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Secretory Pathway (secretory + pathway)
Kinds of Secretory Pathway Selected AbstractsFractional CO2 laser: a novel therapeutic device upon photobiomodulation of tissue remodeling and cytokine pathway of tissue repairDERMATOLOGIC THERAPY, Issue 2009F. Prignano ABSTRACT Minimally ablative fractional laser devices have gained acceptance as a preferred method for skin resurfacing. Notable improvements in facial rhytides, photodamage, acne scarring, and skin laxity have been reported. The aim of the present work was to compare how different CO2 laser fluences, by modulating the secretory pathway of cytokines, are able to influence the wound-healing process, and how these fluences are associated with different clinical results. Eighteen patients, all with photodamaged skin, were treated using a fractional CO2 laser (SmartXide DOT, Deka M.E.L.A., Florence, Italy) with varying laser fluences (2.07, 2.77, and 4.15 J/cm2). An immunocytochemical study was performed at defined end points in order to obtain information about specific cytokines of the microenvironment before and after treatment. The secretory pathway of cytokines changed depending on the re-epithelization and the different laser fluences. Different but significant improvements in wrinkles, skin texture, and hyperpigmentation were definitely obtained when using 2.07, 2.77, and 4.15 J/cm2, indicating fractional CO2 laser as a valuable tool in photorejuvenation with good clinical results, rapid downtime, and an excellent safety profile. [source] Expression of Gpr177, a Wnt trafficking regulator, in mouse embryogenesisDEVELOPMENTAL DYNAMICS, Issue 7 2010Hsiao-Man Ivy Yu Abstract Wls/Evi/Srt encoding a multipass transmembrane protein has been identified as a regulator for proper sorting and secretion of Wnt in flies. We have previously demonstrated that Gpr177 is the mouse ortholog required for axis determination. Gpr177 is a transcriptional target of Wnt that is activated to assist its subcellular distribution in a feedback regulatory loop. We, therefore, proposed that reciprocal regulation of Wnt and Gpr177 is essential for the Wnt-dependent developmental and pathogenic processes. Here, we examine the expression pattern of Gpr177 in mouse development. Gpr177 is expressed in a variety of tissues and cell types during organogenesis. Furthermore, Gpr177 is a glycoprotein primarily accumulating in the Golgi apparatus in signal-producing cells. The glycosylation of Gpr177 is necessary for proper transportation in the secretory pathway. Our findings suggest that the Gpr177-mediated regulation of Wnt is crucial for organogenesis in health and disease. Developmental Dynamics 239:2102,2109, 2010. © 2010 Wiley-Liss, Inc. [source] Efficient copackaging and cotransport yields postsynaptic colocalization of neuromodulators associated with synaptic plasticityDEVELOPMENTAL NEUROBIOLOGY, Issue 10 2008J.E. Lochner Abstract Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are copackaged and cotransported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively copackaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo cotransport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF colocalize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Mutagenesis studies in transgenic Xenopus intermediate pituitary cells reveal structural elements necessary for correct prion protein biosynthesisDEVELOPMENTAL NEUROBIOLOGY, Issue 6 2007Jos W.G. van Rosmalen Abstract The cellular prion protein (PrPC) is generally accepted to be involved in the development of prion diseases, but its physiological role is still under debate. To obtain more insight into PrPC functioning, we here used stable Xenopus transgenesis in combination with the proopiomelanocortin (POMC) gene promoter to express mutated forms of Xenopus PrPC fused to the C-terminus of the green fluorescent protein (GFP) specifically in the neuroendocrine Xenopus intermediate pituitary melanotrope cells. Similar to GFP-PrPC, the newly synthesized GFP-PrPCK81A mutant protein was stepwise mono- and di-N-glycosylated to 48- and 51-kDa forms, respectively, and eventually complex glycosylated to yield a 55-kDa mature form. Unlike GFP-PrPC, the mature GFP-PrPCK81A mutant protein was not cleaved, demonstrating the endoproteolytic processing of Xenopus PrPC at lysine residue 81. Surprisingly, removal of the glycosylphosphatidylinositol (GPI) anchor signal sequence or insertion of an octarepeat still allowed N-linked glycosylation, but the GFP-PrPC,GPI and GFP-PrPCocta mutant proteins were not complex glycosylated and not cleaved, indicating that the GPI/octa mutants did not reach the mid-Golgi compartment of the secretory pathway. The transgene expression of the mutant proteins did not affect the ultrastructure of the melanotrope cells nor POMC biosynthesis and processing, or POMC-derived peptide secretion. Together, our findings reveal the evolutionary conservation of the site of metabolic cleavage and the importance of the presence of the GPI anchor and the absence of the octarepeat in Xenopus PrPC for its correct biosynthesis. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulationFEBS JOURNAL, Issue 16 2009Lilian C. Russo Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 ,m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Structured digital abstract ,,MINT-7148420: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62161) bind (MI:0407) by surface plasmon resonance (MI:0107) ,,MINT-7148437: EP24.15 (uniprotkb:P52888) and Calmodulin (uniprotkb:P62158) colocalize (MI:0403) by surface plasmon resonance (MI:0107) ,,MINT-7148406: Calmodulin (uniprotkb:P62161) binds (MI:0407) to EP24.15 (uniprotkb:P52888) by pull down (MI:0096) [source] Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogenFEBS JOURNAL, Issue 2 2008Outi Itkonen Trypsinogen-1 and -2 are well-characterized enzymes that are expressed in the pancreas and also in several other tissues. Many cancers produce trypsinogen isoenzymes that differ from the pancreatic ones with respect to substrate specificity and isoelectric point. These tumor-associated trypsinogens play a pivotal role in cancer progression and metastasis. The differences between these and the pancreatic isoenzymes have been suggested to be caused by post-translational modification, either sulfation or phosphorylation of a tyrosine residue. We aimed to elucidate the cause of these differences. We isolated trypsinogens from pancreatic juice and conditioned medium from a colon carcinoma cell line. Intact proteins, and tryptic and chymotryptic peptides were characterized by electrospray ionization mass spectrometry. We also used immunoblotting with antibody against phosphotyrosine and N-terminal sequencing. The results show that pancreatic trypsinogen-1 and -2 are sulfated at Tyr154, whereas tumor-associated trypsinogen-2 is not. Detachment of a labile sulfogroup could be demonstrated by both in-source dissociation and low-energy collision-induced dissociation in a tandem mass spectrometer. Tyrosine sulfation is an ubiquitous protein modification occurring in the secretory pathway, but its significance is often underestimated due to difficulties in its analysis. Sulfation is an almost irreversible modification that is thought to regulate protein,protein interactions and the activity of proteolytic enzymes. We conclude that the previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are probably caused by sulfation of Tyr154 in pancreatic trypsinogens. [source] PC1/3, PC2 and PC5/6A are targeted to dense core secretory granules by a common mechanismFEBS JOURNAL, Issue 16 2007Jimmy D. Dikeakos There are seven members of the proprotein convertase (PC) family of secreted serine proteases that cleave their substrates at basic amino acids, thereby activating a variety of hormones, growth factors, and viruses. PC1/3, PC2 and PC5/6A are the only members of the PC family that are targeted to dense core secretory granules, where they carry out the processing of proteins that are secreted from the cell in a regulated manner. Previous studies have identified ,-helices in the C-termini of the PC1/3 and PC2 proteases that are required for this subcellular targeting. In the current study, we demonstrate that a predicted ,-helix in the C-terminus of PC5/6A is also critical for the ability of this domain to target a heterologous protein to the regulated secretory pathway of mouse endocrine AtT-20 cells. Analysis of the subcellular distribution of fusion proteins containing the C-terminal domains of PC1/3, PC2 and PC5/6A confirmed that all three domains have the capacity to redirect a constitutively secreted protein to the granule-containing cytoplasmic extensions. Analysis of the predicted structures formed by these three granule-sorting helices shows a correlation between their granule-sorting efficiency and the clustering of hydrophobic amino acids in their granule-targeting helices. [source] Highly efficient targeting and accumulation of a Fab fragment within the secretory pathway and apoplast of Arabidopsis thalianaFEBS JOURNAL, Issue 15 2001Koen Peeters To further improve antibody production in plants, constructs were designed to minimize transgene silencing and to retain a Fab fragment within the secretory pathway of transgenic Arabidopsis thaliana plants. The levels of antibody accumulation suggest that placing the sequences that encode Fd and light chain under the control of nonidentical 3, regions reduces susceptibility to post-transcriptional gene silencing compared with when the individual polypeptide-encoding sequences are placed under the control of identical 3, regions. High levels of accumulation (up to 6% of total soluble protein) were found for both secreted and intracellularly targeted antibody fragments. Immunofluorescence microscopic analysis showed that Fab fragments devoid of any additional C-terminal sequence were efficiently secreted, whereas retention of Fab fragments within the endomembrane system of the secretory pathway was achieved by C-terminal fusion of the DIKDEL sequence to the antibody light chain. Furthermore, analysis by immunoprecipitation and ELISA showed that intracellular retention of antibody fragments did not affect antigen-binding activity, and more than 80% of the isolated antibody fragments were found to bind antigen. Taken together, our results provide improvements to the technology of recombinant antibody production in transgenic plants. [source] Calcium and magnesium competitively influence the growth of a PMR1 deficient Saccharomyces cerevisiae strainFEMS MICROBIOLOGY LETTERS, Issue 2 2005Réka Szigeti Abstract PMR1, the Ca2+/Mn2+ ATPase of the secretory pathway in Saccharomyces cerevisiae was the first member of the secretory pathway Ca2+ ATPases (SPCA) to be characterized. In the past few years, pmr1, yeast have received more attention due to the recognition that the human homologue of this protein, hSPCA1 is defective in chronic benign pemphigus or Hailey,Hailey disease (HHD). Recent publications have described pmr1, S. cerevisiae as a useful model organism for studying the molecular pathology of HHD. Some observations indicated that the high Ca2+ sensitive phenotype of PMR1 defective yeast strains may be the most relevant in this respect. Here we show that the total cellular calcium response of a pmr1, S. cerevisiae upon extracellular Ca2+ challenge is decreased compared to the wild type strain similarly as observed in keratinocytes. Additionally, the novel magnesium sensitivity of PMR1 defective yeast is revealed, which appears to be a result of competition for uptake between Ca2+ and Mg2+ at the plasma membrane level. Our findings indicate that extracellular Ca2+ and Mg2+ competitively influence the intracellular Ca2+ homeostasis of S. cerevisiae. These observations may further our understanding of HHD. [source] Genetic and functional interaction between Ryh1 and Ypt3: two Rab GTPases that function in S. pombe secretory pathwayGENES TO CELLS, Issue 3 2006Yi He We have previously isolated ypt3-i5 mutant and showed that Ypt3 GTPase functions in the fission yeast secretory pathway. Here, the same genetic screen led to the isolation of ryh1-i6, a mutant allele of the ryh1+ gene encoding a homolog of Rab6. The ryh1-i6 mutant showed phenotypes that support its role in retrograde traffic from endosome to the Golgi. Interestingly, ryh1+ gene deletion was synthetically lethal with ypt3-i5 mutation. Consistently, the over-expression of the GDP-conformational mutant, Ryh1T25 N, inhibited the growth of ypt3-i5 mutant but had no effect on that of wild-type cells. Furthermore, the over-expression of the Ryh1T25N mutant inhibited the acid phosphatase glycosylation and exacerbated the cell wall integrity of ypt3-i5 mutant, but had no effect on those of wild-type cells. GFP-Ryh1 and GFP-Ypt3 both localized at the Golgi/endosome, but showed distinct subcellular localizations. The localization of GFP-Ryh1 in ypt3-i5 mutant and that of GFP-Ypt3 in ryh1-i6 mutant were distinct from those in wild-type cells. In addition, Ryh1 as well as Ypt3 were shown to be involved in acid phosphatase secretion. These results suggest that Ryh1 is involved in the secretory pathway and may have a potential overlapping function with Ypt3 in addition to its role in recycling. [source] Ebp2p, yeast homologue of a human protein that interacts with Epstein,Barr virus Nuclear Antigen 1, is required for pre-rRNA processing and ribosomal subunit assemblyGENES TO CELLS, Issue 7 2000Rota Tsujii Background A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response. Results EBP2, encoding the yeast homologue of a human protein that interacts with Epstein,Barr virus Nuclear Antigen 1, was cloned in a two-hybrid screen using RRS1 as a bait. The rrs1-1 mutation, which produces Rrs1p without the C-terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p-depleted cells, the rate of pre-rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild-type cells. The polysome pattern indicates that Ebp2p-depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half-mer polysomes. Conclusions Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis. [source] Confocal imaging and tracking of the exocytotic routes for D -serine-mediated gliotransmissionGLIA, Issue 12 2008Magalie Martineau Abstract D -Serine is an astrocyte-derived regulator for N -methyl- D -aspartate receptors, but the intracellular routes of its trafficking are still largely unknown. Here, we combined confocal microscopy with colocalization quantification to track the astrocytic organelles that store D -serine. We report that D -serine colocalizes with the transfected eGFP-synaptobrevin/VAMP2 and eGFP-cellubrevin/VAMP3, two v-SNAREs of the regulated secretory pathway. No significant colocalization was found with markers of the endosomal sorting and recycling system: EEA1, eGFP-endobrevin/VAMP8, eGFP-TI-VAMP/VAMP7, LAMP1, and CD63. Blockade of vesicular budding with colchicine shows that secretory vesicles import D -serine downstream to the Golgi apparatus. Finally, treatment of astrocytes with the Ca2+ -ionophore A23187, glutamate agonists, or bradykinin trigger translocation of synaptobrevin/VAMP2 to the plasma membrane with a concomitant disappearance of D -serine from the regulated secretory pathway. Our results provide morphological evidence for a vesicular storage of D -serine in the regulated secretory pathway and the possible recruitment of these stores by Ca2+ mobilization to release D -serine. © 2008 Wiley-Liss, Inc. [source] Primary hemophagocytic syndromes point to a direct link between lymphocyte cytotoxicity and homeostasisIMMUNOLOGICAL REVIEWS, Issue 1 2005Gael Ménasché Summary:, Hemophagocytic syndrome (HS) is a severe and often fatal syndrome resulting from potent and uncontrolled activation and proliferation of T-lymphocytes, leading to excessive macrophage activation and multiple deleterious effects. The onset of HS characterizes several inherited disorders in humans. In each condition, the molecular defect impairs the granule-dependent cytotoxic activity of lymphocytes, thus highlighting the determinant role of this function in driving the immune system to a state of equilibrium following infection. It has also been shown that some of the proteins required for lytic granule secretion are required for melanocyte function, leading to associated hypopigmentation in these conditions. This review focuses on several effectors of this secretory pathway, recently identified, because their defects cause these disorders, and discusses their role and molecular interactions in granule-dependent cytotoxic activity. [source] Storage and secretion of the peritrophic matrix protein Ag-Aper1 and trypsin in the midgut of Anopheles gambiaeINSECT MOLECULAR BIOLOGY, Issue 4 2004M. Devenport Abstract The gene Ag-Aper1 encodes a peritrophic matrix (PM) protein from the mosquito Anopheles gambiae. Ag-Aper1 gene expression and protein localization in the mosquito midgut were studied during the course of a blood meal. Ag-Aper1 mRNA abundance does not change appreciably during the course of blood ingestion and digestion. Prior to a blood meal, the protein is stored in secretory vesicles of midgut epithelial cells. Moreover, Ag-Aper1 colocalizes to the same secretory vesicles as trypsin, indicating that these proteins use a common secretory pathway. Blood feeding triggers the secretion of vesicle contents into the midgut lumen, after which Ag-Aper1 is incorporated into the PM. Newly synthesized Ag-Aper1 protein was again detected within the midgut epithelial cells at 60 h after blood ingestion. [source] Emerging functions of the calpain superfamily of cysteine proteases in neuroendocrine secretory pathwaysJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Joanne S. Evans Abstract The first calpain protease was discovered over 40 years ago now, yet despite the vast amount of literature that has subsequently emerged detailing their involvement in the pathophysiology of a variety of human diseases, it is only in the last decade that calpain-mediated actions along the secretory pathway have begun to emerge. However, the number of secretory pathway substrates identified and their diversity of function continues to grow. This review summarizes our current knowledge of calpain-mediated mechanisms of action that are pertinent to synaptic vesicle assembly and budding, cytoskeletal organization, endosomal recycling, and exocytotic membrane fusion. [source] In Vivo Gene Transfer Studies on the Regulation and Function of the Vasopressin and Oxytocin GenesJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2003D. Murphy Abstract Novel genes can be introduced into the germline of rats and mice by microinjecting fertilized one-cell eggs with fragments of cloned DNA. A gene sequence can thus be studied within the physiological integrity of the resulting transgenic animals, without any prior knowledge of its regulation and function. These technologies have been used to elucidate the mechanisms by which the expression of the two genes in the locus that codes for the neuropeptides vasopressin and oxytocin is confined to, and regulated physiologically within, specific groups of neurones in the hypothalamus. A number of groups have described transgenes, derived from racine, murine and bovine sources, in both rat and mouse hosts, that mimic the appropriate expression of the endogenous vasopressin and genes in magnocellular neurones (MCNs) of the supraoptic and paraventricular nuclei. However, despite considerable effort, a full description of the cis -acting sequences mediating the regulation of the vasopressin-oxytocin locus remains elusive. Two general conclusions have nonetheless been reached. First, that the proximal promoters of both genes are unable to confer any cell-specific regulatory controls. Second, that sequences downstream of the promoter, within the structural gene and/or the intergenic region that separates the two genes, are crucial for appropriate expression. Despite these limitations, sufficient knowledge has been garnered to specifically direct the expression of reporter genes to vasopressin and oxytocin MCNs. Further, it has been shown that reporter proteins can be directed to the regulated secretory pathway, from where they are subject to appropriate physiological release. The use of MCN expression vectors will thus enable the study of the physiology of these neurones through the targeted expression of biologically active molecules. However, the germline transgenic approach has a number of limitations involving the interpretation of phenotypes, as well as the large cost, labour and time demands. High-throughput somatic gene transfer techniques, principally involving the stereotaxic injection of hypothalamic neuronal groups with replication-deficient adenoviral vectors, are now being developed that obviate these difficulties, and which enable the robust, long-lasting expression of biologically active proteins in vasopressin and oxytocin MCNs. [source] Processing of Frameshifted Vasopressin PrecursorsJOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2000Evans Biosynthesis of the vasopressin (VP) prohormone in magnocellular neurones of the hypothalamo-neurohypophysial system comprises endoplasmic reticulum (ER) transit, sorting into the regulated secretory pathway and subsequent processing in the individual proteins VP, neurophysin and a glycoprotein. These processes are severely disrupted in the homozygous diabetes insipidus (di/di) Brattleboro rat, which expresses a mutant VP precursor due to a single nucleotide deletion in the neurophysin region of the VP gene resulting in VP deficiency. Previous studies have shown the presence of additional frameshift mutations in VP transcripts, in solitary magnocellular neurones of the di/di rat due to a GA dinucleotide deletion resulting in two different mutant VP precursors with partly restored reading frame. Frameshifted VP precursors are also expressed in several magnocellular neurones in wild-type rats. In this study, we determined if the +1 frameshifted precursors from di/di and wild-type rats can lead to biosynthesis of the hormone VP. Therefore, eukaryotic expression plasmids containing the frameshifted VP cDNAs were transiently expressed in peptidergic tumour cell lines, and cells were analysed by reversed phase high-performance liquid chromatography and specific radioimmunoassays, and by immunofluoresence. Neuro2A neuroblastoma cells expressing the +1 frameshifted precursors of di/di rats retained products in the cell body. Only precursor or insignificant quantities of neurophysin-immunoreactive products were detected. In contrast, in AtT20 cells, frameshifted VP precursors were at least partly processed to yield the VP peptide, indicating that they have access to the regulated secretory pathway. Comparison between the two cell lines showed a very slow ER transit of the wild-type prohormone combined with inefficient processing in Neuro2A cells. The results show that mutant precursors can reach the regulated secretory pathway if ER transport is sufficiently rapid as in the case of AtT20 cells. This suggests that the di/di rat may regain the capacity to biosynthesize authentic VP through these +1 frameshifted precursors in magnocellular neurones. [source] Endoplasmic reticulum-associated degradation of the NR1 but not the NR2 subunits of the N-methyl-D-aspartate receptor induced by inhibition of the N-glycosylation in cortical neuronsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2007Sergio Gascón Abstract The N-methyl-D-aspartate receptor (NMDAR) is fundamental to normal and pathological functioning of neurons. The receptor subunits are N-glycosylated proteins synthesized in the endoplasmic reticulum (ER) that fold, mature, and oligomerize as they transit through the secretory pathway. Although the early processes of biogenesis are fundamental to NMDAR expression and function, our knowledge of them is nevertheless limited. Additionally, the investigation of NMDAR synthesis is highly relevant, in that ER dysfunction, frequently associated with acute and degenerative brain diseases, might alter this process. We characterize here the effect of ER stress produced by inhibition of N-glycosylation on NMDAR synthesis and function. We use first heterologous systems of NMDAR expression in which NR1 and NR2A subunits are synthesized in nonneuronal cells. The function of these NMDARs as Ca2+ channels is repressed by tunicamycin, because of the inhibition of NR1, but no NR2A, synthesis. The regulation of NR1 is relevant to the central nervous system, in that a dramatic decrease in synthesis of this subunit and assembly of NMDARs is observed in cortical neurons treated with tunicamycin. The inhibition of NR1 synthesis is not due to changes in levels of mRNA but associated with the earliest stages in NMDAR biogenesis. The inhibition of N-glycosylation activates ER-specific stress responses in neurons, which include the ER-associated degradation (ERAD) mechanism responsible for differential and extremely efficient degradation of nonglycosylated NR1 by the proteasome after ubiquitination. Because this is an obligatory NMDAR component, the significant sensitivity of NR1 to ER stress will have important consequences on receptor function. © 2007 Wiley-Liss, Inc. [source] Dietary lithium induces regional increases of mRNA encoding cysteine string protein in rat brainJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2003Mara L. Cordeiro Abstract Lithium salts are used to treat manic-depressive disorders; however, the mechanism by which lithium produces its therapeutic benefit remains obscure. The action of lithium may involve alterations of proteins important for regulating synaptic function. In this context, we observed recently that lithium at therapeutically relevant concentrations enhanced expression of cysteine string protein (csp) at the level of both mRNA and protein, in cell culture and in rat brain. Several lines of evidence have shown that csps are vital components of the regulated secretory pathway. We were interested whether lithium modulates expression of csp in specific brain regions. To study this issue, we analyzed the effects of chronic lithium administration (21 days) on csp mRNA levels in rat brain using in situ hybridization. Densitometric analysis revealed that lithium upregulated csp mRNA in several brain areas that are important for mood and behavior. This effect may be germane to understanding the beneficial action of lithium in mood disorders. © 2003 Wiley-Liss, Inc. [source] Probing platelet factor 4 ,-granule targetingJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 12 2004V. Briquet-Laugier Summary., The storage mechanism of endogenous secretory proteins in megakaryocyte ,-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet ,-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in ,-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify ,-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in ,-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze ,-granule biogenesis and its alterations in the congenital defect gray platelet syndrome. [source] Identification of a cellular protein specifically interacting with the precursor of the hepatitis B e antigenJOURNAL OF VIRAL HEPATITIS, Issue 3 2001S. Salhi In hepatitis B virus (HBV) the precore gene encodes a protein from which derives P22, the precursor of the mature secreted hepatitis B virus e antigen (HBeAg). Circumstantial evidences suggest that HBeAg and/or its precursor P22 are important for establishing persistent infection. Although P22 is essentially present in the secretory pathway, a substantial fraction has been found in the cytosol. In order to get new insights into the biological function of P22, we looked for cellular proteins which could strongly associate with this protein. Using immunoprecipitation studies on human cell extracts, we found that a non-secreted cellular protein of about 32 kDa (P32) bound with a high specificity to P22. P32 associated neither with HBeAg nor with the viral core protein P21 which exhibits the same amino acids sequence as P22 but is N-terminally shorter by 10 residues. We also demonstrated that this interaction depended on the presence of the P22 C-terminal domain. Our data argues for a potential biological function of P22. [source] Fluorescent proteins as markers in the plant secretory pathwayMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2006Sally L. Hanton Abstract The use of fluorescent proteins and live cell imaging has greatly increased our knowledge of cell biology in recent years. Not only can these technologies be used to study protein trafficking under different conditions, but they have also been of use in elucidating the relationships between different organelles in a noninvasive manner. The use of multiple different fluorochromes allows the observation of interactions between organelles and between proteins, making this one of the fastest-developing and exciting fields at this time. In this review, we discuss the multitude of fluorescent markers that have been generated to study the plant secretory pathway. Although these markers have been used to solve many mysteries in this field, some areas that require further discussion remain. Microsc. Res. Tech. 69:152,159, 2006. © 2006 Wiley-Liss, Inc. [source] Investigating lipoprotein biogenesis and function in the model Gram-positive bacterium Streptomyces coelicolorMOLECULAR MICROBIOLOGY, Issue 4 2010Benjamin J. Thompson Summary Lipoproteins are a distinct class of bacterial membrane proteins that are translocated across the cytoplasmic membrane primarily by the Sec general secretory pathway and then lipidated on a conserved cysteine by the enzyme lipoprotein diacylglycerol transferase (Lgt). The signal peptide is cleaved by lipoprotein signal peptidase (Lsp) to leave the lipid-modified cysteine at the N-terminus of the mature lipoprotein. In all Gram-positive bacteria tested to date this pathway is non-essential and the lipid attaches the protein to the outer leaflet of the cytoplasmic membrane. Here we identify lipoproteins in the model Gram-positive bacterium Streptomyces coelicolor using bioinformatics coupled with proteomic and downstream analysis. We report that Streptomyces species translocate large numbers of lipoproteins out via the Tat (twin arginine translocase) pathway and we present evidence that lipoprotein biogenesis might be an essential pathway in S. coelicolor. This is the first analysis of lipoproteins and lipoprotein biogenesis in Streptomyces and provides the first evidence that lipoprotein biogenesis could be essential in a Gram-positive bacterium. This report also provides the first experimental evidence that Tat plays a major role in the translocation of lipoproteins in a specific bacterium. [source] An endocytic mechanism for haemoglobin-iron acquisition in Candida albicansMOLECULAR MICROBIOLOGY, Issue 1 2008Ziva Weissman Summary The fungal pathogen Candida albicans is able to utilize haemin and haemoglobin as iron sources. Haem-iron utilization is facilitated by Rbt5, an extracellular, glycosylphophatidylinositol (GPI)-anchored, haemin- and haemoglobin-binding protein. Here, we show that Rbt5 and its close homologue Rbt51 are short-lived plasma membrane proteins, degradation of which depends on vacuolar activity. Rbt5 facilitates the rapid endocytosis of haemoglobin into the C. albicans vacuole. We relied on recapitulation of the Rbt51-dependent haem-iron utilization in Saccharomyces cerevisiae to identify mutants defective in haemoglobin utilization. Homologues of representative mutants in S. cerevisiae were deleted in C. albicans and tested for haemoglobin-iron utilization and haemoglobin uptake. These mutants define a novel endocytosis-mediated haemoglobin utilization mechanism that depends on acidification of the lumen of the late secretory pathway, on a type I myosin and on the activity of the ESCRT pathway. [source] Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteinsMOLECULAR MICROBIOLOGY, Issue 5 2004Karine Kaiser Summary Invasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage-specific pre-erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite-specific genes encoding the circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low-molecular-mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite,red blood cell interactions. [source] Identification of a novel Gsp-related pathway required for secretion of the manganese-oxidizing factor of Pseudomonas putida strain GB-1MOLECULAR MICROBIOLOGY, Issue 4 2003Johannes De Vrind Summary The manganese-oxidizing factor of Pseudomonas putida strain GB-1 is associated with the outer membrane. One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp). The gsp genes are called xcp in Pseudomonas species. In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor. In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1). We show that xcmT1 is part of a two-gene operon that includes an xcpS homologue (designated xcmS). No other xcp -like genes are present in the regions flanking the xcmT1/xcmS cluster. We also characterized the site of transposon insertion of another transport mutant of P. putida GB-1. This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin-related protein. This xcmX is clustered with two other xcpT -related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side. The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium. A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P. putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well-described ,regular'xcp/gsp clusters. They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking. The results suggest a novel Xcp-related system for the transport of manganese-oxidizing enzymes to the cell surface. [source] The protein secretory pathway of Candida albicansMYCOSES, Issue 4 2009William A. Fonzi Summary Virulence of the opportunistic pathogen, Candida albicans, relies on an assemblage of attributes. These include the secretion of hydrolytic enzymes, cell surface adhesins, morphological transition between yeast and hyphae, phenotypic switching and biofilm formation. These diverse features are united by their dependence on the protein secretory apparatus for expression. Although the secretory apparatus of C. albicans has been studied limitedly, it appears to conform to the well-conserved eukaryotic system of vesicle-mediated transport between intracellular compartments and the cell surface. Genome comparison with Saccharomyces cerevisiae, however, shows multiple differences whose functional significance is yet unstudied. A unique aspect of the secretory pathway of C. albicans is its structural, and perhaps functional, rearrangement in hyphal vs. yeast cells. This, and evidence of non-conserved secretion mechanism(s), suggest that there is much fundamental knowledge to be derived from the analysis of secretion in C. albicans, which will be relevant to its ability to cause disease. [source] Anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plantsPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2008Alessandra Barbante Summary The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions. [source] Subcellular proteomics of Trypanosoma cruzi reservosomesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2009Celso Sant'Anna Abstract Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC-MS/MS analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi -specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles. [source] Characterisation of Plasmodium invasive organelles; an ookinete microneme proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2009Kalpana Lal Dr. Abstract Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14-fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC-MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion. [source] | |||||||||||||||||||||||||||||||