440706

  17. Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules.

    PubMed

    Santodomingo, Jaime; Vay, Laura; Camacho, Marcial; Hernández-Sanmiguel, Esther; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2008-10-01

    The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.

  18. Calcium dynamics in catecholamine-containing secretory vesicles.

    PubMed

    Moreno, Alfredo; Lobatón, Carmen D; Santodomingo, Jaime; Vay, Laura; Hernández-SanMiguel, Esther; Rizzuto, Rosario; Montero, Mayte; Alvarez, Javier

    2005-06-01

    We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.

  19. High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris.

    PubMed

    Peng, Lisheng; Zhong, Xiaofen; Ou, Jingxing; Zheng, Suilan; Liao, Jian; Wang, Lei; Xu, Anlong

    2004-03-04

    Enterokinase (EC 3.4.21.9) is a serine proteinase with a specific digest sequence (Asp)4-Lys in the duodenum. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for an in vitro cleavage of fusion proteins. In this work, an active bovine enterokinase light chain (EK(L)) was produced in secretory form by a recombinant strain of the methylotrophic yeast Pichia pastoris. The influences of methanol utilization phenotype of the host strain, induction pH, and carbon source on the recombinant production were studied. The production of recombinant EK(L) by Mut(s) strain was much higher than that by Mut+ strain. When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EK(L) (rEK(L)) reached 350 mg l(-1), which was 20-fold higher than that reported previously. The recombinant EK(L) was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EK(L) were obtained from 100 ml culture broth supernatant. The specific activity of purified rEK(L) was approximately 9000 u mg(-1). To facilitate purification and removal of rEKL after cleavage of fusion protein, the C-terminal His-tagged EK(L) (EK(L)/His) was also expressed in P. pastoris, and this His-tagged EK(L) exhibited a similar enzymatic activity to the untagged EK(L).

  20. Detecting the Senescence-Associated Secretory Phenotype (SASP) by High Content Microscopy Analysis.

    PubMed

    Hari, Priya; Acosta, Juan Carlos

    2017-01-01

    The diverse arrays of proteins secreted by senescent cells have been described to influence aging and to have both pro-tumorigenic and anti-tumorigenic influences on the surrounding microenvironment. Further characterization of these proteins, known as the senescence-associated secretory phenotype (SASP), and their regulators is required to understand and further manipulate such activities. The use of high-throughput technology allows us to obtain visual and quantitative data on a large number of samples quickly and easily. Not only is this an invaluable tool for conducting large-scale RNAi or compound screenings, but also allows rapid validation of candidates of interest. Here, we describe how we use the Widefield High-Content Analysis Systems to characterize the phenotypes of cells following modulation by potential regulators of Oncogene-Induced Senescence (OIS) by measuring numerous markers of senescence, including the SASP. This approach can be also used to screen for siRNA able to perturb the expression of SASP components during OIS.

  1. Morphology in conjunction with immunohistochemistry is sufficient for the diagnosis of mammary analogue secretory carcinoma.

    PubMed

    Shah, Akeesha A; Wenig, Bruce M; LeGallo, Robin D; Mills, Stacey E; Stelow, Edward B

    2015-03-01

    The recently described mammary analogue secretory carcinoma (MASC) is a low-grade salivary gland malignancy that harbors the recurrent cytogenetic abnormality t(12;15) (p13;q25) ETV6-NTRK3. Confirmation of this is currently considered the gold standard for diagnosis. Some have postulated that morphology together with supporting immunohistochemistry is sufficient to diagnose MASC. In this study we retrospectively review a series of 19 MASCs diagnosed based on histology in conjunction with immunohistochemistry; subsequently we performed in situ hybridization using an ETV6 break-apart probe. Immunohistochemistry for S100 protein and mammaglobin as well as fluorescence in situ hybridization using the Vysis ETV6 Dual Color Break-Apart FISH Probe Kit were performed on all cases. The 19 cases were from 12 females and 7 males with ages ranging from 16 to 76 years (mean = 45 years). Sixteen cases were from the parotid gland, 1 case was from a periparotid lymph node and 2 cases were from the submandibular gland. All 19 cases demonstrated moderate to strong expression of S100 protein. Eighteen cases demonstrated strong, diffuse expression of mammaglobin, while one case had only rare tumor cells that strongly expressed mammaglobin. Eighteen of 19 cases (95 %) demonstrated the ETV6 rearrangement by fluorescence in situ hybridization. Given that morphology together with immunohistochemistry is highly correlated with the ETV6 gene rearrangement, we conclude that molecular confirmation is not required to diagnose MASC.

  2. Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells.

    PubMed

    Chaiyadet, Sujittra; Smout, Michael; Laha, Thewarach; Sripa, Banchob; Loukas, Alex; Sotillo, Javier

    2016-02-09

    The association between liver fluke infection caused by Opisthorchis viverrini and cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium) has been well established. Multiple mechanisms play a role in the development of CCA, but the excretory/secretory products released by O. viverrini (OvES) represent the major interface between the parasite and its host, and their uptake by biliary epithelial cells has been suggested to be responsible for proliferation of cholangiocytes, the cells that line the biliary epithelium. Despite recent progress in the study of the molecular basis of O. viverrini-host interactions, little is known about the effects that OvES induces upon internalization by host cells. In the present study we incubated non-cancerous human cholangiocytes (H69) and human colon cancer (CaCo-2) cells with OvES and performed a time-course quantitative proteomic analysis on the cells to determine the early changes induced by the parasite. Different KEGG pathways were altered in H69 cells compared to Caco-2 cells: glycolysis/gluconeogenesis and protein processing in the endoplasmic reticulum. In addition, the Reactome pathway analysis showed a predominance of proteins involved in cellular pathways related to apoptosis and apoptotic execution phase in H69 cells after incubation with OvES. The present study provides the first proteomic analysis to address the molecular mechanisms by which OvES products interact with host cells, and Sheds light on the cellular processes involved in O. viverrini-induced CCA.

  3. Involvement of Oxidative Pathways in Cytokine-induced Secretory Phospholipase A2-IIA in Astrocytes

    PubMed Central

    Jensen, Michael D.; Sheng, Wenwen; Simonyi, Agnes; Johnson, Gary S.; Sun, Albert Y.; Sun, Grace Y.

    2009-01-01

    Recent studies have suggested the involvement of secretory phospholipase A2-IIA (sPLA2-IIA) in neuroinflammatory diseases. Although sPLA2-IIA is transcriptionally induced through the NF-κB pathway by pro-inflammatory cytokines, whether this induction pathway is affected by other intracellular signaling pathways has not been investigated in detail. In this study, we demonstrated the induction of sPLA2-IIA mRNA and protein expression in astrocytes by cytokines and detected the protein in the culture medium after stimulation. We further investigated the effects of oxidative pathways and botanical antioxidants on the induction pathway and observed that IL-1β-induced sPLA2-IIA mRNA expression in astrocytes is dependent on ERK1/2 and PI-3 kinase, but not p38 MAPK. In addition to apocynin, a known NADPH oxidase inhibitor, botanical antioxidants, such as resveratrol and epigallocatechin gallate, also inhibited IL-1β-induced sPLA2-IIA mRNA expression. These compounds also suppressed IL-1β-induced ERK1/2 activation and translocation of the NADPH oxidase subunit p67 phox from cytosol to membrane fraction. Taken together, these results support the involvement of reactive oxygen species from NADPH oxidase in cytokine induction of sPLA2-IIA in astrocytes and promote the use of botanical antioxidants as protective agents for inhibition of inflammatory responses in these cells. PMID:19375465

  4. A Nibbling Mechanism for Clathrin-mediated Retrieval of Secretory Granule Membrane after Exocytosis*

    PubMed Central

    Bittner, Mary A.; Aikman, Rachel L.; Holz, Ronald W.

    2013-01-01

    Clathrin-mediated endocytosis is the major pathway for recycling of granule membrane components after strong stimulation and high exocytotic rates. It resembles “classical” receptor-mediated endocytosis but has a trigger that is unique to secretion, the sudden appearance of the secretory granule membrane in the plasma membrane. The spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of the nucleation events are unknown. Furthermore, a size mismatch between chromaffin granules (∼300-nm diameter) and typical clathrin-coated vesicles (∼90 nm) makes it unlikely that clathrin-mediated endocytosis internalizes as a unit the entire fused granule membrane. We have used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins along with a fluid-phase marker to address these issues. We demonstrate that the fused granule membrane remains a distinct entity and serves as a nucleation site for clathrin- and dynamin-mediated endocytosis that internalizes granule membrane components in small increments. PMID:23386611

  5. OKT3-induced nephrotoxicity is associated with release of group II secretory phospholipase A2.

    PubMed

    Wever, P C; Roest, R W; Wolbink-Kamp, A M; Wolbink, G J; Weening, J J; Hack, C E; ten Berge, J M

    1996-10-01

    Administration of the murine IgG2a CD3 monoclonal antibody OKT3 exerts a transient nephrotoxic effect. Increased levels of group II secretory phospholipase A2 (sPLA2-II) might account for this nephrotoxicity as sPLA2-II induces the biosynthesis of prostaglandins, vasoactive lipid mediators that influence glomerular haemodynamics and renal function. Furthermore, extracellular phospholipases seem to be involved in proximal tubular cell injury. We studied plasma sPLA2-II levels in relation to circulating creatinine, tumour necrosis factor alpha, interleukin 6 and C-reactive protein levels in 15 renal allograft recipients receiving rejection treatment with OKT3. As a control group, we studied 15 renal allograft recipients receiving rejection treatment with methylprednisolone. A maximal fourfold increase in sPLA2-II levels was observed 48 h after the first OKT3 administration, preceded by increased tumour necrosis factor alpha and interleukin 6 levels and accompanied by increased C-reactive protein levels. Creatinine levels reached a maximal increase 72 h after initiation of treatment. During methylprednisolone treatment no increase in any of the studied parameters was observed. Thus, administration of OKT3 induces increased sPLA2-II levels, presumably via generation of cytokines. We hypothesize that sPLA2-II may contribute to the nephrotoxic effect of OKT3 by inducing vasoconstrictive prostaglandins and renal tubular cell injury.

  6. Production of Recombinant CCN Proteins by Brevibacillus choshinensis.

    PubMed

    Hanagata, Hiroshi; Mizukami, Makoto

    2017-01-01

    Brevibacillus choshinensis is an excellent host for the production of secretory proteins. This host has also been applied successfully to efficient production of CCN proteins. Described herein are methods of constructing plasmids for CCN protein production (IGFBP-, VWC-, TSP-, and CT-domain) with Brevibacillus as a host, cultivation methods for protein production, and methods of purification for domain proteins using his-tag.

  7. Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

    PubMed Central

    1985-01-01

    Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins. PMID:3988801

  8. Pancreatic secretory and trophic response to caerulein in rats: effect of proglumide and lorglumide.

    PubMed

    Varga, G; Papp, M; Scarpignato, C

    1989-01-01

    The effect of proglumide and lorglumide, two CCK-receptor antagonists, on caerulein-induced pancreatic secretion and growth was studied in the rat. In anaesthetised animals, caerulein (1 microgram/kg) significantly increased the volume of pancreatic juice and protein output. Lorglumide (5 and 10 mg/kg), administered intraperitoneally 15 min before stimulation, reduced peptide-induced pancreatic exocrine secretion. By contrast, proglumide (100 and 400 mg/kg) was completely ineffective. In experiments dealing with the trophic effect of caerulein, both drugs were administered alone or combined with the peptide (1 microgram/kg) 3 times daily for 5 d. Saline-treated rats served as controls. At the end of the experiment, rats were sacrificed, and growth and composition of pancreatic tissue were determined. Pretreatment of the animals with either proglumide or lorglumide did not affect pancreatic size and composition. Caerulein increased the weight of the pancreas, the total pancreatic protein, trypsin, amylase, and DNA content. After pretreatment with proglumide, all these parameters were not significantly different from those obtained with caerulein alone. In contrast, when lorglumide was given together with caerulein, it significantly reduced caerulein-induced pancreatic growth and decreased enzymatic protein content of the gland. These results show that lorglumide is a much more potent and effective CCK-receptor antagonist than proglumide. Its ability to antagonize the pancreatic secretory and trophic action of a CCK-analogue (i.e. caerulein) supports the view that these physiological actions of CCK are mediated through an interaction of the hormone with specific receptors.

  9. Expression and production of human chorionic gonadotropin (hCG) in the normal secretory endometrium: evidence of CGB7 and/or CGB6 beta hCG subunit gene expression.

    PubMed

    Zimmermann, Gerolf; Ackermann, Wilfried; Alexander, Henry

    2012-03-01

    We have previously confirmed glandular cell CGB and CGA subunit mRNA gene expression as well as the expression of their dimeric and single-subunit