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At least 19 recordsLinked to original sources

PubMed2006

Protein secretion and secreted proteins in pathogenic Neisseriaceae.

Secreted proteins of pathogenic bacteria are often essential virulence factors. They are involved, for example, in the adherence of the bacteria to host cells or required to suppress the host's defence mechanisms. Until recently, only IgA1 protease had been studied in detail in the NeisseriaceaeNeisseria meningitidis and Neisseria gonorrhoeae. The availability of their genome sequences, however, has boosted research in this area. Here, we present a survey of the secretome of the pathogenic Neisseriaceae, based on the available genome sequences, and the current knowledge of the functions and structures of the secreted proteins. Of the six protein-secretion pathways that are widely disseminated among Gram-negative bacteria, three pathways appear to be present among the Neisseriaceae, i.e. the autotransporter-, the two-partner- and the type I-secretion mechanisms. Comparison of the predicted secretomes reveals a considerable flexibility. As compared with N. meningitidis and the nonpathogen N. lactamica, N. gonorrhoeae appears to have a considerably degenerated secretome, which may reflect its altered niche occupancy. The flexibility of the secretome may be enhanced by the presence of ORFs in the genomes potentially encoding fragments of secreted proteins. We hypothesize that these ORFs may substitute for the corresponding fragments in the full-length genes through genetic recombination, thereby changing the host-cell receptor specificity of the secreted protein.

Bacterial Proteins↗

PubMed1993

Corticotropin-induced secreted protein, an ACTH-induced protein secreted by adrenocortical cells, is structurally related to thrombospondins.

The treatment of primary cultures of bovine adrenocortical cells with nanomolar concentrations of ACTH induces a 10-fold increase in the synthesis of a secreted protein of apparent molecular mass 195 kDa on reducing SDS-polyacrylamide gels. This corticotropin-induced secreted protein (CISP) appears to be an oligomeric calcium-binding protein. Its secretion under serum-free culture conditions is sustained over 4 days in the continuous presence of ACTH. Induction of CISP secretion by ACTH is mimicked by cAMP analogs and adenylate cyclase activators. We report here the purification of CISP to apparent homogeneity with an overall yield of 43% using a combination of heparin-agarose and Mono-Q chromatographies. The NH2-terminal amino acid sequence and the sequence of several tryptic peptides revealed that CISP is structurally related to the members of the thrombospondin (TSP) family. Among these members, bovine CISP appeared to be more homologous to mouse TSP2 (85% identity in the 29 amino acid long NH2-terminal sequence) than to TSP1 (18% identity in the same region). We also observed that CISP binds Ca2+ and is an adhesive protein for bovine adrenocortical cells. Thus, CISP possesses both structural and functional properties of thrombospondins. Whether CISP represents the bovine form of TSP2 or a novel member of the expanding thrombospondin family will need to be elucidated by cloning and sequencing of a larger portion of the molecule.

Adrenal Cortex↗

PubMed2003

The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells.

To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca(2+)-ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter.

Biological Transport↗

PubMed2006

Genetic screen for signal peptides in Hydra reveals novel secreted proteins and evidence for non-classical protein secretion.

We have screened a Hydra cDNA library for sequences encoding N-terminal signal peptides using the yeast invertase secretion vector pSUC [Jacobs et al., 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198, 289-296]. We isolated and sequenced 907 positive clones; 88% encoded signal peptides; 12% lacked signal peptides. By searching the Hydra EST database we identified full-length sequences for the selected clones. These encoded 37 known proteins with signal peptides and 40 novel Hydra-specific proteins with signal peptides. Localization of two signal peptide-containing sequences, VEGF and ferritin, to the secretory pathway was confirmed with GFP fusion proteins. In addition, we isolated 105 clones which lacked signal peptides but which supported invertase secretion from yeast. Isolation of plasmids from these clones and retransformation in invertase-negative yeast cells confirmed the phenotype. A GFP fusion protein of one such clone encoding the foot morphogen pedibin was localized to the cytoplasm in transfected Hydra cells and did not enter the ER/Golgi secretory pathway. Secretion of pedibin and other proteins lacking signal peptides appears to occur by a non-classical protein secretion route.

Amino Acid Sequence↗

PubMed1985

Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion.

We have constructed a series of plasmids containing a modified form of the phoA gene of Escherichia coli K-12 that have general utility for studies of protein secretion. In these plasmids, the promoter and signal sequence-encoding region of the phoA gene have been deleted; thus, expression of the gene, giving rise to active alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1], is absolutely dependent upon fusion in the correct reading frame to DNA containing a promoter, a translational start site, and a complete signal sequence-encoding region. Alkaline phosphatase, which is normally located in the periplasm of E. coli, is efficiently secreted to the periplasm when fused either to a signal sequence from another periplasmic protein, beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6), or to signal sequences from the outer membrane proteins LamB and OmpF. These heterologous signal sequences are processed during secretion. In the absence of a complete signal sequence, phosphatase becomes localized in the cytoplasm and is inactive. Phosphatase fusion proteins lacking up to 13 amino-terminal amino acids beyond the signal sequence show the same specific activity as that of the wild-type enzyme. However, a significant decrease in activity is seen when 39 or more amino-terminal amino acids are deleted. Addition of approximately 150 amino acids from the enzyme beta-lactamase to the amino terminus of alkaline phosphatase has little effect on the specific activity of the enzyme. The ability to change the amino terminus of phosphatase without altering its activity makes the enzyme particularly useful for construction of protein fusions. The fact that phosphatase is designed for transport across the cytoplasmic membrane makes it an ideal tool for study of protein secretion.

Alkaline Phosphatase↗

PubMed1996

Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins.

Enteropathogenic Escherichia coli (EPEC) secretes at least five proteins. Two of these proteins, EspA and EspB (previously called EaeB), activate signal transduction pathways in host epithelial cells. While the role of the other three proteins (39, 40, and 110 kDa) remains undetermined, secretion of all five proteins is under the control of perA, a known positive regulator of several EPEC virulence factors. On the basis of amino-terminal protein sequence data, we cloned and sequenced the gene which encodes the 110-kDa secreted protein and examined its possible role in EPEC signaling and interaction with epithelial cells. In accordance with the terminology used for espA and espB, we called this gene espC, for EPEC-secreted protein C. We found significant homology between the predicted EspC protein sequence and a family of immunoglobulin A (IgA) protease-like proteins which are widespread among pathogenic bacteria. Members of this protein family are found in avian pathogenic Escherichia coli (Tsh), Haemophilus influenzae (Hap), and Shigella flexneri (SepA). Although these proteins and EspC do not encode IgA protease activity, they have considerable homology with IgA protease from Neisseria gonorrhoeae and H. influenzae and appear to use a export system for secretion. We found that genes homologous to espC also exist in other pathogenic bacteria which cause attaching and effacing lesions, including Hafnia alvei biotype 19982, Citrobacter freundii biotype 4280, and rabbit diarrheagenic E. coli (RDEC-1). Although these strains secrete various proteins similar in molecular size to the proteins secreted by EPEC, we did not detect secretion of a 110-kDa protein by these strains. To examine the possible role of EspC in EPEC interactions with epithelial cells, we constructed a deletion mutant in espC by allelic exchange and characterized the mutant by standard tissue culture assays. We found that EspC is not necessary for mediating EPEC-induced signal transduction in HeLa epithelial cells and does not play a role in adherence or invasion of tissue culture cells.

Amino Acid Sequence↗

PubMed1999

Loss of insulin-like growth factor I receptor-dependent expression of p107 and cyclin A in cells that lack the extracellular matrix protein secreted protein acidic and rich in cysteine.

The extracellular matrix-associated glycoprotein secreted protein acidic and rich in cysteine (SPARC) has been implicated in the control of cell proliferation during tissue remodeling, wound healing, and malignant development. Here, we describe a novel mechanism through which SPARC influences cell cycle progression in embryonic fibroblasts derived from Sparc-nullizygous (-/-) mice. SPARC-deficient cells were indistinguishable from wild-type cells in their ability to initiate DNA synthesis after treatment with either fetal bovine serum or platelet-derived growth factor. In contrast, Sparc -/- cells responded poorly to activation of the insulin-like growth factor receptor (IGFI-R) by insulin. This defect was traced to reduced expression of the IGFI-R in Sparc -/- cells. Consistent with impaired cell cycle progression through S-phase, insulin-stimulated Sparc -/- cells also revealed reduced expression of two key regulators of S phase progression (cyclin A and thymidine kinase), whereas expression of the G1 phase progression regulators cmyc or cyclin D1 was unaffected. An examination of the status of retinoblastoma family pocket proteins in Sparc -/- cells revealed a selective and dramatic reduction in levels of the retinoblastoma-related protein p107. Exogenous platelet-derived growth factor restored expression of the IGFI-R and IGFI-R dependent DNA synthesis as well as induction of cyclin A, thymidine kinase, and p107 in insulin-stimulated Sparc -/- cells. These results suggest that SPARC-dependent matrix to cell interactions contribute to the regulation of p107 and cyclin A through IGFI-R dependent pathway(s).

Animals↗

PubMed2004

Plasmodium ookinete-secreted proteins secreted through a common micronemal pathway are targets of blocking malaria transmission.

The mosquito midgut ookinete stage of the malaria parasite, Plasmodium, possesses microneme secretory organelles that mediate locomotion and midgut wall egress to establish sporogonic stages and subsequent transmission. The purpose of this study was 2-fold: 1) to determine whether there exists a single micronemal population with respect to soluble and membrane-associated secreted proteins; and 2) to evaluate the ookinete micronemal proteins chitinase (PgCHT1), circumsporozoite and TRAP-related protein (CTRP), and von Willebrand factor A domain-related protein (WARP) as immunological targets eliciting sera-blocking malaria parasite infectivity to mosquitoes. Indirect immunofluorescence localization studies in Plasmodium gallinaceum using specific antisera showed that all three proteins are distributed intracellularly with a similar granular cytoplasmic appearance and with focal concentration of PgCHT1 and PgCTRP, but not PgWARP, at the ookinete apical end. Immunogold double-labeling electron microscopy, using antisera against the membrane-associated protein CTRP and the soluble WARP, showed that these two proteins co-localized to the same micronemal population. Within the microneme CTRP was associated peripherally at the microneme membrane, whereas PgCHT1 and WARP were diffuse within the micronemal lumen. Sera produced against Plasmodium falciparum WARP significantly reduced the infectivity of P. gallinaceum to Aedes aegypti and P. falciparum to Anopheles mosquitoes. Antisera against PgCTRP and PgCHT1 also significantly reduced the infectivity of P. gallinaceum for A. aegypti. These results support the concept that ookinete micronemal proteins may constitute a general class of malaria transmission-blocking vaccine candidates.

Amino Acid Sequence↗

PubMed1996

Protein secretion by heterologous bacterial ABC-transporters: the C-terminus secretion signal of the secreted protein confers high recognition specificity.

Pseudomonas aeruginosa releases several extracellular proteins which are secreted via two independent secretion pathways. Alkaline protease (AprA) Is released by its own specific secretion machinery which is an ABC-transporter. Despite sequence similarities between components of ABC-transporters in different bacteria, each transporter is dedicated to the secretion of a particular protein or a family of closely related proteins. Heterologous complementation between ABC-transporters for unrelated polypeptides can occur, but only at a very low level. We show that the 50 C-terminal amino acids of AprA constitute an autonomous secretion signal. By heterologous complementation experiments between the unrelated alpha-haemolysin (HlyA) and Apr secretion systems we demonstrated that it is only the recognition of the secretion signal by the translocator which confers specificity to the secretion process. Secretion was size-dependent. However inclusion of glycine-rich repeats from HlyA in AprA seems to overcome the size limitation exerted by the Apr secretion apparatus such that the machinery secreted a hybrid protein 20 kDa larger than the normal maximal size.

ATP-Binding Cassette Transporters↗

PubMed2006

A novel recently evolved gene C19orf24 encodes a non-classical secreted protein.

Secreted proteins play important roles in many crucial biological processes, and can be new agents or targets for drug therapies. Here, we report on the isolation and characterization of a novel human non-classical secreted protein which is encoded by the hypothetical gene C19orf24 (chromosome 19 open reading frame 24). It has no signal peptide, but can still secrete extracellularly despite the presence of the inhibitor brefeldin A (BFA), proving its non-classical secreted protein status. Via subcellular localization using C19orf24 in vivo and transfected pEYFP-Golgi plasmid in Hela cells, C19orf24 was shown not to co-localize in the Golgi apparatus, which suggested that it secretes via a new and unknown pathway. Deglycosylation analysis with PNGase F verified that it has no N-glycosylation modification sites. Via the reverse transcription-PCR method, it was found to be expressed only in the human liver, and preferentially in normal tissue. In addition, C19orf24 was shown to be a recently evolved gene, found only in Homo sapiens and Pan troglodytes. By calculating its synonymous and non-synonymous substitution rate (d (S)/d (N)), we found that it experienced a purifying selection, which suggests that C19orf24 may have a special, irreplaceable biological function in the human organism.

Amino Acid Sequence↗

PubMed2004

Amblyomma americanum salivary glands: double-stranded RNA-mediated gene silencing of synaptobrevin homologue and inhibition of PGE2 stimulated protein secretion.

Protein secretion into the saliva from the tick salivary glands is due to exocytosis of vesicular membrane bound granular material regulated by SNARE complex proteins after salivary gland stimulation by PGE2 [Insect Biochem. Mol. Biol. 32 (2002) 1711]. Proteins associated with vesicles (v-SNAREs) are essential components of the exocytotic process. Synaptobrevin is a key v-SNARE in all secreting cells studied to date. A vesicle-associated synaptobrevin cDNA fragment homologue from the salivary glands of partially fed lone star tick females was cloned and sequenced. Double-stranded (ds) RNA interference (RNAi) is an effective method to silence specific gene expression. The functional role of synaptobrevin in protein secretion in partially fed tick salivary glands was studied with an in vitro RNAi method. Incubation of isolated salivary glands with double-stranded RNA (dsRNA) transcribed from a tick salivary gland synaptobrevin cDNA fragment resulted in decreased expression of the transcript, a reduction in the level of synaptobrevin protein and inhibition of PGE2 stimulated anticoagulant protein secretion by isolated salivary glands. We demonstrate the applicability of RNAi for studying individual steps in the mechanism of PGE2 stimulated exocytosis in the salivary glands of ixodid ticks.

Amino Acid Sequence↗

PubMed2002

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

Protein secretion from Bacillus species is a major industrial production tool with a market of over $1 billion per year. However, standard export technologies, based on the well-characterised general secretory (Sec) pathway, are frequently inapplicable for the production of proteins. The recently discovered twin-arginine translocation (Tat) pathway offers additional potential to transport proteins. Here we review the use of functional genomic and proteomic approaches to explore the Tat pathway of Bacillus subtilis. The properties of Tat pathway components and the twin-arginine signal peptides that direct proteins into this pathway are discussed. Where appropriate, a comparison is made with Tat systems from other organism, such as Escherichia coli. Recent findings with the latter organism in particular provide proof-of-principle that the Tat pathway can be exploited for the production of Sec-incompatible proteins.

Amino Acid Sequence↗

PubMed2004

N-terminal-capturing screening system for the isolation of Brucella abortus genes encoding surface exposed and secreted proteins.

Secreted as well as surface exposed proteins are assumed to play major roles in bacterial virulence. In this report we describe the construction of an N-terminal protein-capturing system and its use for the isolation of Brucella abortus S2308 genes coding for putative surface exposed or secreted proteins. For this purpose, a cloning vector that generates gene fusions to a ribosome binding site and start codon deficient Chloramphenicol Acetyl Transferase (CAT) reporter gene was constructed and the resulting library introduced into B. abortus S2308 and virB mutant strains. Secreted translational fusions were identified by determining CAT activity in culture supernatants. Secretion was confirmed by Western Blot using a polyclonal anti-CAT antibody. A total of 864 clones were screened and 10 genes encoding putative secreted/surface exposed proteins were identified. Seven are Brucella proteins with an assigned function, whereas three are hypothetical proteins. The number of amino acid residues that promotes CAT secretion varies from 5 to 386 and no conserved motifs were detected. Secretion in a virB mutant background of some of the isolated fusion proteins was also determined. Interestingly, some hybrid proteins seemed to require a full VirB system for their secretion.

Bacterial Proteins↗

PubMed1992

Dissociation of CCK-8-induced fluid secretion from protein secretion by ion-transport blockers in rat pancreas.

The effects of ion-transport blockers on CCK-8-induced protein output and concomitant fluid secretion were compared in isolated, perfused normal and hypertrophied rat pancreata. In the normal pancreas, perfusion with ouabain (1 mM), amiloride (1 mM), furosemide (1 mM), or SITS (0.1 mM) caused corresponding inhibition of both fluid and protein secretion that was induced by 100 pM CCK-8. Hypertrophy of the pancreas was produced by oral administration of a synthetic protease inhibitor (FOY-305) once a day for 3 wk. In the hypertrophied pancreas, perfusion with ouabain (0.1 or 1 mM) or amiloride (0.1 mM or 1 mM) decreased CCK-8-induced fluid secretion without changing CCK-8-induced protein output. Perfusion with furosemide (1 mM) inhibited both fluid and protein secretion induced by CCK-8, but the amount of inhibition of fluid secretion was much greater than that of protein secretion. Perfusion with SITS (0.1 mM) significantly decreased CCK-8-induced fluid secretion but not protein secretion. These results indicate that in contrast to a normal rat pancreas, the coupling of fluid and protein secretion induced by CCK-8 can be disrupted by experimental procedures that induce hypertrophy in the rat pancreas.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed2005

Analysis of secreted proteins from Aspergillus flavus.

MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus.

Aspergillus flavus↗

PubMed1998

A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture.

The Hrp (type III protein secretion) system is essential for the plant parasitic ability of Pseudomonas syringae and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two P. syringae proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp+ bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in Escherichia coli to secrete AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The hrp gene cluster, carried on cosmid pCPP2156, was cloned from Erwinia chrysanthemi, a pathogen that differs from P. syringae in being host promiscuous. E. coli DH5alpha carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in Nicotiana clevelandii only when also expressing avrB in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion, enabled immunoblot detection of the secretion of these proteins to E. coli(pCPP2156) culture media. Secretion was Hrp dependent, occurred without leakage of a cytoplasmic marker, and did not occur with E. coli(pHIR11), which encodes a functional P. syringae Hrp system. E. coli(pCPP2156) will promote investigation of Avr protein secretion and systematic prospecting for the effector proteins underlying bacterial plant pathogenicity.

Bacterial Proteins↗

PubMed2005

Type II secretion: a protein secretion system for all seasons.

In Gram-negative bacteria, type II secretion (T2S) is one of five protein secretion systems that permit the export of proteins from within the bacterial cell to the extracellular milieu and/or into target host cells. An analysis of numerous sequenced genomes now reveals that T2S genes are common, but by no means universal, in Gram-negative bacteria. Recent functional studies indicate that T2S can promote the virulence of human, animal and plant pathogens, as well as the physiology of various environmental bacteria. Thus, it is an opportune time to highlight the new and different ways in which T2S serves bacterial function.

Bacterial Proteins↗

PubMed2004

Development of secreted proteins as biotherapeutic agents.

As one of the most important classes of proteins, secreted factors account for about one-tenth of the human genome, 3000 - 4000 in total, including factors of signalling pathways, blood coagulation and immune defence, as well as digestive enzymes and components of the extracellular matrix. Secreted proteins are a rich source of new therapeutics and drug targets, and are currently the focus of major drug discovery programmes throughout the industry. Many of the most important novel drugs developed in biotechnology have resulted from the application of secreted proteins as therapeutics. Secreted proteins often circulate throughout the body and, therefore, have access to most organs and tissues. Because of that, many of the factors are themselves therapeutic agents. This paper gives an overview on the features and functions of human secreted proteins and peptides, as well as strategies by which to discover additional therapeutic proteins from the human 'secretome'. Furthermore, a variety of examples are provided for the therapeutic use of recombinant secreted proteins as 'biologicals', including features and applications of recombinant antibodies, erythropoietin, insulin, interferon, plasminogen activators, growth hormone and colony-stimulating factors.

Drug Design↗