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William Y. Chey - One of the best experts on this subject based on the ideXlab platform.
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Secretin: historical perspective and current status.
Pancreas, 2014Co-Authors: William Y. Chey, Ta-min ChangAbstract:This review describes the history of Secretin discovery, identification, purification, and structural determination; cloning of Secretin and its receptor; synthetic Secretin; and highly specific and sensitive radioimmunoassay to define the characteristic physiological role on postprandial pancreatic fluid and bicarbonate secretion, which requires robust potentiation by cholecystokinin. Secretin plays a key role in the negative and positive regulatory mechanisms of exocrine pancreatic secretion. Secretin-releasing peptides were discovered in duodenal acid perfusates of both rat and dog and in canine pancreatic juice. The release and action of Secretin and Secretin-releasing peptides are in part mediated via vagovagal reflex mechanism involving afferent sensory neurons in proximal intestine and efferent cholinergic neurons in the pancreas. Besides acetylcholine, many neurotransmitters or neuromodulators influence release and action of Secretin. The action of Secretin in the pancreas depends on insulin, which also suppresses local release of somatostatin and pancreatic polypeptide. Thus, release and action of Secretin are mediated via neurohormonal interaction. Clinical conditions with hyperSecretinemia and hypoSecretinemia are discussed. Synthetic human Secretin is used for studies of exocrine pancreatic secretion, Secretin-enhanced magnetic resonance cholangiopancreatography combined with exocrine pancreatic function test and diagnosis of gastrinoma syndrome. Therapeutic use of Secretin is considered for the relief of severe pain in chronic pancreatitis.
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CHAPTER 153 – Secretin
Handbook of Biologically Active Peptides, 2006Co-Authors: William Y. Chey, Ta-min ChangAbstract:Secretin is the first peptide hormone ever discovered. Secretin is a 27-amino-acid peptide of Secretin-glucagon-vasoactive intestinal polypeptide superfamily and is localized mainly in the upper small intestinal mucosa. The gene structures of Secretin and its receptor have been determined. Compared with several bio-active proSecretins, Secretin is the most active form. The physiological functions of Secretin include the stimulation of pancreatic exocrine secretion of water and electrolytes, inhibition of gastric acid secretion, and motility. The physiological release and actions of Secretin are subjected to hormone-hormonal and neural-hormonal regulations, among which the vagal afferent pathway plays a significant role. Secretin is used clinically to assess pancreatic function and test for pancreatic malignancy. Pathological states of both hypoSecretinemia and hyperSecretinemia have been well documented.
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Secretin, 100 years later
Journal of Gastroenterology, 2003Co-Authors: William Y. Chey, Ta-min ChangAbstract:One hundred years have elapsed since the discovery of Secretin by Bayliss and Starling in 1902. In the past century, the research of Secretin has gone by many milestones including isolation, purification and structural determination, chemical synthesis, establishment of its hormonal status by radioimmunoassay and immunoneutralization, identification of the specific receptor, cloning of Secretin and its receptor, and identification of a Secretin-releasing peptide. It has become clear that Secretin is a hormone-regulating pancreatic exocrine secretion of fluid and bicarbonate, gastric acid secretion, and gastric motility. The release and actions of Secretin is regulated by hormone–hormonal and neurohormonal interactions. The vagus nerve, particularly its afferent pathway, plays an essential role in the physiological actions of Secretin. Substantial information about the property of the Secretin receptor has been accumulated, but a potent Secretin receptor-specific antagonist remains to be formulated. The neural regulatory mechanisms of the release and action of Secretin await further elucidation. The physiological role of Secretin in intestinal secretions and motility and extragastrointestinal organs remains to be defined. The presence of Secretin and its receptor in the central nervous system is well documented, but its function as a neuropeptide has been recognized gradually and requires extensive study in the future.
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A Secretin releasing peptide exists in dog pancreatic juice.
Life sciences, 2000Co-Authors: Yu Song, Ta-min Chang, Kae Yol Lee, William Y. CheyAbstract:Abstract Canine pancreatic juice has been shown to stimulate exocrine pancreatic secretion in the dog. In the present study we investigated whether there is a Secretin-releasing peptide in canine pancreatic juice. Pancreatic juice was collected from the dogs with Thomas gastric and duodenal cannulas while pancreatic secretion was stimulated by intravenous administration of Secretin at 0.5 μ/gk/gh and CCK-8 at 0.2 μg/kg/h, respectively. The pancreatic juice was separated into three different molecular weight (MW) fractions (Fr) by ultrafiltration (Fr 1; MW > 10,000, Fr 2; MW = 10,000 − 4,000 and Fr 3; MW mg ml for 60 min at 37 ° C) but not by heating (100 ° C, 10 min). Intravenous injection of a rabbit anti-Secretin serum, which rendered plasma Secretin almost undetectable in rat plasma, also abolished Fr 3-stimulated pancreatic secretion of fluid and bicarbonate secretion. These observations suggest that a Secretin-releasing peptide exists in the canine pancreatic juice. It is trypsin-sensitive and heat-resistant. This peptide may play a significant physiological role on the release of Secretin and regulation of exocrine pancreatic secretion.
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significant cholinergic role in Secretin stimulated exocrine secretion in isolated rat pancreas
American Journal of Physiology-gastrointestinal and Liver Physiology, 1998Co-Authors: H S Park, Hyeok Yil Kwon, William Y. CheyAbstract:Effects of intrapancreatic cholinergic activation by electrical field stimulation (EFS) on Secretin-stimulated pancreatic exocrine secretion were investigated in the totally isolated perfused rat pancreas. EFS at 15 V, 2 ms, and 8 Hz for 45 min markedly increased spontaneous pancreatic secretion. This increase was completely inhibited by tetrodotoxin (1 μM) but not by hexamethonium (100 μM). Atropine (2 μM) significantly reduced the EFS-evoked volume flow and amylase output by 52% and 80%, respectively. EFS further increased the Secretin (12 pM)-stimulated pancreatic secretion of fluid and amylase. The increases of the two parameters were significantly suppressed by atropine by 28% and 72%, respectively. Interestingly, EFS significantly increased concentrations of somatostatin-like immunoreactivity in portal venous effluents. When pertussis toxin (200 ng/ml) or rabbit antisomatostatin serum (0.1 ml/10 ml; titer of 1:50,000) was intra-arterially administered, EFS further increased the Secretin-stimulated pancreatic secretion. In conclusion, the activation of intrapancreatic cholinergic neurons potentiated the Secretin action on pancreatic exocrine secretion in the rat. This potentiating effect was significantly reduced by local somatostatin released during EFS that activated intrapancreatic cholinergic tone.
Andréa Dessen - One of the best experts on this subject based on the ideXlab platform.
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Bacterial Secretins: Mechanisms of assembly and membrane targeting
Protein Science, 2020Co-Authors: Yuri Rafael De Oliveira Silva, Carlos Contreras-martel, Pauline Macheboeuf, Andréa DessenAbstract:Secretion systems are employed by bacteria to transport macromolecules across membranes without compromising their integrities. Processes including virulence, colonization, and motility are highly dependent on the secretion of effector molecules toward the immediate cellular environment, and in some cases, into the host cytoplasm. In Type II and Type III secretion systems, as well as in Type IV pili, homomultimeric complexes known as Secretins form large pores in the outer bacterial membrane, and the localization and assembly of such 1 MDa molecules often relies on pilotins or accessory proteins. Significant progress has been made toward understanding details of interactions between Secretins and their partner proteins using approaches ranging from bacterial genetics to cryo electron microscopy. This review provides an overview of the mode of action of pilotins and accessory proteins for T2SS, T3SS, and T4PS Secretins, highlighting recent near-atomic resolution cryo-EM Secretin complex structures and underlining the importance of these interactions for Secretin functionality.
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Structure and assembly of pilotin-dependent and -independent Secretins of the type II secretion system
PLoS Pathogens, 2019Co-Authors: S Peter Howard, Leandro F Estrozi, Viviana Job, Guy Schoehn, Carlos Contreras-martel, Quentin Bertrand, Timothy Strozen, Alexandre Martins, Daphna Fenel, Andréa DessenAbstract:The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the Secretin. Localization and assembly of Secretins in the outer membrane requires recognition of Secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential Secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the Secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent Secretin assembly by performing modelling with the C-terminus of EpsD. We also show that Secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD Secretin. Since Secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.
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Structural similarity of Secretins from type II and type III secretion systems.
Structure, 2014Co-Authors: Tommaso Tosi, Leandro F Estrozi, Viviana Job, Ingrid Guilvout, Anthony P Pugsley, Guy Schoehn, Andréa DessenAbstract:Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system Secretin, and PulD, the Klebsiella oxytoca type II secretion system Secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both Secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both Secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that Secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.
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outer membrane targeting of Secretin puld protein relies on disordered domain recognition by a dedicated chaperone
Journal of Biological Chemistry, 2011Co-Authors: Andréa Dessen, Nicholas N Nickerson, Tommasso Tosi, Bruno BaronAbstract:Interaction of bacterial outer membrane Secretin PulD with its dedicated lipoprotein chaperone PulS relies on a disorder-to-order transition of the chaperone binding (S) domain near the PulD C terminus. PulS interacts with purified S domain to form a 1:1 complex. Circular dichroism, one-dimensional NMR, and hydrodynamic measurements indicate that the S domain is elongated and intrinsically disordered but gains secondary structure upon binding to PulS. Limited proteolysis and mass spectrometry identified the 28 C-terminal residues of the S domain as a minimal binding site with low nanomolar affinity for PulS in vitro that is sufficient for outer membrane targeting of PulD in vivo. The region upstream of this binding site is not required for targeting or multimerization and does not interact with PulS, but it is required for Secretin function in type II secretion. Although other Secretin chaperones differ substantially from PulS in sequence and secondary structure, they have all adopted at least superficially similar mechanisms of interaction with their cognate Secretins, suggesting that intrinsically disordered regions facilitate rapid interaction between Secretins and their chaperones.
Ta-min Chang - One of the best experts on this subject based on the ideXlab platform.
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Secretin: historical perspective and current status.
Pancreas, 2014Co-Authors: William Y. Chey, Ta-min ChangAbstract:This review describes the history of Secretin discovery, identification, purification, and structural determination; cloning of Secretin and its receptor; synthetic Secretin; and highly specific and sensitive radioimmunoassay to define the characteristic physiological role on postprandial pancreatic fluid and bicarbonate secretion, which requires robust potentiation by cholecystokinin. Secretin plays a key role in the negative and positive regulatory mechanisms of exocrine pancreatic secretion. Secretin-releasing peptides were discovered in duodenal acid perfusates of both rat and dog and in canine pancreatic juice. The release and action of Secretin and Secretin-releasing peptides are in part mediated via vagovagal reflex mechanism involving afferent sensory neurons in proximal intestine and efferent cholinergic neurons in the pancreas. Besides acetylcholine, many neurotransmitters or neuromodulators influence release and action of Secretin. The action of Secretin in the pancreas depends on insulin, which also suppresses local release of somatostatin and pancreatic polypeptide. Thus, release and action of Secretin are mediated via neurohormonal interaction. Clinical conditions with hyperSecretinemia and hypoSecretinemia are discussed. Synthetic human Secretin is used for studies of exocrine pancreatic secretion, Secretin-enhanced magnetic resonance cholangiopancreatography combined with exocrine pancreatic function test and diagnosis of gastrinoma syndrome. Therapeutic use of Secretin is considered for the relief of severe pain in chronic pancreatitis.
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CHAPTER 153 – Secretin
Handbook of Biologically Active Peptides, 2006Co-Authors: William Y. Chey, Ta-min ChangAbstract:Secretin is the first peptide hormone ever discovered. Secretin is a 27-amino-acid peptide of Secretin-glucagon-vasoactive intestinal polypeptide superfamily and is localized mainly in the upper small intestinal mucosa. The gene structures of Secretin and its receptor have been determined. Compared with several bio-active proSecretins, Secretin is the most active form. The physiological functions of Secretin include the stimulation of pancreatic exocrine secretion of water and electrolytes, inhibition of gastric acid secretion, and motility. The physiological release and actions of Secretin are subjected to hormone-hormonal and neural-hormonal regulations, among which the vagal afferent pathway plays a significant role. Secretin is used clinically to assess pancreatic function and test for pancreatic malignancy. Pathological states of both hypoSecretinemia and hyperSecretinemia have been well documented.
-
Secretin, 100 years later
Journal of Gastroenterology, 2003Co-Authors: William Y. Chey, Ta-min ChangAbstract:One hundred years have elapsed since the discovery of Secretin by Bayliss and Starling in 1902. In the past century, the research of Secretin has gone by many milestones including isolation, purification and structural determination, chemical synthesis, establishment of its hormonal status by radioimmunoassay and immunoneutralization, identification of the specific receptor, cloning of Secretin and its receptor, and identification of a Secretin-releasing peptide. It has become clear that Secretin is a hormone-regulating pancreatic exocrine secretion of fluid and bicarbonate, gastric acid secretion, and gastric motility. The release and actions of Secretin is regulated by hormone–hormonal and neurohormonal interactions. The vagus nerve, particularly its afferent pathway, plays an essential role in the physiological actions of Secretin. Substantial information about the property of the Secretin receptor has been accumulated, but a potent Secretin receptor-specific antagonist remains to be formulated. The neural regulatory mechanisms of the release and action of Secretin await further elucidation. The physiological role of Secretin in intestinal secretions and motility and extragastrointestinal organs remains to be defined. The presence of Secretin and its receptor in the central nervous system is well documented, but its function as a neuropeptide has been recognized gradually and requires extensive study in the future.
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A Secretin releasing peptide exists in dog pancreatic juice.
Life sciences, 2000Co-Authors: Yu Song, Ta-min Chang, Kae Yol Lee, William Y. CheyAbstract:Abstract Canine pancreatic juice has been shown to stimulate exocrine pancreatic secretion in the dog. In the present study we investigated whether there is a Secretin-releasing peptide in canine pancreatic juice. Pancreatic juice was collected from the dogs with Thomas gastric and duodenal cannulas while pancreatic secretion was stimulated by intravenous administration of Secretin at 0.5 μ/gk/gh and CCK-8 at 0.2 μg/kg/h, respectively. The pancreatic juice was separated into three different molecular weight (MW) fractions (Fr) by ultrafiltration (Fr 1; MW > 10,000, Fr 2; MW = 10,000 − 4,000 and Fr 3; MW mg ml for 60 min at 37 ° C) but not by heating (100 ° C, 10 min). Intravenous injection of a rabbit anti-Secretin serum, which rendered plasma Secretin almost undetectable in rat plasma, also abolished Fr 3-stimulated pancreatic secretion of fluid and bicarbonate secretion. These observations suggest that a Secretin-releasing peptide exists in the canine pancreatic juice. It is trypsin-sensitive and heat-resistant. This peptide may play a significant physiological role on the release of Secretin and regulation of exocrine pancreatic secretion.
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dual inhibitory mechanism of Secretin action on acid secretion in totally isolated vascularly perfused rat stomach
Gastroenterology, 1994Co-Authors: Ping Li, William Y. Chey, Ta-min Chang, Insik ChungAbstract:Abstract Background/Aims: Secretin is an inhibitory hormone of gastric acid secretion. However, its inhibitory mechanism has not been well understood. Possible roles of both somatostatin and prostaglandins were investigated. Methods: Totally isolated rat stomachs were vascularty perfused with Krebs-Ringer buffer containing 50 μmol/L isobutyl methylxanthine at 1.4 mL·min −1 . Gastric lumen was perfused with 0.15 mol/L NaCl at 1.0 min·min −1 . Effect of Secretin in three different doses given intra-arterially on basal acid secretion and acid secretion stimulated by pentagastrin was studied. To determine roles of somatostatin and prostaglandins in the Secretin-induced inhibition, an antisomatostatin serum and indomethacin were tested, and both somatostatin and prostaglandin E 2 concentrations in portal venous effluent were determined by radioimmunoassay. Results: Both basal- and pentagastrin-stimulated acid secretion were significantly inhibited by Secretin. The inhibition was completely reversed by either indomethacin or antisomatostatin serum. Secretin significantly increased concentrations of both somatostatin and prostaglandin E 2 . Although indomethacin blocked the increase in prostaglandin E 2 , Secretin-induced increase in prostaglandin E 2 was not affected by antisomatostatin serum or was indomethacin influenced by somatostatin level. Finally, the inhibition by somatostatin of acid secretion was not affected by indomethacin. Conclusions: The inhibition of gastric acid secretion by Secretin in rats is mediated by simultaneous releases of both somatostatin and prostaglandin E 2 , which independently inhibit gastric acid secretion.
Badreddine Douzi - One of the best experts on this subject based on the ideXlab platform.
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Unraveling the Self-Assembly of the Pseudomonas aeruginosa XcpQ Secretin Periplasmic Domain Provides New Molecular Insights into Type II Secretion System Secreton Architecture and Dynamics
mBio, 2017Co-Authors: Badreddine Douzi, Nhung Trinh, Sandra Michel-souzy, Aline Desmyter, Genevieve Ball, Pascale Barbier, Artemis Kosta, Eric Durand, Katrina Forest, Christian CambillauAbstract:The type II secretion system (T2SS) releases large folded exoproteins across the envelope of many Gram-negative pathogens. This secretion process therefore requires specific gating, interacting, and dynamics properties mainly operated by a bipartite outer membrane channel called Secretin. We have a good understanding of the structure-function relationship of the pore-forming C-terminal domain of Secretins. In contrast, the high flexibility of their periplasmic N-terminal domain has been an obstacle in obtaining the detailed structural information required to uncover its molecular function. InPseudomonas aeruginosa, the Xcp T2SS plays an important role in bacterial virulence by its capacity to deliver a large panel of toxins and degradative enzymes into the surrounding environment. Here, we revealed that the N-terminal domain of XcpQ Secretin spontaneously self-assembled into a hexamer of dimers independently of its C-terminal domain. Furthermore, and by using multidisciplinary approaches, we elucidate the structural organization of the XcpQ N domain and demonstrate that Secretin flexibility at interdimer interfaces is mandatory for its function.IMPORTANCEBacterial Secretins are large homooligomeric proteins constituting the outer membrane pore-forming element of several envelope-embedded nanomachines essential in bacterial survival and pathogenicity. They comprise a well-defined membrane-embedded C-terminal domain and a modular periplasmic N-terminal domain involved in substrate recruitment and connection with inner membrane components. We are studying the XcpQ Secretin of the T2SS present in the pathogenic bacteriumPseudomonas aeruginosaOur data highlight the ability of the XcpQ N-terminal domain to spontaneously oligomerize into a hexamer of dimers. Furtherin vivoexperiments revealed that this domain adopts different conformations essential for the T2SS secretion process. These findings provide new insights into the functional understanding of bacterial T2SS Secretins.
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Towards a better understanding of the bacterial type II secretion pathway
2014Co-Authors: Badreddine Douzi, Genevieve Ball, Christian Cambillau, - Sandra Michel, Frédéric Cadoret, - Bérengère Ize, Sophie Bleves, Chantal Soscia, Mariella TegoniAbstract:The bacterial type II secretion system (T2SS) is unique in its ability to promote the transport of large folded and sometimes multimeric proteins. In this secretion process, exoproteins are first translocated into the periplasm. The final release into the medium requires a multiprotein complex called the secreton. Although the 12 individual components of the secreton have been identified, its mode of action remains obscure. We set up various dedicated in vitro and in vivo protein-protein interaction experiments to identify the Pseudomonas aeruginosa Xcp T2SS periplasmic interactome. BIAcore experiments revealed that three Xcp components, XcpP, the Secretin XcpQ, and the pseudopilus tip, directly and specifically interact with secreted exoproteins. Affinity chromatography co-purification indicated that the XcpY periplasmic domain interacts with the secreted substrate and a component of the pseudopilus tip XcpW. Interestingly, the periplasmic domain of another member of the Xcp inner membrane platform, XcpZ co-elutes with the XcpY/substrate and the XcpY/ XcpW complexes during affinity chromatography. Finally the direct interaction between the secreted substrate and XcpY was confirmed by in situ photo-crosslinking. All together, our results allowed us to propose the most advanced integrative model of Xcp T2SS assembly and function.
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Deciphering the Xcp Pseudomonas aeruginosa type II secretion machinery through multiple interactions with substrates.
Journal of Biological Chemistry, 2011Co-Authors: Badreddine Douzi, Genevieve Ball, Christian Cambillau, Mariella Tegoni, Romé VoulhouxAbstract:The type II secretion system enables gram-negative bacteria to secrete exoproteins into the extracellular milieu. We performed biophysical and biochemical experiments to identify systematic interactions between Pseudomonas aeruginosa Xcp type II secretion system components and their substrates. We observed that three Xcp components, XcpP(C), the Secretin XcpQ(D), and the pseudopilus tip, directly and specifically interact with secreted exoproteins. We established that XcpP(C), in addition to its interaction with the substrate, likely shields the entire periplasmic portion of the secreton. It can therefore be considered as the recruiter of the machinery. Moreover, the direct interaction observed between the substrate and the pseudopilus tip validates the piston model hypothesis, in which the pseudopilus pushes the substrate through the Secretin pore during the secretion process. All together, our results allowed us to propose a model of the different consecutive steps followed by the substrate during the type II secretion process.
Anthony P Pugsley - One of the best experts on this subject based on the ideXlab platform.
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Structural similarity of Secretins from type II and type III secretion systems.
Structure, 2014Co-Authors: Tommaso Tosi, Leandro F Estrozi, Viviana Job, Ingrid Guilvout, Anthony P Pugsley, Guy Schoehn, Andréa DessenAbstract:Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system Secretin, and PulD, the Klebsiella oxytoca type II secretion system Secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both Secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both Secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that Secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.
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type iv like pili formed by the type ii secreton specificity composition bundling polar localization and surface presentation of peptides
Journal of Bacteriology, 2003Co-Authors: Guillaume Vignon, Rolf Kohler, Eric Larquet, Stephanie Giroux, Mariechristine Prevost, Pascal Roux, Anthony P PugsleyAbstract:The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through Secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).
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domain structure of Secretin puld revealed by limited proteolysis and electron microscopy
The EMBO Journal, 2000Co-Authors: Anthony P Pugsley, Nico Nouwen, Henning Stahlberg, Andreas EngelAbstract:Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gram-negative bacteria. Limited proteolysis of Secretin PulD from the Klebsiella oxytoca pullulanase secretion pathway showed that it consists of an N-terminal domain and a protease-resistant C-terminal domain that remains multimeric after proteolysis. The stable C-terminal domain starts just before the region in PulD that is highly conserved in the Secretin superfamily and apparently lacks the region at the C-terminal end to which the Secretin-specific pilot protein PulS binds. Electron microscopy showed that the stable fragment produced by proteolysis is composed of two stacked rings that encircle a central channel and that it lacks the peripheral radial spokes that are seen in the native complex. Moreover, the electron microscopic images suggest that the N-terminal domain folds back into the large cavity of the channel that is formed by the C-terminal domain of the native complex, thereby occluding the channel, consistent with previous electrophysiological studies showing that the channel is normally closed.
