Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

Energy Technology Data Exchange (ETDEWEB)

Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Roszak, Aleksander W. [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel, E-mail: daniel.walker@glasgow.ac.uk [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom)

2015-06-30

The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

Spheres of influence: Porphyromonas gingivalis outer membrane vesicles.

Science.gov (United States)

Gui, M J; Dashper, S G; Slakeski, N; Chen, Y-Y; Reynolds, E C

2016-10-01

Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram-negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P. gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A-LPS) and associated C-terminal domain (CTD) -family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A-LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD-family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non-motile bacteria by motile bacteria. The P. gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro- and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Periplasmic quality control in biogenesis of outer membrane proteins.

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Lyu, Zhi Xin; Zhao, Xin Sheng

2015-04-01

The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

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Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

2011-02-11

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

A Subset of Membrane-Altering Agents and γ-Secretase Modulators Provoke Nonsubstrate Cleavage by Rhomboid Proteases

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Siniša Urban

2014-09-01

Full Text Available Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

Topological analysis of Chlamydia trachomatis L2 outer membrane protein 2

DEFF Research Database (Denmark)

Mygind, P; Christiansen, Gunna; Birkelund, Svend

1998-01-01

Using monospecific polyclonal antisera to different parts of Chlamydia trachomatis L2 outer membrane protein 2 (Omp2), we show that the protein is localized at the inner surface of the outer membrane. Omp2 becomes immunoaccessible when Chlamydia elementary bodies are treated with dithiothreitol...

Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB

OpenAIRE

Okuda, Suguru; Tokuda, Hajime

2009-01-01

Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detai...

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

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Harner, Max; Neupert, Walter; Deponte, Marcel

2011-07-15

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

Substrate specificity within a family of outer membrane carboxylate channels.

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Elif Eren

2012-01-01

Full Text Available Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

Covalent glycoinositolphospholipid (GPI binding to hemoglobin is associated with insulin-activation of erythrocyte membrane protease

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VESNA NIKETIC

2004-05-01

Full Text Available Recently, it was demonstrated that prolonged hyperinsulinism associated with hypoglycemia, both in vivo and in vitro, caused covalent glycoinositolphospholipid (GPI binding to the C termini of both hemoglobin b-chains, which resulted in the formation of a novel, hitherto unrecognized, minor hemoglobin fraction (GPI-Hb (Niketic et al., Biochem. Biophys. Res. Commun. 239 (1997 435. In this study it was demonstrated that exposure of erythrocyte membranes to insulin causes the activation of membrane protease as well as that the formation of GPI-Hb parallels its activity. It is suggested that the insulin-activated protease is able to catalyze, albeit slowly, the transpeptidation, i.e., the replacement of the carboxy-terminal amino acid(s residues of the Hb b-chains with GPI as an exogenous nucleophile. To our knowledge the present results show for the first time that insulin stimulates protease activity in erythrocyte membranes, as well as that insulin-activated protease may be involved in post-translational GPI binding to proteins.

Outer membrane biogenesis in Helicobacter pylori: A deviation from the paradigm

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George W. Liechti

2012-04-01

Full Text Available The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM. Lipopolysaccharide (LPS and numerous outer membrane proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its outer membrane profile limits the effectiveness of vaccines that use any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε- proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε-proteobacteria, while the inner and outer membrane associated apparatus of LPS, lipoprotein, and OM protein transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to

Selective imipenem resistance in Pseudomonas aeruginosa associated with diminished outer membrane permeability.

OpenAIRE

Studemeister, A E; Quinn, J P

1988-01-01

The permeability of the outer membranes of imipenem-susceptible and imipenem-resistant clinical isolates of Pseudomonas aeruginosa was investigated by the liposome swelling assay. Sugars and cephaloridine penetrated rapidly, whereas imipenem penetrated poorly into liposomes constructed from porin-rich outer membrane fractions of the resistant isolates.

The properties of the outer membrane localized Lipid A transporter LptD

International Nuclear Information System (INIS)

Haarmann, Raimund; Ibrahim, Mohamed; Stevanovic, Mara; Bredemeier, Rolf; Schleiff, Enrico

2010-01-01

Gram-negative bacteria are surrounded by a cell wall including the outer membrane. The outer membrane is composed of two distinct monolayers where the outer layer contains lipopolysaccharides (LPS) with the non-phospholipid Lipid A as the core. The synthesis of Lipid A is initiated in the cytosol and thereby the molecule has to be transported across the inner and outer membranes. The β-barrel lipopolysaccharide-assembly protein D (LptD) was discovered to be involved in the transfer of Lipid A into the outer membrane of Gram-negative bacteria. At present the molecular procedure of lipid transfer across the outer membrane remains unknown. Here we approached the functionality of the transfer system by an electrophysiological analysis of the outer membrane protein from Escherichia coli named ecLptD. In vitro the protein shows cation selectivity and has an estimated pore diameter of about 1.8 nm. Addition of Lipid A induces a transition of the open state to a sub-conductance state with two independent off-rates, which might suggest that LptD is able to bind and transport the molecule in vitro. To generalize our findings with respect to the Lipid A transport system of other Gram-negative bacteria we have explored the existence of the proteins involved in this pathway by bioinformatic means. We were able to identify the membrane-inserted components of the Lipid A transport system in all Gram-negative bacteria, whereas the periplasmic components appear to be species-specific. The LptD proteins of different bacteria are characterized by their periplasmic N-terminal domain and a C-terminal barrel region. The latter shows distinct sequence properties, particularly in LptD proteins of cyanobacteria, and this specific domain can be found in plant proteins as well. By electrophysiological experiments on LptD from Anabaena sp. PCC 7120 we are able to confirm the functional relation of anaLptD to Lipid A transport.

The outer membrane protein assembly machinery of Neisseria meningitidis

NARCIS (Netherlands)

Volokhina, E.B.|info:eu-repo/dai/nl/304837202

2009-01-01

Gram-negative bacteria are characterized by a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM), which are separated by the peptidoglycan-containing periplasm. While the integral IM proteins are alpha-helical, all but one known integral OM proteins (OMPs) are

A progenitor of the outer membrane LamB trimer.

OpenAIRE

Stader, J; Silhavy, T J

1988-01-01

During its localization to the outer membrane, LamB possesses distinctive biochemical properties as it passes through the cytoplasmic membrane. Because LamB entered this dynamic state with an attached signal sequence and leaves after cleavage, we call this export-related form of LamB the early-translocation form (et-LamB).

The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

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Pierce, Brittany K; Voegel, Tanja; Kirkpatrick, Bruce C

2014-01-01

Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

Identification of immunogenic outer membrane proteins of Haemophilus influenzae type b in the infant rat model system

International Nuclear Information System (INIS)

Hansen, E.J.; Frisch, C.F.; McDade, R.L. Jr.; Johnston, K.H.

1981-01-01

Outer membrane proteins of Haemophilus influenzae type b which are immunogenic in infant rats were identified by a radioimmunoprecipitation method. Intact cells of H. influenzae type b were radioiodinated by a lactoperoxidase-catalyzed procedure, and an outer membrane-containing fraction was prepared from these cells. These radioiodinated outer membranes were mixed with sera obtained from rats convalescing from systemic H. influenzae type b disease induced at 6 days of age, and the resultant (antibody-outer membrane protein antigen) complexes were extracted from these membranes by treatment with nonionic detergent and ethylenediaminetetraacetic acid. These soluble antibody-antigen complexes were isolated by means of adsorption to protein A-bearing staphylococci, and the radioiodinated protein antigens were identified by gel electrophoresis followed by autoradiography. Infant rats were shown to mount a readily detectable antibody response to several different proteins present in the outer membrane of H. influenzae type b. Individual infant rats were found to vary both qualitatively and quantitatively in their immune response to these immunogenic outer membrane proteins

Porphyromonas gingivalis Outer Membrane Vesicles Mediate Coaggregation and Piggybacking of Treponema denticola and Lachnoanaerobaculum saburreum

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Daniel Grenier

2013-01-01

Full Text Available Porphyromonas gingivalis sheds outer membrane vesicles that contain several virulence factors, including adhesins. In this study, we investigated the ability of P. gingivalis outer membrane vesicles to mediate the coaggregation and piggybacking of Treponema denticola and Lachnoanaerobaculum saburreum. Marked coaggregation between T. denticola and L. saburreum occurred in the presence of P. gingivalis outer membrane vesicles. Sucrose was an effective chemoattractant for the motile species T. denticola. The addition of outer membrane vesicles to a mixture of T. denticola and L. saburreum significantly increased the number of nonmotile bacteria that migrated into a sucrose-filled capillary tube immersed in the bacterial mixture. Under optimal conditions, the number of nonmotile L. saburreum in the capillary tube increased approximately 5-fold, whereas no increase occurred when boiled vesicles were used. This study showed that P. gingivalis outer membrane vesicles mediate coaggregation between T. denticola and L. saburreum and that nonmotile bacteria can be translocated by piggybacking on spirochetes.

Expression and distribution of leptospiral outer membrane components during renal infection of hamsters

NARCIS (Netherlands)

Barnett, J. K.; Barnett, D.; Bolin, C. A.; Summers, T. A.; Wagar, E. A.; Cheville, N. F.; Hartskeerl, R. A.; Haake, D. A.

1999-01-01

The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

Science.gov (United States)

Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

2016-12-06

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein.

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Deirdre R Ducken

Full Text Available Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to

Redefining the essential trafficking pathway for outer membrane lipoproteins

OpenAIRE

Grabowicz, Marcin; Silhavy, Thomas J.

2017-01-01

In Gram-negative bacteria, most lipoproteins synthesized in the inner membrane (IM) are trafficked to the outer membrane (OM). The Lol pathway is the trafficking paradigm: LolCDE releases lipoproteins from the IM; LolA shuttles them between membranes to LolB in the OM. Several OM lipoproteins are essential for viability. In apparent concordance, the Lol proteins are each essential in wild-type cells. However, we show that Escherichia coli grows well without LolA and LolB in the absence of one...

Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii

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Soni Priya Valeru

2014-01-01

Full Text Available Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive inside Acanthamoeba castellanii. It has been shown that V. cholerae expresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA and outer membrane vesicles (OMVs in survival of V. cholerae alone and during its interaction with A. castellanii. The results showed that an OmpA mutant of V. cholerae survived longer than wild-type V. cholerae when cultivated alone. Cocultivation with A. castellanii enhanced the survival of both bacterial strains and OmpA protein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of the OmpA mutant of V. cholerae decreased the viability of A. castellanii and this bacterial strain released more OMVs than wild-type V. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from the OmpA mutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule for OmpA in survival of V. cholerae and OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.

Genomic analysis indicates the presence of an asymmetric bilayer outer membrane in Planctomycetes and Verrucomicrobia

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Daan R Speth

2012-08-01

Full Text Available Bacteria of the phylum Planctomycetes are of special interest for the study of compartmental cellular organization. Members of this phylum share a very unusual prokaryotic cell plan, featuring several membrane-bound compartments. Recently, it was shown that this cellular organization might extend to certain members of the phylum Verrucomicrobia. The Planctomycete cell plan has been defined as featuring a proteinaceous cell wall, a cytoplasmic membrane surrounding the paryphoplasm and an intracytoplasmic membrane defining the riboplasm. So far it was presumed that Planctomycetes did not have an asymmetric bilayer outer membrane as observed in Gram-negative bacteria. However, recent work on outer membrane biogenesis has provided several marker genes in the outer membrane protein (OMP assembly and the lipopolysaccharide (LPS insertion complexes. Additionally, advances in computational prediction of OMPs provided new tools to perform more accurate genomic screening for such proteins.Here we searched all 22 Planctomycetes and Verrucomicrobia genomes available in Genbank, plus the recently published genome of ‘Candidatus Scalindua profunda’, for markers of outer membrane biogenesis and OMPs. We were able to identify the key components of LPS insertion, OMP assembly and at least eight OMPs in all genomes tested. Additionally, we have analyzed the transcriptome and proteome data of the Planctomycetes ‘Candidatus Kuenenia stuttgartiensis’ and ‘Ca. S. profunda’ and could confirm high expression of several predicted OMPs, including the biomarkers of outer membrane biogenesis.

Outer membrane components of the Tad (tight adherence) secreton of Aggregatibacter actinomycetemcomitans.

Science.gov (United States)

Clock, Sarah A; Planet, Paul J; Perez, Brenda A; Figurski, David H

2008-02-01

Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins.

Roles of secretory leukocyte protease inhibitor amniotic membrane in oral wound healing

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Elly Munadziroh

2006-12-01

Full Text Available Secretory Leukocyte Protease Inhibitor (SLPI is serine protease inhibitor. Secretory Leukocyte Protease Inhibitor is a protein found in secretions such as whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears, and cerebral spinal fluid, as in secretions from the nose and bronchi, amniotic fluid and amniotic membrane etc. These findings demonstrate that SLPI function as a potent anti protease, anti inflammatory, bactericidal, antifungal, tissue repair, extra cellular synthesis. Impaired healing states are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that SLPI may have a role in the process. The objectives of this article are to investigate the role of SLPI in oral inflammation and how it contributes to tissue repair in oral mucosa. The oral wound healing responses are impaired in the SLPI sufficient mice and matrix synthesis and collagen deposition are delayed. This study indicated that SLPI is a povital factor necessary for optimal wound healing.

Structural basis for alginate secretion across the bacterial outer membrane

Energy Technology Data Exchange (ETDEWEB)

Whitney, J.C.; Robinson, H.; Hay, I. D.; Li, C.; Eckford, P. D. W.; Amaya, M. F.; Wood, L. F.; Ohman, D. E.; Bear, C. E.; Rehm, B. H.; Howell, P. L.

2011-08-09

Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

Structural Basis for Alginate Secretion Across the Bacterial Outer Membrane

Energy Technology Data Exchange (ETDEWEB)

J Whitney; I Hay; C Li; P Eckford; H Robinson; M Amaya; L Wood; D Ohman; C Bear; et al.

2011-12-31

Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 {angstrom} crystal structure of AlgE, which reveals a monomeric 18-stranded {beta}-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 ({Delta}T8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or {Delta}L2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with {Delta}T8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.

Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

Science.gov (United States)

Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

2017-08-01

The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

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Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

2016-01-22

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin

Energy Technology Data Exchange (ETDEWEB)

Nobre, Thatyane M.; Martynowycz, Michael W.; Andreev, Konstantin; Kuzmenko, Ivan; Nikaido, Hiroshi; Gidalevitz, David

2015-12-01

Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted asmonolayers at the air-water interface, and their properties, aswell as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region,butwas prevented fromthis penetration intothemodified lipopolysaccharides.Results correlatewith behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains.

Comprehensive in silico prediction and analysis of chlamydial outer membrane proteins reflects evolution and life style of the Chlamydiae

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Myers Garry

2009-12-01

Full Text Available Abstract Background Chlamydiae are obligate intracellular bacteria comprising some of the most important bacterial pathogens of animals and humans. Although chlamydial outer membrane proteins play a key role for attachment to and entry into host cells, only few have been described so far. We developed a comprehensive, multiphasic in silico approach, including the calculation of clusters of orthologues, to predict outer membrane proteins using conservative criteria. We tested this approach using Escherichia coli (positive control and Bacillus subtilis (negative control, and applied it to five chlamydial species; Chlamydia trachomatis, Chlamydia muridarum, Chlamydia (a.k.a. Chlamydophila pneumoniae, Chlamydia (a.k.a. Chlamydophila caviae, and Protochlamydia amoebophila. Results In total, 312 chlamydial outer membrane proteins and lipoproteins in 88 orthologous clusters were identified, including 238 proteins not previously recognized to be located in the outer membrane. Analysis of their taxonomic distribution revealed an evolutionary conservation among Chlamydiae, Verrucomicrobia, Lentisphaerae and Planctomycetes as well as lifestyle-dependent conservation of the chlamydial outer membrane protein composition. Conclusion This analysis suggested a correlation between the outer membrane protein composition and the host range of chlamydiae and revealed a common set of outer membrane proteins shared by these intracellular bacteria. The collection of predicted chlamydial outer membrane proteins is available at the online database pCOMP http://www.microbial-ecology.net/pcomp and might provide future guidance in the quest for anti-chlamydial vaccines.

In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli.

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Larsen, Ray A; Letain, Tracy E; Postle, Kathleen

2003-07-01

Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino-terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino-terminus, located in the cytoplasm, ever became accessible to the cys-specific, cytoplasmic membrane-impermeant molecule, Oregon Green(R) 488 maleimide (OGM) in vivo. A full-length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full-length TonB and truncated TonB (consisting of the amino-terminal two-thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross-link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full-length TonB could be amino-terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full-length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.

An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

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Marcin Michalik

Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

Entry and exit of bacterial outer membrane proteins.

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Misra, Rajeev

2015-08-01

The sites of new outer membrane protein (OMP) deposition and the fate of pre-existing OMPs are still enigmatic despite numerous concerted efforts. Rassam et al. identified mid-cell regions as the primary entry points for new OMP insertion in clusters, driving the pre-existing OMP clusters towards cell poles for long-term storage. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

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Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

2010-12-01

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

Science.gov (United States)

Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

2002-08-01

Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

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Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

2016-04-15

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

Localization of cytochromes in the outer membrane of Desulfovibrio vulgaris (Hildenborough) and their role in anaerobic biocorrosion.

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Van Ommen Kloeke, F; Bryant, R D; Laishley, E J

1995-12-01

A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50 kDa, and 77.5 kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.

Identification of outer membrane proteins of Yersinia pestis through biotinylation

NARCIS (Netherlands)

Smither, S.J.; Hill, J.; Baar, B.L.M. van; Hulst, A.G.; Jong, A.L. de; Titball, R.W.

2007-01-01

The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with

Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium Xanthomonas campestris pv. campestris

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Niehaus Karsten

2008-06-01

Full Text Available Abstract Background Outer membrane vesicles (OMVs are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli. Results We have demonstrated that Xanthomonas campestris pv. campestris (Xcc releases OMVs into the culture supernatant during growth. A proteome study identified 31 different proteins that associate with the OMV fraction of which half are virulence-associated. A comparison with the most abundant outer membrane (OM proteins revealed that some proteins are enriched in the OMV fraction. This may be connected to differences in the LPS composition between the OMVs and the OM. Furthermore, a comparison of the OMV proteomes from two different culture media indicated that the culture conditions have an impact on the protein composition. Interestingly, the proteins that are common to both culture conditions are mainly involved in virulence. Conclusion Outer membrane vesicles released from the OM of Xcc contain membrane- and virulence-associated proteins. Future experiments will prove whether these structures can serve as "vehicles" for the transport of virulence factors into the host membrane.

Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1.

Science.gov (United States)

Yun, Sung Ho; Lee, Sang-Yeop; Choi, Chi-Won; Lee, Hayoung; Ro, Hyun-Joo; Jun, Sangmi; Kwon, Yong Min; Kwon, Kae Kyoung; Kim, Sang-Jin; Kim, Gun-Hwa; Kim, Seung Il

2017-01-01

Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMV Novo ) are spherical in shape, and the average diameter of OMV Novo is 25-70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMV Novo . Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMV Novo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.

Detergent organisation in crystals of monomeric outer membrane phospholipase A

NARCIS (Netherlands)

Snijder, HJ; Timmins, PA; Kalk, KH; Dijkstra, BW

The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H2O/D2O contrast. From the neutron diffraction at five contrasts,

Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production

DEFF Research Database (Denmark)

Pedersen, K.; Gram, Lone; Austin, D.A.

1997-01-01

The virulence of 18 strains of Vibrio anguillarum serogroup 01 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only str...

Green Modification of Outer Selective P84 Nanofiltration (NF) Hollow Fiber Membranes for Cadmium Removal

KAUST Repository

Gao, Jie

2015-10-26

Outer-selective thin-film composite (TFC) hollow fiber membranes are normally made from interfacial polymerization of m-phenylenediamine (MPD) and trimesoyl chloride (TMC). However, the removal of excess MPD solution and the large consumption of alkane solvents are their technical bottlenecks. In this study, green methods to prepare the outer selective TFC hollow fiber membranes were explored by firstly modifying the membrane substrate with polyethyleneimine (PEI) and then by water soluble small molecules such as glutaraldehyde (GA) and epichlorohydrin (ECH). Using P84 polyimide as the substrate, not only do these modifications decrease substrate\\'s pore size, but also vary surface charge by making the membranes less positively charged. As a result, the resultant membranes have higher rejections against salts such as Na2SO4, NaCl and MgSO4. The PEI and then GA modified membrane has the best separation performance with a NaCl rejection over 90% and a pure water permeability (PWP) of 1.74±0.01 Lm−2bar−1h−1. It also shows an impressive rejection to CdCl2 (94%) during long-term stability tests. The CdCl2 rejection remains higher than 90% at operating temperatures from 5 to 60 °C. This study may provide useful insights for green manufacturing of outer-selective nanofiltration (NF) hollow fiber membranes.

Outer Mitochondrial Membrane Localization of Apoptosis-Inducing Factor: Mechanistic Implications for Release

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Seong-Woon Yu

2009-10-01

Full Text Available Poly(ADP-ribose polymerase-1-dependent cell death (known as parthanatos plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor, but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

Isolation and identification of java race amniotic membrane secretory leukocyte protease inhibitor gene

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Elly Munadziroh

2008-09-01

Full Text Available Background: Secretory leukocyte protease inhibitor (SLPI has been found to facilitate epithelialization, maintain a normal epithelial phenotype, reduce inflammation, secrete growth factors such as IL-4, IL-6, IL-10, EGF, FGF, TGF, HGFand 2-microbulin. SLPI is serine protease inhibitor, which found in secretions such as whole saliva, seminal fluid, cervical mucus, synovial fluid, breast milk, tears, amniotic fluid and amniotic membrane. Impaired healing states are characterized by excessive proteolysis and oftenbacterial infection, leading to the hypothesis that SLPI may have a role in the healing process in oral inflammation and contributes to tissue repair in oral mucosa. The oral wound healing response is impaired in the SLPI sufficient mice since matrix synthesis and collagen deposition delayed. The objective of this research is to isolate and identify the amniotic membrane of Java Race SLPI Gene. Methods: SLPI RNA was isolated from Java Race amniotic membrane and the cDNA was amplified by polymerase chain reaction (PCR. Result: Through sequence analyses, SLPI cDNA was 530 nucleotide in length with a predicted molecular mass about 12 kDa. The nucleotide sequence showed that human SLPI from sample was 98% identical with human SLPI from gene bank. PCR analysis revealed that the mRNA of SLPI was highly expressed in the amniotic membrane from Java Race sample. Conclusion: it is demonstrated that human SLPI are highly conserved in sequence content as compared to the human SLPI from gene.

Quantitative kinetic analysis of blood vessels in the outer membranes of chronic subdural hematomas

International Nuclear Information System (INIS)

Mori, Kentaro; Adachi, Keiji; Cho, Kajin; Ishimaru, Sumio; Maeda, Minoru

1998-01-01

Dynamic biologic modeling was used to calculate the transfer rate constant for gadolinium-diethylenetriaminepenta-acetic acid (Gd-DTPA) and capillary permeability in the outer membrane of chronic subdural hematomas and effusions. Following intravenous Gd-DTPA injection, Gd concentrations in the subdural fluid and in timed arterial blood samples were measured by ion-coupled plasma emission spectrometry in 53 chronic subdural hematomas and 18 chronic subdural effusions. The capillary surface area in outer membrane was assessed morphometrically. Transfer rate constants for subdural hematomas and subdural effusions were 12.4±1.0 and 20.6±1.7 (x 10 -4 )min -1 , respectively. Capillary permeabilities for subdural hematomas and subdural effusions were 16±1.2 and 19±3.7 ml·min -1 (mm 2 /mm 3 ) -1 , respectively. The capillary surface areas for subdural hematomas and subdural effusions were 48±3 and 77±10 mm 2 /mm 3 , respectively. The high degree of infiltration of Gd into subdural effusions reflects the high capillary surface area in the outer membrane rather than greater permeability of individual capillaries. The value of transfer rate constant was correlated inversely with the duration of the chronic subdural fluid collection. Immature outer membrane has a high transfer rate constant which allows extravasation of plasma components into the subdural space, resulting in increasing volume of the subdural effusion. Delayed magnetic resonance imaging following Gd administration may be clinically useful for estimating the age of chronic subdural fluid accumulations. (author)

Next-generation outer membrane vesicle vaccines from concept to clinical trials

NARCIS (Netherlands)

Waterbeemd, van de B.

2013-01-01

Only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. The OMV vaccines, however, provide limited coverage and are difficult to produce. This is caused by an obligatory detergent treatment, which removes lipopolysaccharide

Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB.

Science.gov (United States)

Okuda, Suguru; Tokuda, Hajime

2009-04-07

Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detail. However, it remained unclear how Lol factors interact with each other to conduct very efficient lipoprotein transfer in the periplasm where ATP is not available. To address this issue, a photo-reactive phenylalanine analogue, p-benzoyl-phenylalanine, was introduced at various positions of LolA and LolB, of which the overall structures are very similar and comprise an incomplete beta-barrel with a hydrophobic cavity inside. Cells expressing LolA or LolB derivatives containing the above analogue were irradiated with UV for in vivo photo-cross-linking. These analyses revealed a hot area in the same region of LolA and LolB, through which LolA and LolB interact with each other. This area is located at the entrance of the hydrophobic cavity. Moreover, this area in LolA is involved in the interaction with a membrane subunit, LolC, whereas no cross-linking occurs between LolA and the other membrane subunit, LolE, or ATP-binding subunit LolD, despite the structural similarity between LolC and LolE. The hydrophobic cavities of LolA and LolB were both found to bind lipoproteins inside. These results indicate that the transfer of lipoproteins through Lol proteins occurs in a mouth-to-mouth manner.

Analysis of long-chain fatty acid binding activity in vesicles of the outer membrane generated from Escherchia coli

International Nuclear Information System (INIS)

Black, P.N.

1987-01-01

Escherichia coli transports long-chain fatty acids across the dual membrane by a high affinity, saturable, energy-dependent process. The fadL gene codes for an outer membrane protein which appears to act specifically as a long-chain fatty acid binding protein when fatty acid utilization is blocked by mutation. In an effort to understand the function of the fadL gene product, FLP, membranes have been isolated from fadL + and fadL - strains following osmotic lysis. Following isolation, total membranes were separated into inner and outer membrane fractions and assayed for long-chain fatty acid binding activity. Outer membrane vesicles were incubated 2-5 min at 37 0 C with 3 H oleate (C/sub 18:1/), cooled to 0 0 C, and centrifuged through a Lipidex 100 column for 3 min to remove the unbound fatty acid. The level of fatty acid binding was quantitated by scintillation counting of the eluate. Outer membrane vesicles generated from a fadL + strain bind 325 pmol fatty acid/mg protein whereas vesicles generated for a mutant strain bind 175 pmol fatty acid/mg protein. These data suggest that FLP acts at least as a long-chain fatty acid binding protein on the surface of the cell

The participation of outer membrane proteins in the bacterial sensitivity to nanosilver

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Anna Kędziora

2016-06-01

Full Text Available The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI‑TOF MS are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico‑chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.

Meningococcal outer membrane vesicle composition-dependent activation of the innate immune response

NARCIS (Netherlands)

Zariri, Afshin; Beskers, Joep; van de Waterbeemd, Bas; Hamstra, Hendrik Jan; Bindels, Tim H E; van Riet, Elly; van Putten, Jos P M; van der Ley, Peter

2016-01-01

Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen associated molecular patterns including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an

Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

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Tatini Rakshit

Full Text Available Rhodopsin forms nanoscale domains (i.e., nanodomains in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.

Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin.

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Collin, Séverine; Guilvout, Ingrid; Nickerson, Nicholas N; Pugsley, Anthony P

2011-05-01

The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane. © 2011 Blackwell Publishing Ltd.

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

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Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

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Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

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Miranda Lo

Full Text Available BACKGROUND: Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. METHODOLOGY/PRINCIPAL FINDINGS: To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS. We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. CONCLUSIONS/SIGNIFICANCE: This is the first study to compare transcriptional and translational responses to temperature

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

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Frederico Guilherme Coutinho Abath

1990-03-01

Full Text Available Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76 were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page. Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted.

Analysis of proteins in Chlamydia trachomatis L2 outer membrane complex, COMC

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Birkelund, Svend; Morgan-Fisher, Marie; Timmerman, Evy

2009-01-01

The protein composition and N-terminal sequences of proteins in the outer membrane of Chlamydia trachomatis L2 were analysed following isolation of N-terminal peptides using combined fractional diagonal chromatography and identification by liquid chromatography tandem MS. Acetylation of primary a...

The role of outer membrane in Serratia marcescens intrinsic resistance to antibiotics.

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Sánchez, L; Ruiz, N; Leranoz, S; Viñas, M; Puig, M

1997-09-01

Three different porins from Serratia marcescens were described. They were named Omp1, Omp2 and Omp3 and their molecular weights were 42, 40 and 39 kDa respectively. Omp2 and Omp3 showed osmoregulation and thermoregulation in a similar way to OmpC and OmpF of Escherichia coli. Permeability coefficients of the outer membrane of this species were calculated following the Zimmermann and Rosselet method. P values were similar to those obtained in Escherichia coli, which suggests that the chromosomal beta-lactamase would play a major role in the resistance of Serratia marcescens to beta-lactam antibiotics. Both MIC values and permeabilities were modified by salycilates and acetylsalycilate. Synergism between the outer membrane and the beta-lactamase was also evaluated. When bacteria grew in the presence of a beta-lactam in the medium, the beta-lactamase accounted for most of the resistance.

Helicobacter pylori Outer Membrane Protein-Related Pathogenesis

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Yuichi Matsuo

2017-03-01

Full Text Available Helicobacter pylori colonizes the human stomach and induces inflammation, and in some cases persistent infection can result in gastric cancer. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and outer membrane proteins (OMPs play a pivotal role in binding to human cells. Some OMP interaction molecules are known in H. pylori, and their associated host cell responses have been gradually clarified. Many studies have demonstrated that OMPs are essential to CagA translocation into gastric cells via the Type IV secretion system of H. pylori. This review summarizes the mechanisms through which H. pylori utilizes OMPs to colonize the human stomach and how OMPs cooperate with the Type IV secretion system.

An ABC-transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa

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Casabona, Maria G.; Silverman, Julie M.; Sall, Khady M.; Boyer, Frédéric; Couté, Yohann; Poirel, Jessica; Grunwald, Didier; Mougous, Joseph D.; Elsen, Sylvie; Attree, Ina

2012-01-01

Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SS). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Thr kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and export of Hcp1 and Tse1. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signaling pathway that promotes H1-T6SS activity under optimal environmental conditions. PMID:22765374

Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

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Dhandapani, Gunasekaran; Sikha, Thoduvayil; Rana, Aarti; Brahma, Rahul; Akhter, Yusuf; Gopalakrishnan Madanan, Madathiparambil

2018-04-10

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies. © 2018 Wiley Periodicals, Inc.

Targeting and Assembly of Components of the TOC Protein Import Complex at the Chloroplast Outer Envelope Membrane

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Lynn G.L. Richardson

2014-06-01

Full Text Available The translocon at the outer envelope membrane of chloroplasts (TOC initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

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Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Components of SurA required for outer membrane biogenesis in uropathogenic Escherichia coli.

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Kristin M Watts

2008-10-01

Full Text Available SurA is a periplasmic peptidyl-prolyl isomerase (PPIase and chaperone of Escherichia coli and other Gram-negative bacteria. In contrast to other PPIases, SurA appears to have a distinct role in chaperoning newly synthesized porins destined for insertion into the outer membrane. Previous studies have indicated that the chaperone activity of SurA rests in its "core module" (the N- plus C-terminal domains, based on in vivo envelope phenotypes and in vitro binding and protection of non-native substrates.In this study, we determined the components of SurA required for chaperone activity using in vivo phenotypes relevant to disease causation by uropathogenic E. coli (UPEC, namely membrane resistance to permeation by antimicrobials and maturation of the type 1 pilus usher FimD. FimD is a SurA-dependent, integral outer membrane protein through which heteropolymeric type 1 pili, which confer bladder epithelial binding and invasion capacity upon uropathogenic E. coli, are assembled and extruded. Consistent with prior results, the in vivo chaperone activity of SurA in UPEC rested primarily in the core module. However, the PPIase domains I and II were not expendable for wild-type resistance to novobiocin in broth culture. Steady-state levels of FimD were substantially restored in the UPEC surA mutant complemented with the SurA N- plus C-terminal domains. The addition of PPIase domain I augmented FimD maturation into the outer membrane, consistent with a model in which domain I enhances stability of and/or substrate binding by the core module.Our results confirm the core module of E. coli SurA as a potential target for novel anti-infective development.

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32.

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Eshghi, Azad; Pinne, Marija; Haake, David A; Zuerner, Richard L; Frank, Ami; Cameron, Caroline E

2012-03-01

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

Infectious polymorphic toxins delivered by outer membrane exchange discriminate kin in myxobacteria.

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Vassallo, Christopher N; Cao, Pengbo; Conklin, Austin; Finkelstein, Hayley; Hayes, Christopher S; Wall, Daniel

2017-08-18

Myxobacteria are known for complex social behaviors including outer membrane exchange (OME), in which cells exchange large amounts of outer membrane lipids and proteins upon contact. The TraA cell surface receptor selects OME partners based on a variable domain. However, traA polymorphism alone is not sufficient to precisely discriminate kin. Here, we report a novel family of OME-delivered toxins that promote kin discrimination of OME partners. These SitA lipoprotein toxins are polymorphic and widespread in myxobacteria. Each sitA is associated with a cognate sitI immunity gene, and in some cases a sitB accessory gene. Remarkably, we show that SitA is transferred serially between target cells, allowing the toxins to move cell-to-cell like an infectious agent. Consequently, SitA toxins define strong identity barriers between strains and likely contribute to population structure, maintenance of cooperation, and strain diversification. Moreover, these results highlight the diversity of systems evolved to deliver toxins between bacteria.

An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa.

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Casabona, Maria G; Silverman, Julie M; Sall, Khady M; Boyer, Frédéric; Couté, Yohann; Poirel, Jessica; Grunwald, Didier; Mougous, Joseph D; Elsen, Sylvie; Attree, Ina

2013-02-01

Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

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Yu, Johnson Chung Sing

Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

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Fujita Naoya

2011-01-01

Full Text Available Abstract Background The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. Results We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Conclusions Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.

Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern

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Snoussi Sarra

2012-02-01

Full Text Available Abstract Background Salmonella enterica serovar Hadar (S. Hadar is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments. Results The present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h and the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE and LC-MS/MS spectrometry, we compared the proteome of enriched-outer membrane fraction before and after exposure to a magnetic field. A total of 11 proteins, displaying more than a two-fold change, were differentially expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated. These proteins were involved in the integrity of cell envelope (TolB, Pal, in the response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF, in the oxidative stress status (bacterioferritin, in virulence (OmpX, Yfgl or in motility (FlgE and UspF. Complementary experiments associated the down-regulation of FlgE and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore, the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility in exposed cells. This decrease could be partly associated with the up-regulation of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein, was up-regulated. Conclusions SMF (200 mT seems to maintain the cell envelope integrity and to submit the exposed cells to an oxidative stress. Some alterations suggest an increase of the ability of exposed cells to form biofilms.

Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

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Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

2016-05-01

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

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McClafferty Heather

2005-01-01

Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

Entry of Porphyromonas gingivalis Outer Membrane Vesicles into Epithelial Cells Causes Cellular Functional Impairment▿

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Furuta, Nobumichi; Takeuchi, Hiroki; Amano, Atsuo

2009-01-01

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis. PMID:19737899

Quantitative Proteomics Reveals Distinct Differences in the Protein Content of Outer Membrane Vesicle Vaccines

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Waterbeemd, van de B.; Mommen, G.P.M.; Pennings, J.L.A.; Eppink, M.H.M.; Wijffels, R.H.; Pol, van der L.A.; Jong, de A.P.J.M.

2013-01-01

At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in

NMR structure of temporin-1 ta in lipopolysaccharide micelles: mechanistic insight into inactivation by outer membrane.

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Rathi Saravanan

Full Text Available BACKGROUND: Antimicrobial peptides (AMPs play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS. The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane. PRINCIPAL FINDINGS: Using NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles. SIGNIFICANCE: We propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a

A conserved small RNA promotes silencing of the outer membrane protein YbfM

DEFF Research Database (Denmark)

Rasmussen, Anders Aamann; Johansen, Jesper; Nielsen, Jesper S

2009-01-01

important physiological role of regulatory RNA molecules in Gram-negative bacteria is to modulate the cell surface and/or to prevent accumulation of OMPs in the envelope. Here, we extend the OMP-sRNA network by showing that the expression of the outer membrane protein YbfM is silenced by a conserved sRNA......In the past few years an increasing number of small non-coding RNAs (sRNAs) in enterobacteria have been found to negatively regulate the expression of outer membrane proteins (OMPs) at the post-transcriptional level. These RNAs act under various growth and stress conditions, suggesting that one......, designated MicM (also known as RybC/SroB). The regulation is strictly dependent on the RNA chaperone Hfq, and mutational analysis indicates that MicM sequesters the ribosome binding site of ybfM mRNA by an antisense mechanism. Furthermore, we provide evidence that Hfq strongly enhances the on-rate of duplex...

Monoclonal antibodies against the iron regulated outer membrane Proteins of Acinetobacter baumannii are bactericidal

Directory of Open Access Journals (Sweden)

Goel Vikas

2001-08-01

Full Text Available Abstract Background Iron is an important nutrient required by all forms of life.In the case of human hosts,the free iron availability is 10-18M,which is far less than what is needed for the survival of the invading bacterial pathogen.To survive in such conditions, bacteria express new proteins in their outer membrane and also secrete iron chelators called siderophores. Results/ Discussion Acinetobacter baumannii ATCC 19606, a nosocomial pathogen which grows under iron restricted conditions, expresses four new outer membrane proteins,with molecular weight ranging from 77 kDa to 88 kDa, that are called Iron Regulated Outer Membrane Proteins (IROMPs. We studied the functional and immunological properties of IROMPs expressed by A.baumanii ATCC 19606.The bands corresponding to IROMPs were eluted from SDS-PAGE and were used to immunize BALB/c mice for the production of monoclonal antibodies. Hybridomas secreting specific antibodies against these IROMPs were selected after screening by ELISA and their reactivity was confirmed by Western Blot. The antibodies then generated belonged to IgM isotype and showed bactericidical and opsonising activities against A.baumanii in vitro.These antibodies also blocked siderophore mediated iron uptake via IROMPs in bacteria. Conclusion This proves that iron uptake via IROMPs,which is mediated through siderophores,may have an important role in the survival of A.baumanii inside the host,and helps establishing the infection.

Bacterial proteases and virulence

DEFF Research Database (Denmark)

Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

2013-01-01

signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell...

Decreased outer membrane permeability in imipenem-resistant mutants of Pseudomonas aeruginosa.

OpenAIRE

Trias, J; Dufresne, J; Levesque, R C; Nikaido, H

1989-01-01

The outer membrane of imipenem-resistant mutants of Pseudomonas aeruginosa was shown to have decreased permeability to imipenem but not to cephaloridine. These experiments were performed with intact cells and liposomes containing imipenem-hydrolyzing beta-lactamase derived from Pseudomonas maltophilia, in both cases utilizing an imipenem concentration of 50 microM. In contrast, liposome swelling assays using imipenem at 8 mM detected no significant difference between the imipenem-resistant mu...

The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD

Science.gov (United States)

Calmettes, Charles; Ing, Christopher; Buckwalter, Carolyn M.; El Bakkouri, Majida; Chieh-Lin Lai, Christine; Pogoutse, Anastassia; Gray-Owen, Scott D.; Pomès, Régis; Moraes, Trevor F.

2015-01-01

Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops. PMID:26282243

Exposure of outer membrane proteins on the surface of Pseudomonas aeruginosa PA01 revealed by labelling with [125I]lactoperoxidase

International Nuclear Information System (INIS)

Lambert, P.A.; Booth, B.R.

1982-01-01

The authors have investigated the exposure of the major outer membrane proteins on the cell surface by treating whole cells of P. aeruginosa with [ 125 I]lactoperoxidase. This reagent catalyses the iodination of tyrosine and histidine residues of proteins in the presence of hydrogen peroxide. It is too large to penetrate the outer membrane (Msub(r) 77500), therefore it is assumed to label only those proteins which have such residues exposed on the cell surface and has been applied to a number of Gram-negative organisms. It is found that F was the major labelled protein, D1 and/or D2 were less heavily labelled, and G was very faintly labelled. In addition, two proteins (Msub(r) 72500 and 38000) which did not appear to be major outer membrane proteins were labelled. (Auth.)

Outer-selective pressure-retarded osmosis hollow fiber membranes from vacuum-assisted interfacial polymerization for osmotic power generation

KAUST Repository

Sun, Shipeng; Chung, Neal Tai-Shung

2013-01-01

In this paper, we report the technical breakthroughs to synthesize outer-selective thin-film composite (TFC) hollow fiber membranes, which is in an urgent need for osmotic power generation with the pressure-retarded osmosis (PRO) process. In the first step, a defect-free thin-film composite membrane module is achieved by vacuum-assisted interfacial polymerization. The PRO performance is further enhanced by optimizing the support in terms of pore size and mechanical strength and the TFC layer with polydopamine coating and molecular engineering of the interfacial polymerization solution. The newly developed membranes can stand over 20 bar with a peak power density of 7.63 W/m2, which is equivalent to 13.72 W/m2 of its inner-selective hollow fiber counterpart with the same module size, packing density, and fiber dimensions. The study may provide insightful guidelines for optimizing the interfacial polymerization procedures and scaling up of the outer-selective TFC hollow fiber membrane modules for PRO power generation. © 2013 American Chemical Society.

Outer-selective pressure-retarded osmosis hollow fiber membranes from vacuum-assisted interfacial polymerization for osmotic power generation.

Science.gov (United States)

Sun, Shi-Peng; Chung, Tai-Shung

2013-11-19

In this paper, we report the technical breakthroughs to synthesize outer-selective thin-film composite (TFC) hollow fiber membranes, which is in an urgent need for osmotic power generation with the pressure-retarded osmosis (PRO) process. In the first step, a defect-free thin-film composite membrane module is achieved by vacuum-assisted interfacial polymerization. The PRO performance is further enhanced by optimizing the support in terms of pore size and mechanical strength and the TFC layer with polydopamine coating and molecular engineering of the interfacial polymerization solution. The newly developed membranes can stand over 20 bar with a peak power density of 7.63 W/m(2), which is equivalent to 13.72 W/m(2) of its inner-selective hollow fiber counterpart with the same module size, packing density, and fiber dimensions. The study may provide insightful guidelines for optimizing the interfacial polymerization procedures and scaling up of the outer-selective TFC hollow fiber membrane modules for PRO power generation.

Outer-selective pressure-retarded osmosis hollow fiber membranes from vacuum-assisted interfacial polymerization for osmotic power generation

KAUST Repository

Sun, Shipeng

2013-11-19

In this paper, we report the technical breakthroughs to synthesize outer-selective thin-film composite (TFC) hollow fiber membranes, which is in an urgent need for osmotic power generation with the pressure-retarded osmosis (PRO) process. In the first step, a defect-free thin-film composite membrane module is achieved by vacuum-assisted interfacial polymerization. The PRO performance is further enhanced by optimizing the support in terms of pore size and mechanical strength and the TFC layer with polydopamine coating and molecular engineering of the interfacial polymerization solution. The newly developed membranes can stand over 20 bar with a peak power density of 7.63 W/m2, which is equivalent to 13.72 W/m2 of its inner-selective hollow fiber counterpart with the same module size, packing density, and fiber dimensions. The study may provide insightful guidelines for optimizing the interfacial polymerization procedures and scaling up of the outer-selective TFC hollow fiber membrane modules for PRO power generation. © 2013 American Chemical Society.

Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens.

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Karin Aistleitner

Full Text Available The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydiaouter membrane proteins, PomS (pc1489 and PomT (pc1077, are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts.

Molecular transformers in the cell: lessons learned from the DegP protease-chaperone.

Science.gov (United States)

Sawa, Justyna; Heuck, Alexander; Ehrmann, Michael; Clausen, Tim

2010-04-01

Structure-function analysis of DegP revealed a novel mechanism for protease and chaperone regulation. Binding of unfolded proteins induces the oligomer reassembly from the resting hexamer (DegP6) into the functional protease-chaperone DegP12/24. The newly formed cage exhibits the characteristics of a proteolytic folding chamber, shredding those proteins that are severely misfolded while stabilizing and protecting proteins present in their native state. Isolation of native DegP complexes with folded outer membrane proteins (OMPs) highlights the importance of DegP in OMP biogenesis. The encapsulated OMP beta-barrel is significantly stabilized in the hydrophobic chamber of DegP12/24 and thus DegP seems to employ a reciprocal mechanism to those chaperones assisting the folding of water soluble proteins via polar interactions. In addition, we discuss in this review similarities to other complex proteolytic machines that, like DegP, are under control of a substrate-induced or stress-induced oligomer conversion.

Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones

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Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

2012-01-01

Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586

Roles of the Protruding Loop of Factor B Essential for the Localization of Lipoproteins (LolB) in the Anchoring of Bacterial Triacylated Proteins to the Outer Membrane*

Science.gov (United States)

Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

2014-01-01

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed. PMID:24569999

Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

Science.gov (United States)

Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

2014-04-11

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.

Large-scale preparation of the homogeneous LolA–lipoprotein complex and efficient in vitro transfer of lipoproteins to the outer membrane in a LolB-dependent manner

OpenAIRE

Watanabe, Shoji; Oguchi, Yuki; Yokota, Naoko; Tokuda, Hajime

2007-01-01

An ATP-binding cassette transporter LolCDE complex of Escherichia coli releases lipoproteins destined to the outer membrane from the inner membrane as a complex with a periplasmic chaperone, LolA. Interaction of the LolA–lipoprotein complex with an outer membrane receptor, LolB, then causes localization of lipoproteins to the outer membrane. As far as examined, formation of the LolA–lipoprotein complex strictly depends on ATP hydrolysis by the LolCDE complex in the presence of LolA. It has be...

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

Science.gov (United States)

Pirbadian, Sahand; Barchinger, Sarah E; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Shi, Liang; Gorby, Yuri A; Golbeck, John H; El-Naggar, Mohamed Y

2014-09-02

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

RC1339/APRc from Rickettsia conorii is a novel aspartic protease with properties of retropepsin-like enzymes.

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Rui Cruz

2014-08-01

Full Text Available Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a

Multifunctional Mitochondrial AAA Proteases.

Science.gov (United States)

Glynn, Steven E

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

A novel Geobacteraceae-specific outer membrane protein J (OmpJ is essential for electron transport to Fe (III and Mn (IV oxides in Geobacter sulfurreducens

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Schiffer Marianne

2005-07-01

Full Text Available Abstract Background Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III reduction in the Geobacter species that are the predominant Fe(III reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. Results When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J, was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe (III and insoluble Mn (IV oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

Science.gov (United States)

Sun, Zhan; Bao, Lang; Li, DaoKun; Huang, Bi; Wu, Bingting

2010-09-01

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

Location of macular xanthophylls in the most vulnerable regions of photoreceptor outer-segment membranes.

Science.gov (United States)

Subczynski, Witold K; Wisniewska, Anna; Widomska, Justyna

2010-12-01

Lutein and zeaxanthin are two dietary carotenoids that compose the macular pigment of the primate retina. Another carotenoid, meso-zeaxanthin, is formed from lutein in the retina. A membrane location is one possible site where these dipolar, terminally dihydroxylated carotenoids, named macular xanthophylls, are accumulated in the nerve fibers and photoreceptor outer segments. Macular xanthophylls are oriented perpendicular to the membrane surface, which ensures their high solubility, stability, and significant effects on membrane properties. It was recently shown that they are selectively accumulated in membrane domains that contain unsaturated phospholipids, and thus are located in the most vulnerable regions of the membrane. This location is ideal if they are to act as lipid antioxidants, which is the most accepted mechanism through which lutein and zeaxanthin protect the retina from age-related macular degeneration. In this mini-review, we examine published data on carotenoid-membrane interactions and present our hypothesis that the specific orientation and location of macular xanthophylls maximize their protective action in membranes of the eye retina. Copyright © 2010 Elsevier Inc. All rights reserved.

Identification of two novel genes encoding 97- to 99-kilodalton outer membrane proteins of Chlamydia pneumoniae.Infect Immun. 1999 Jan;67(1):375-83

DEFF Research Database (Denmark)

Knudsen, K; Madsen, AS; Mygind, P

1999-01-01

Two genes encoding 97- to 99-kDa Chlamydia pneumoniae VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the C. pneumoniae outer membrane complex, and the deduced amino acid sequences were similar to those...

Mechanism and function of the outer membrane channel TolC in multidrug resistance and physiology of enterobacteria

Directory of Open Access Journals (Sweden)

Helen I. Zgurskaya

2011-09-01

Full Text Available TolC is an archetypal member of the Outer membrane Efflux Protein (OEP family. These proteins are involved in export of peptide and small molecule toxins across the outer membrane of Gram-negative bacteria. Genomes of some bacteria such as Pseudomonas species contain multiple copies of OEPs. In contrast, enterobacteria contain a single tolC gene, the product of which functions with multiple transporters. Inactivation of tolC has a major impact on enterobacterial physiology and virulence. Recent studies suggest that the role of TolC in physiology of enterobacteria is very broad and affects almost all aspects of cell adaptation to adverse enviroments. We review the current state of understanding TolC structure and present an integrated view of TolC function in enterobacteria. We propose that seemingly unrelated phenotypes of tolC mutants are linked together by a single most common condition – an oxidative damage to membranes.

Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism.

Science.gov (United States)

Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng

2011-09-15

The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.

Outer membrane lipoprotein biogenesis: Lol is not the end.

Science.gov (United States)

Konovalova, Anna; Silhavy, Thomas J

2015-10-05

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology. © 2015 The Author(s).

Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles

Science.gov (United States)

Xie, H

2015-01-01

Porphyromonas gingivalis is one of the keystone pathogens associated with chronic periodontitis. All P. gingivalis strains examined thus far produce outer membrane vesicles. Recent studies have found that vesicles possess some well-known virulence factors of P. gingivalis such as adhesins, toxins and proteolytic enzymes. Carrying most of the characteristic features of their parent P. gingivalis cells, vesicles communicate with host cells and other members of microbial biofilms, resulting in the transmission of virulence factors into these host cells and the formation of pathogenic bacteria-dominated microbial communities. An in-depth understanding of both the nature and role of vesicles in the pathogenicity of P. gingivalis is both important and timely, particularly when speaking of periodontitis and its related systemic effects. PMID:26343879

Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

Science.gov (United States)

Panigrahi, Rashmi; Lemieux, M Joanne

2015-01-01

Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

Antigen sequence typing of outer membrane protein (fetA gene of Neisseria meningitidis serogroup A from Delhi & adjoining areas

Directory of Open Access Journals (Sweden)

S Dwivedi

2014-01-01

Full Text Available Background & objectives: Meningitis caused by Neisseria meningitidis is a fatal disease. Meningococcal meningitis is an endemic disease in Delhi and irregular pattern of outbreaks has been reported in India. All these outbreaks were associated with serogroup A. Detailed molecular characterization of N. meningitidis is required for the management of this fatal disease. In this study, we characterized antigenic diversity of surface exposed outer membrane protein (OMP FetA antigen of N. meningitidis serogroup A isolates obtained from cases of invasive meningococcal meningitis in Delhi, India. Methods: Eight isolates of N. meningitidis were collected from cerebrospinal fluid during October 2008 to May 2011 from occasional cases of meningococcal meningitis. Seven isolates were from outbreaks of meningococcal meningitis in 2005-2006 in Delhi and its adjoining areas. These were subjected to molecular typing of fetA gene, an outer membrane protein gene. Results: All 15 N. meningitides isolates studied were serogroup A. This surface exposed porin is putatively under immune pressure. Hence as a part of molecular characterization, genotyping was carried out to find out the diversity in outer membrane protein (FetA gene among the circulating isolates of N. meningitidis. All 15 isolates proved to be of the same existing allele type of FetA variable region (VR when matched with global database. The allele found was F3-1 for all the isolates. Interpretation & conclusions: There was no diversity reported in the outer membrane protein FetA in the present study and hence this protein appeared to be a stable molecule. More studies on molecular characterization of FetA antigen are required from different serogroups circulating in different parts of the world.

Einfluss von Legionella pneumophila outer membrane vesicles auf die bakterielle Replikation in Makrophagen

OpenAIRE

Jung, Anna Lena; Schmeck, Bernd (Prof. Dr.)

2016-01-01

Gramnegative Bakterien treten über die Sekretion verschiedenster Moleküle mit ihrer Umwelt in Kontakt. Die Freisetzung von Proteinen und Nukleinsäuren kann aber nicht nur über die bakteriellen Sekretionssysteme vermittelt werden, sondern auch über outer membrane vesicles (OMVs) erfolgen. Diese kleinen, sphäroiden Membranvesikel werden von allen gramnegativen Bakterien gebildet und können über weite Entfernung wirken, da die zu tra...

Changes in outer membrane proteins of nontypable Haemophilus influenzae in patients with chronic obstructive pulmonary disease

NARCIS (Netherlands)

Groeneveld, K.; van Alphen, L.; Eijk, P. P.; Jansen, H. M.; Zanen, H. C.

1988-01-01

Five individual colonies of Haemophilus influenzae were isolated from each of one to three cultures of sputum collected from 18 patients with chronic obstructive pulmonary disease (COPD). The isolates were studied to investigate whether the major outer membrane proteins (MOMPs) changed during

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

DEFF Research Database (Denmark)

Peters, Diane E; Szabo, Roman; Friis, Stine

2014-01-01

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. ...

Robust outer-selective thin-film composite polyethersulfone hollow fiber membranes with low reverse salt flux for renewable salinity-gradient energy generation

KAUST Repository

Cheng, Zhen Lei; Li, Xue; Liu, Ying Da; Chung, Neal Tai-Shung

2016-01-01

This study reports outer-selective thin-film composite (TFC) hollow fiber membranes with extremely low reverse salt fluxes and robustness for harvesting salinity-gradient energy from pressure retarded osmosis (PRO) processes. Almost defect-free polyamide layers with impressive low salt permeabilities were synthesized on top of robust polyethersulfone porous supports. The newly developed TFC-II membrane shows a maximum power density of 7.81 W m−2 using 1 M NaCl and DI water as feeds at 20 bar. Reproducible data obtained in the 2nd and 3rd runs confirm its stability under high hydraulic pressure differences. Comparing to other PRO membranes reported in the literature, the newly developed membrane exhibits not only the smallest slope between water flux decline and ΔPΔP increase but also the lowest ratio of reverse salt flux to water flux. Thus, the effective osmotic driving force could be well maintained even under high pressure operations. For the first time, the effect of feed pressure buildup induced by feed flowrate was evaluated towards PRO performance. A slight increment in feed pressure buildup was found to be beneficial to water flux and power density up to 10.06 W m−2 without comprising the reverse salt flux. We believe this study may open up new perspectives on outer-selective PRO hollow fiber membranes and provide useful insights to understand and design next-generation outer-selective TFC hollow fiber membranes for osmotic power generation.

Robust outer-selective thin-film composite polyethersulfone hollow fiber membranes with low reverse salt flux for renewable salinity-gradient energy generation

KAUST Repository

Cheng, Zhen Lei

2016-01-08

This study reports outer-selective thin-film composite (TFC) hollow fiber membranes with extremely low reverse salt fluxes and robustness for harvesting salinity-gradient energy from pressure retarded osmosis (PRO) processes. Almost defect-free polyamide layers with impressive low salt permeabilities were synthesized on top of robust polyethersulfone porous supports. The newly developed TFC-II membrane shows a maximum power density of 7.81 W m−2 using 1 M NaCl and DI water as feeds at 20 bar. Reproducible data obtained in the 2nd and 3rd runs confirm its stability under high hydraulic pressure differences. Comparing to other PRO membranes reported in the literature, the newly developed membrane exhibits not only the smallest slope between water flux decline and ΔPΔP increase but also the lowest ratio of reverse salt flux to water flux. Thus, the effective osmotic driving force could be well maintained even under high pressure operations. For the first time, the effect of feed pressure buildup induced by feed flowrate was evaluated towards PRO performance. A slight increment in feed pressure buildup was found to be beneficial to water flux and power density up to 10.06 W m−2 without comprising the reverse salt flux. We believe this study may open up new perspectives on outer-selective PRO hollow fiber membranes and provide useful insights to understand and design next-generation outer-selective TFC hollow fiber membranes for osmotic power generation.

Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

Directory of Open Access Journals (Sweden)

Marlena M Wilson

Full Text Available Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

Natural inhibitors of tumor-associated proteases

International Nuclear Information System (INIS)

Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

2002-01-01

The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

Safety and Immunogenicity Testing of an Intranasal Group B Meningococcal Native Outer Membrane Vesicle Vaccine in Healthy Volunteers

National Research Council Canada - National Science Library

Drabick, Joseph

1998-01-01

An intranasal vaccine composed of native outer membrane vesicles (NOMV) not exposed to detergent or denaturing agents was prepared from the group B meningococcal strain and tested in 32 healthy adult volunteers...

Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

Science.gov (United States)

Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

2017-12-01

Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei. Copyright © 2017 Elsevier Inc. All rights reserved.

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria.

Science.gov (United States)

Sperandeo, Paola; Martorana, Alessandra M; Polissi, Alessandra

2017-11-01

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016. Published by Elsevier B.V.

Comparison of dot-ELISA and standard ELISA for detection of Neisseria meningitidis outer membrane complex-specific antibodies

Directory of Open Access Journals (Sweden)

Elza FT Belo

Full Text Available Dot-ELISA using the outer membrane complex antigens of Neisseria meningitidis as a target was standardized for rapid detection of meningococcal-specific antibodies in human serum. We investigated the level of meningococcal-specific IgG, IgA, and IgM in serum using dot-ELISA with outer membrane antigens prepared from Neisseria meningitidis serotype B:4.19:P1.15,3,7,9 (a strain isolated from a Brazilian epidemic. The dot-ELISA is based on the same principles as the standard ELISA and is useful for detection of anti-N. meningitidis B antibodies in serum of patients with meningococcal infections. For the assay, outer membrane complexes (OMCs were absorbed by nitrocellulose membrane and blocked with a 5% skim milk solution. Serum samples were drawn upon hospital admission and during convalescence from patients with meningococcal septicemia, and single samples were drawn from uninfected controls. We retrospectively examined a total of 57 serum samples: 35 from patients infected with N. meningitidis B, 12 from patients infected with Haemophilus influenzae b, and 10 from health individuals. When performed at room temperature, dot-ELISA took approximately four hours to perform, and the optimum antigen concentration was 0.42 µg per dot. The specificity of IgG, IgM, and IgA demonstrates that dot-ELISA using OMCs from N. meningitidis B as a target is suitable for serologic verification of clinically suspected meningococcal disease in patients and for titer determination of antibodies produced during different phases of natural infection. Furthermore, the sensitivity of dot-ELISA was comparable to that of standard ELISA. Overall, dot-ELISA is simple to perform, rapid, and low cost. Further validation of the test as a screening tool is required.

Outer-selective thin film composite (TFC) hollow fiber membranes for osmotic power generation

KAUST Repository

Le, Ngoc Lieu

2016-01-14

The pressure-retarded osmosis (PRO) process is a green technique for power generation to respond the world\\'s need of energy sustainability. In this study, we have developed the vital component of the process, i.e. membrane, in the configuration of the outer-selective thin-film composite (TFC) hollow fiber, which is more practical than other configurations in the real applications. The support layer morphology and the formation of the selective polyamide layer have been optimized for a good PRO performance. The results show that the bore fluid with higher amount of the solvent N-methyl-2-pyrrolidone leads to full finger-like hollow fibers, which provide higher flux but lower pressure tolerance. The addition of higher amount of diethylene glycol into the dope solution, improves the pore formation and suppresses the macrovoid formation, while properly lowering the take-up speed increases their wall thickness and pressure tolerance. A simple alcohol-pre-wetting approach on the fiber support leads to a smooth and thin polyamide layer, which is favorable for a high water flux and power density. Its efficiency follows this order: n-propanol>ethanol>methanol>water. The n-propanol pre-wetted TFC membrane can tolerate 17 bar with a peak power density of 9.59 W/m2 at room temperature, using 1 M NaCl solution as the draw solution and DI water as feed. This work demonstrates the potential of outer-selective TFC hollow fiber membranes for energy conversion via PRO process, provides useful database to fabricate suitable support morphology and raise a simple technique to practically form a thin and smooth polyamide layer.

Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

Energy Technology Data Exchange (ETDEWEB)

Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong, E-mail: weicando@ipbcams.ac.cn; Jin, Qi, E-mail: zdsys@vip.sina.com

2014-10-31

Highlights: • We utilized mTRAQ-based quantification to study protein changes in Congo red-induced OMVs. • A total of 148 proteins were identified in S. flexneri-derived OMVs. • Twenty-eight and five proteins are significantly up- and down-regulated in the CR-induced OMV, respectively. • The result implied that a special sorting mechanism of particular proteins into OMVs may exist. • Key node proteins in the protein interaction network might be important for pathogenicity. - Abstract: The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

International Nuclear Information System (INIS)

Chen, Yong; Liu, Liguo; Fu, Hua; Wei, Candong; Jin, Qi

2014-01-01

Highlights: • We utilized mTRAQ-based quantification to study protein changes in Congo red-induced OMVs. • A total of 148 proteins were identified in S. flexneri-derived OMVs. • Twenty-eight and five proteins are significantly up- and down-regulated in the CR-induced OMV, respectively. • The result implied that a special sorting mechanism of particular proteins into OMVs may exist. • Key node proteins in the protein interaction network might be important for pathogenicity. - Abstract: The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis. In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria

The voltage-dependent anion selective channel 1 (VDAC1 topography in the mitochondrial outer membrane as detected in intact cell.

Directory of Open Access Journals (Sweden)

Marianna F Tomasello

Full Text Available Voltage-Dependent Anion selective Channel maintains the permeability of the outer mitochondrial membrane and is relevant in bioenergetic metabolism and apoptosis. The structure of the protein was shown to be a β-barrel formed by 19 strands. The topology or sideness of the pore has been predicted with various approaches but a general consensus was never reached. This is an important issue since VDAC is considered receptor of Hexokinase and Bcl-2. We fused at VDAC1 C-terminus two tags separated by a caspase cleavage site. Activation in cellulo of caspases was used to eventually separate the two reporters. This experiment did not require the isolation of mitochondria and limited the possibility of outer membrane rupture due to similar procedures. Our results show that the C-terminus end of VDAC faces the mitochondrial inter-membrane space.

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer membrane protein OmpL32

Science.gov (United States)

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer membrane proteins has been shown to modulate the effectiveness of the host immu...

The major outer membrane proteins of enterobacteriaceae. Their immunological relatedness and their possible role in bacterial opsonization

NARCIS (Netherlands)

Hofstra, Harmen

1981-01-01

This thesis deals with immunological investigations of the major outer membrane proteins of the Enterobacteriaceae as a new group of enterobacterial common envelope antigens, and with some aspects of the possible role of antibodies, prepared against these proteins, in host defense mechanisms. ...

Distribution of macular xanthophylls between domains in a model of photoreceptor outer segment membranes.

Science.gov (United States)

Wisniewska, Anna; Subczynski, Witold K

2006-10-15

A model of photoreceptor outer segment (POS) membranes has been proposed, consisting of an equimolar ternary mixture of 1-palmitoyl-2-docosahexaenoylphosphatidylcholine/distearoylphosphatidylcholine/cholesterol. It was shown that, as in membranes made from the raft-forming mixture, in the model of POS membranes, two domains are formed: the raft domain (detergent resistant membranes, DRM), and the bulk domain (detergent soluble membranes, DSM). Saturation-recovery EPR discrimination by oxygen transport method also demonstrated the presence of two domains in this model system in situ at a wide range of temperatures (10-55 degrees C), showing additionally that neither lutein nor zeaxanthin at 1 mol% affect the formation of these domains. These membrane domains have been separated using cold Triton X-100 extraction from a model of POS membranes containing 1 mol% of either lutein or zeaxanthin. The results indicated that the macular xanthophylls lutein and zeaxanthin are substantially excluded from DRM and remain concentrated in DSM, a domain enriched in highly unsaturated docosahexaenoyl acid which is abundant in retina membranes. The concentration of xanthophylls in DRM and DSM calculated as the mol ratio of either xanthophyll to total lipid (phospholipid+cholesterol) was 0.0028 and 0.0391, respectively. Thus, xanthophylls are about 14 times more concentrated in DSM than in DRM. No significant difference in the distribution of lutein and zeaxanthin was found. The obtained results suggest that in POS membranes macular xanthophylls should also be concentrated in domains enriched in polyunsaturated chains.

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

International Nuclear Information System (INIS)

Reid, D.M.; Molday, R.S.; Friedel, U.; Cook, N.J.

1990-01-01

After neuraminidase treatment the Na + /Ca 2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of M r 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na + /Ca 2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na + /Ca 2+ exchange activation by sodium. The authors further investigated the density of the Na + /Ca 2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na + /Ca 2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as they have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na + /Ca 2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriae

International Nuclear Information System (INIS)

Brillet, Karl; Meksem, Ahmed; Thompson, Andrew; Cobessi, David

2009-01-01

ShuA from S. dysenteriae was crystallized in several crystallization conditions containing detergents. Adding heavy atoms during crystallization strongly improved the crystal quality and the resolution limits. Diffraction data were collected at an energy remote from the Pb M absorption edges. As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His 6 tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5 Å resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3 Å resolution using one crystal of ShuA-Pb, and at 3.2 Å resolution at an energy remote from the Pb M absorption edges for phasing on PROXIMA-1 at SOLEIL

The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM.

Directory of Open Access Journals (Sweden)

Daniel O Frank

Full Text Available The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM. In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20 by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

Science.gov (United States)

Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

2015-01-01

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

Science.gov (United States)

Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H; Klopfenstein, Jeffrey D; Rao, Jasti S

2012-02-01

Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch the glycolytic metabolism of glioma cells to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated OXPHOS to glycolysis switch. The OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to changes in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from the glycolytic to the OXPHOS pathway through an inhibitory effect on Akt, ROS induction and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for the treatment of glioma.

Porphyromonas gingivalis Outer Membrane Vesicles Enter Human Epithelial Cells via an Endocytic Pathway and Are Sorted to Lysosomal Compartments ▿

Science.gov (United States)

Furuta, Nobumichi; Tsuda, Kayoko; Omori, Hiroko; Yoshimori, Tamotsu; Yoshimura, Fuminobu; Amano, Atsuo

2009-01-01

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including major fimbriae and proteases termed gingipains, although it is not confirmed whether MVs enter host cells. In this study, we analyzed the mechanisms involved in the interactions of P. gingivalis MVs with human epithelial cells. Our results showed that MVs swiftly adhered to HeLa and immortalized human gingival epithelial cells in a fimbria-dependent manner and then entered via a lipid raft-dependent endocytic pathway. The intracellular MVs were subsequently routed to early endosome antigen 1-associated compartments and then were sorted to lysosomal compartments within 90 min, suggesting that intracellular MVs were ultimately degraded by the cellular digestive machinery. However, P. gingivalis MVs remained there for over 24 h and significantly induced acidified compartment formation after being taken up by the cellular digestive machinery. In addition, MV entry was shown to be mediated by a novel pathway for transmission of bacterial products into host cells, a Rac1-regulated pinocytic pathway that is independent of caveolin, dynamin, and clathrin. Our findings indicate that P. gingivalis MVs efficiently enter host cells via an endocytic pathway and survive within the endocyte organelles for an extended period, which provides better understanding of the role of MVs in the etiology of periodontitis. PMID:19651865

Identification and characterization of a novel outer membrane protein receptor required for hemin utilization in Vibrio vulnificus

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Datta, Shreya

2011-01-01

Vibrio vulnificus, the cause of septicemia and serious wound infection in humans and fishes, require iron for its pathogenesis. Hemin uptake through the outer membrane receptor, HupA, is one of its many mechanisms by which it acquires iron. We report here the identification of an additional TonB-dependent hemin receptor HvtA, that is needed in conjunction with the HupA protein for optimal hemin utilization. The HvtA protein is significantly homologous to other outer membrane hemin receptors and its expression in trans restored the uptake of hemin and hemoglobin, the latter to a weaker extent, in a mutant strain that was defective in both receptors. Quantitative RT-PCR suggested that transcription of the hvtA gene was iron regulated. The operon containing the hvtA gene is homologous to the operon in V. cholerae containing the hemin receptor gene hutR suggesting a vertical transmission of the hvtA cluster from V. cholerae to V. vulnificus. PMID:22015545

Cross-reactivity of antibodies against PorA after vaccination with a meningococcal B outer membrane vesicle vaccine

NARCIS (Netherlands)

Vermont, C. L.; van Dijken, H. H.; Kuipers, A. J.; van Limpt, C. J. P.; Keijzers, W. C. M.; van der Ende, A.; de Groot, R.; van Alphen, L.; van den Dobbelsteen, G. P. J. M.

2003-01-01

The cross-reactivity of PorA-specific antibodies induced by a monovalent P1.7-2,4 (MonoMen) and/or a hexavalent (HexaMen) meningococcal B outer membrane vesicle vaccine (OMV) in toddlers and school children was studied by serum bactericidal assays (SBA). First, isogenic vaccine strains and

Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

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Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

2016-10-01

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation.

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Gamalier, Juliana P; Silva, Thiago P; Zarantonello, Victor; Dias, Felipe F; Melo, Rossana C N

2017-01-01

Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation. Copyright

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Science.gov (United States)

Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

1992-01-01

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the

Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

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Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

2016-01-01

Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

Overexpression of MicA induces production of OmpC-enriched outer membrane vesicles that protect against Salmonella challenge.

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Choi, Hyun-Il; Kim, Moonjeong; Jeon, Jinseong; Han, Jin Kwan; Kim, Kwang-Sun

2017-08-26

Outer membrane vesicles (OMVs) derived from bacteria are promising candidates for subunit vaccines. Stresses that modulate the composition of outer membrane proteins (OMPs) are important for OMV synthesis. Small RNAs (sRNAs) expressed in response to stress regulate OMPs, although the mechanism underlying sRNA-mediated OMV biogenesis and its utility for developing vaccine platforms remains to be elucidated. Here, we characterized the role of a sRNA, MicA, which regulates OmpA, a major OMP involved in both production of OMVs and reactive immunity against Salmonella challenge. A Salmonella strain overexpressing MicA generated more OMVs than a control strain. In addition, OmpC was the major component of MicA-derived OMV proteins. MicA-derived OMVs induced Th1- and Th17-type immune responses in vitro and reduced Salmonella-mediated lethality in a mouse model. Thus, OmpA-regulatory sRNA-derived OMVs may facilitate production of Salmonella-protective vaccines. Copyright © 2017 Elsevier Inc. All rights reserved.

Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

Science.gov (United States)

Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

2014-01-01

Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

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Hemavathy Harikrishnan

Full Text Available Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

Getting to the Outer Leaflet: Physiology of Phosphatidylserine Exposure at the Plasma Membrane.

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Bevers, Edouard M; Williamson, Patrick L

2016-04-01

Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure. Copyright © 2016 the American Physiological Society.

Unusual Self-Assembly of the Recombinant Chlamydia trachomatis Major Outer Membrane Protein-Based Fusion Antigen CTH522 Into Protein Nanoparticles

DEFF Research Database (Denmark)

Rose, Fabrice; Karlsen, Kasper; Jensen, Pernille

2018-01-01

Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic but is a c......Sexually transmitted Chlamydia trachomatis (Ct) infects more than 100 million people annually, and untreated chlamydia infections can cause severe complications. Therefore, there is an urgent need for a chlamydia vaccine. The Ct major outer membrane protein (MOMP) is highly immunogenic...... but is a challenging vaccine candidate by being an integral membrane protein, and the immunogenicity depends on a correctly folded structure. We investigated the biophysical properties of the recombinant MOMP-based fusion antigen CTH522, which is tested in early human clinical trials. It consists of a truncated......-defined secondary structural elements, and no thermal transitions were measurable. Chemical unfolding resulted monomers that upon removal of the denaturant self-assembled into higher order structures, comparable to the structure of the native protein. The conformation of CTH522 in nanoparticles is thus not entirely...

Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4.

Science.gov (United States)

Cheng, Minyi; Takenaka, Shinji; Aoki, Shunsuke; Murakami, Shuichiro; Aoki, Kenji

2009-04-01

A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

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Bunai Christine L

2009-02-01

Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

Refolding, purification and crystallization of the FrpB outer membrane iron transporter from Neisseria meningitidis

International Nuclear Information System (INIS)

Saleem, Muhammad; Prince, Stephen M.; Patel, Hema; Chan, Hannah; Feavers, Ian M.; Derrick, Jeremy P.

2012-01-01

The refolding, purification and crystallization of FrpB from the meningitis pathogen Neisseria meningitidis is described. FrpB is an integral outer membrane protein from the human pathogen Neisseria meningitidis. It is a member of the TonB-dependent transporter family and promotes the uptake of iron across the outer membrane. There is also evidence that FrpB is an antigen and hence a potential component of a vaccine against meningococcal meningitis. FrpB incorporating a polyhistidine tag was overexpressed in Escherichia coli into inclusion bodies. The protein was then solubilized in urea, refolded and purified to homogeneity. Two separate antigenic variants of FrpB were crystallized by sitting-drop vapour diffusion. Crystals of the F5-1 variant diffracted to 2.4 Å resolution and belonged to space group C2, with unit-cell parameters a = 176.5, b = 79.4, c = 75.9 Å, β = 98.3°. Crystal-packing calculations suggested the presence of a monomer in the asymmetric unit. Crystals of the F3-3 variant also diffracted to 2.4 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 85.3, b = 104.6, c = 269.1 Å. Preliminary analysis suggested the presence of an FrpB trimer in the asymmetric unit

Allostery Is an Intrinsic Property of the Protease Domain of DegS Implications for Enzyme Function and Evolution

Energy Technology Data Exchange (ETDEWEB)

Sohn, Jungsan; Grant, Robert A.; Sauer, Robert T. (MIT)

2010-12-02

DegS is a periplasmic Escherichia coli protease, which functions as a trimer to catalyze the initial rate-limiting step in a proteolytic cascade that ultimately activates transcription of stress response genes in the cytoplasm. Each DegS subunit consists of a protease domain and a PDZ domain. During protein folding stress, DegS is allosterically activated by peptides exposed in misfolded outer membrane porins, which bind to the PDZ domain and stabilize the active protease. It is not known whether allostery is conferred by the PDZ domains or is an intrinsic feature of the trimeric protease domain. Here, we demonstrate that free DegS{sup {Delta}PDZ} equilibrates between active and inactive trimers with the latter species predominating. Substrate binding stabilizes active DegS{sup {Delta}PDZ} in a positively cooperative fashion. Mutations can also stabilize active DegS{sup {Delta}PDZ} and produce an enzyme that displays hyperbolic kinetics and degrades substrate with a maximal velocity within error of that for fully activated, intact DegS. Crystal structures of multiple DegS{sup {Delta}PDZ} variants, in functional and non-functional conformations, support a two-state model in which allosteric switching is mediated by changes in specific elements of tertiary structure in the context of an invariant trimeric base. Overall, our results indicate that protein substrates must bind sufficiently tightly and specifically to the functional conformation of DegS{sup {Delta}PDZ} to assist their own degradation. Thus, substrate binding alone may have regulated the activities of ancestral DegS trimers with subsequent fusion of the protease domain to a PDZ domain, resulting in ligand-mediated regulation.

Immunogenicity of Nontypeable Haemophilus influenzae Outer Membrane Vesicles and Protective Ability in the Chinchilla Model of Otitis Media.

Science.gov (United States)

Winter, Linda E; Barenkamp, Stephen J

2017-10-01

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are enriched in several outer membrane components, including major and minor outer membrane proteins and lipooligosaccharide. We assessed the functional activity of nontypeable Haemophilus influenzae (NTHi) OMV-specific antisera and the protective ability of NTHi OMVs as vaccine antigens in the chinchilla otitis media model. OMVs were purified from three HMW1/HMW2-expressing NTHi strains, two of which were also engineered to overexpress Hia proteins. OMV-specific antisera raised in guinea pigs were assessed for their ability to mediate killing of representative NTHi in an opsonophagocytic assay. The three OMV-specific antisera mediated killing of 18 of 65, 24 of 65, and 30 of 65 unrelated HMW1/HMW2-expressing NTHi strains. Overall, they mediated killing of 39 of 65 HMW1/HMW2-expressing strains. The two Hia-expressing OMV-specific antisera mediated killing of 17 of 25 and 14 of 25 unrelated Hia-expressing NTHi strains. Overall, they mediated killing of 20 of 25 Hia-expressing strains. OMVs from prototype NTHi strain 12 were used to immunize chinchillas and the course of middle ear infection was monitored following intrabullar challenge with the homologous strain. All control animals developed culture-positive otitis media, as did two of three HMW1/HMW2-immunized animals. All OMV-immunized animals, with or without supplemental HMW1/HMW2 immunization, were completely protected against otitis media. NTHi OMVs are the first immunogens examined in this model that provided complete protection with sterile immunity after NTHi strain 12 challenge. These data suggest that NTHi OMVs hold significant potential as components of protective NTHi vaccines, possibly in combination with HMW1/HMW2 proteins. Copyright © 2017 American Society for Microbiology.

Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

International Nuclear Information System (INIS)

Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

2006-01-01

The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials

Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

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Thomas Kieselbach

Full Text Available Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT and leukotoxin (LtxA into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs. To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e using liquid chromatography-tandem mass spectrometry (LC-MS/MS. This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.

Uncoupler resistance in E. coli Tuv and Cuv is due to the exclusion of uncoupler by the outer membrane

DEFF Research Database (Denmark)

Haworth, Robert S.; Jensen, Peter Ruhdal; Michelsen, Ole

1990-01-01

The uncoupler resistant bacterial strains E. coli Tuv and Cuv share the high deoxycholate sensitivity of the parent strain, Doc S. However, both Tuv and Cuv show greater resistance than Doc S to other detergents. Measurement of the periplasmic volume indicates that the outer membrane of Doc S is ...

Epoxide-mediated differential packaging of Cif and other virulence factors into outer membrane vesicles.

Science.gov (United States)

Ballok, Alicia E; Filkins, Laura M; Bomberger, Jennifer M; Stanton, Bruce A; O'Toole, George A

2014-10-01

Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Mapping of a Protective Epitope of the CopB Outer Membrane Protein of Moraxella catarrhalis

OpenAIRE

Aebi, Christoph; Cope, Leslie D.; Latimer, Jo L.; Thomas, Sharon E.; Slaughter, Clive A.; McCracken, George H.; Hansen, Eric J.

1998-01-01

A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003–2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the ...

A Novel Trp-rich Model Antimicrobial Peptoid with Increased Protease Stability

International Nuclear Information System (INIS)

Bang, Jeong Kyu; Nan, Yong Hai; Lee, Eun Kyu; Shin, Song Yub

2010-01-01

In order to increase protease stability of a novel Trp-rich model antimicrobial peptide, K 6 L 2 W 3 (KLWKKWKKWLKNH 2 ) and investigate the effect of L-amino acid to peptoid residue conversion on biological functions, we synthesized its antimicrobial peptoid, k 6 l 2 w 3 . Peptoid k 6 l 2 w 3 had similar bacterial selectivity compared to peptide K 6 L 2 W 3 . The bactericidal rate of k 6 l 2 w 3 was somewhat slower than that of K 6 L 2 W 3 . Peptoid k 6 l 2 w 3 exhibited very little dye leakage from bacterial outer-membrane mimicking PE/PG liposomes, as observed in K 6 L 2 W 3 , indicating that the major target site of K 6 L 2 W 3 and k 6 l 2 w 3 may be not the cell membrane but the cytoplasm of bacteria. Trypsin treatment of K 6 L 2 W 3 completely abolished antimicrobial activities against Escherichia coli and Staphylococcus aureus. In contrast, the antimicrobial activity of k 6 l 2 w 3 was completely preserved after trypsin treatment. Taken together, our results suggested that antimicrobial peptoid k 6 l 2 w 3 can potentially serves as a promising therapeutic agent for the treatment of microbial infection

Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

Science.gov (United States)

Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

2007-06-12

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

The Motion of a Single Molecule, the Lambda-Receptor, in the Bacterial Outer Membrane

DEFF Research Database (Denmark)

Oddershede, Lene; Dreyer, Jakob Kisbye; Grego, Sonia

2002-01-01

Using optical tweezers and single particle tracking, we have revealed the motion of a single protein, the lambda-receptor, in the outer membrane of living Escherichia coli bacteria. We genetically modified the lambda-receptor placing a biotin on an extracellular site of the receptor in vivo....... The efficiency of this in vivo biotinylation is very low, thus enabling the attachment of a streptavidin-coated bead binding specifically to a single biotinylated lambda-receptor. The bead was used as a handle for the optical tweezers and as a marker for the single particle tracking routine. We propose a model...

Studies on the membrane-associated proteolytic activities of Escherichia coli

International Nuclear Information System (INIS)

Palmer, S.M.

1986-01-01

The membrane fraction contains three proteolytic activities which can be resolved from whole membrane detergent extracts by DEAE-cellulose chromatography. The first two eluting activities have been previously reported as protease V and protease IV. These two enzymes were further purified by gel permeation HPLC. Protease V has a M. W. of 31,000 in SDS-PAGE gels. Protease IV has a M. W. of 62,000 and exists in two distinct isoforms of pl ≅ 6.7 and 6.9. The third enzyme eluting from the DEAE-cellulose column was further purified by affinity chromatography on Benzamidine-Sepharose 6B. This enzyme, referred to herein as protease VI, is a 43 kdal protein which has not been previously characterized. Protease VI was sensitive to inhibition by the serine protease inhibitors phenylmethylsulfony fluoride (PMSF), diisopropylfluorophosphate (DFP) and p-amino-benzamidine (PAB). Additionally, this enzyme exhibited maximal activity at pH ≅ 8.0. Incubation of whole membrane preparations with [ 3 H] DFP resulted in 11 specific proteins acquiring the radioactive label, included in this group of proteins were proteases IV, V, and VI. Several of the DFP-reactive proteins were also shown to bind [ 125 I] ampicillin

UV-C Adaptation of Shigella: Morphological, Outer Membrane Proteins, Secreted Proteins, and Lipopolysaccharides Effects.

Science.gov (United States)

Chourabi, Kalthoum; Campoy, Susana; Rodriguez, Jesus A; Kloula, Salma; Landoulsi, Ahmed; Chatti, Abdelwaheb

2017-11-01

Water UV disinfection remains extremely important, particularly in developing countries where drinking and reclaimed crop irrigation water may spread devastating infectious diseases. Enteric bacterial pathogens, among which Shigella, are possible contaminants of drinking and bathing water and foods. To study the effect of UV light on Shigella, four strains were exposed to different doses in a laboratory-made irradiation device, given that the ultraviolet radiation degree of inactivation is directly related to the UV dose applied to water. Our results showed that the UV-C rays are effective against all the tested Shigella strains. However, UV-C doses appeared as determinant factors for Shigella eradication. On the other hand, Shigella-survived strains changed their outer membrane protein profiles, secreted proteins, and lipopolysaccharides. Also, as shown by electron microscopy transmission, morphological alterations were manifested by an internal cytoplasm disorganized and membrane envelope breaks. Taken together, the focus of interest of our study is to know the adaptive mechanism of UV-C resistance of Shigella strains.

A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

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Susannah ePiek

2012-12-01

Full Text Available The Gram-negative bacterial cell envelope consists of an inner membrane (IM that surrounds the cytoplasm, and an asymmetrical outer-membrane (OM that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS, phospholipids, outer membrane proteins (OMPs and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the correct biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation and isomerisation pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, these conserved pathways have been modified to suit the lifestyle of each organism.

Redefining the essential trafficking pathway for outer membrane lipoproteins

Science.gov (United States)

Grabowicz, Marcin; Silhavy, Thomas J.

2017-01-01

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins’ key function is to prevent lethal accumulation of mislocalized lipoproteins. PMID:28416660

Redefining the essential trafficking pathway for outer membrane lipoproteins.

Science.gov (United States)

Grabowicz, Marcin; Silhavy, Thomas J

2017-05-02

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins' key function is to prevent lethal accumulation of mislocalized lipoproteins.

Crystallization and preliminary crystallographic characterization of the iron-regulated outer membrane lipoprotein FrpD from Neisseria meningitidis

Czech Academy of Sciences Publication Activity Database

Sviridova, E.; Bumba, Ladislav; Řezáčová, Pavlína; Procházková, Kateřina; Kavan, Daniel; Bezouška, Karel; Kutý, Michal; Šebo, Peter; Kutá-Smatanová, Ivana

2010-01-01

Roč. 66, - (2010), s. 1119-1123 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LC06010; GA ČR GP310/06/P150 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50520514; CEZ:AV0Z60870520; CEZ:AV0Z40550506 Keywords : Fe-regulated protein D * iron-regulated proteins * outer membrane lipoproteins Subject RIV: EC - Immunology Impact factor: 0.563, year: 2010

Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

Energy Technology Data Exchange (ETDEWEB)

Overton, K. Wesley; Park, Dan M.; Yung, Mimi C.; Dohnalkova, Alice; Smit, John; Jiao, Yongqin

2016-09-23

Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaF_aand RsaF_b, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaF_aand RsaF_bare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaF_aand RsaF_bare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaF_aand RsaF_bled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaF_aand RsaF_bled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaF_aand RsaF_bin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in

Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

DEFF Research Database (Denmark)

Koehler, JF; Birkelund, Svend; Stephens, RS

1992-01-01

The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis....... The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including...

Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens

DEFF Research Database (Denmark)

Pors, Susanne Elisabeth; Pedersen, Ida Just; Skjerning, Ragnhild Bager

2016-01-01

Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have...... ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re...

Outer membrane biogenesis in Escherichia coli, Neisseria meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori.

Science.gov (United States)

Liechti, George; Goldberg, Joanna B

2012-01-01

The bacterial pathogen Helicobacter pylori is capable of colonizing the gastric mucosa of the human stomach using a variety of factors associated with or secreted from its outer membrane (OM). Lipopolysaccharide (LPS) and numerous OM proteins have been shown to be involved in adhesion and immune stimulation/evasion. Many of these factors are essential for colonization and/or pathogenesis in a variety of animal models. Despite this wide array of potential targets present on the bacterial surface, the ability of H. pylori to vary its OM profile limits the effectiveness of vaccines or therapeutics that target any single one of these components. However, it has become evident that the proteins comprising the complexes that transport the majority of these molecules to the OM are highly conserved and often essential. The field of membrane biogenesis has progressed remarkably in the last few years, and the possibility now exists for targeting the mechanisms by which β-barrel proteins, lipoproteins, and LPS are transported to the OM, resulting in loss of bacterial fitness and significant altering of membrane permeability. In this review, the OM transport machinery for LPS, lipoproteins, and outer membrane proteins (OMPs) are discussed. While the principal investigations of these transport mechanisms have been conducted in Escherichia coli and Neisseria meningitidis, here these systems will be presented in the genetic context of ε proteobacteria. Bioinformatic analysis reveals that minimalist genomes, such as that of Helicobacter pylori, offer insight into the smallest number of components required for these essential pathways to function. Interestingly, in the majority of ε proteobacteria, while the inner and OM associated apparatus of LPS, lipoprotein, and OMP transport pathways appear to all be intact, most of the components associated with the periplasmic compartment are either missing or are almost unrecognizable when compared to their E. coli counterparts. Eventual

Analysis of the humoral immune response to Chlamydia outer membrane protein 2

DEFF Research Database (Denmark)

Mygind, P; Christiansen, Gunna; Persson, K

1998-01-01

The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated...... fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2aa23-aa93, Chlamydia psittaci-Omp2aa23-aa94, and Chlamydia trachomatis-Omp2aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547). By an enzyme-linked immunosorbent assay with serologically defined...... patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P species-specific anti-Omp2 immunoglobulins were detected....

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

Science.gov (United States)

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-04-28

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

Science.gov (United States)

Castagliuolo, I; LaMont, J T; Nikulasson, S T; Pothoulakis, C

1996-01-01

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans. PMID:8945570

Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori.

Science.gov (United States)

Chitcholtan, Kenny; Hampton, Mark B; Keenan, Jacqueline I

2008-12-01

Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.

Reversible Unfolding of Rhomboid Intramembrane Proteases.

Science.gov (United States)

Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

2016-03-29

Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

Directory of Open Access Journals (Sweden)

Muhammad Ibrahim

Full Text Available Outer membrane (OM proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

Science.gov (United States)

Ibrahim, Muhammad; Shi, Yu; Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Blondel, Carlos; Santiviago, Carlos A; Contreras, Ines; Sun, Guochang

2012-01-01

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles.

Directory of Open Access Journals (Sweden)

Jennifer M Bomberger

2009-04-01

Full Text Available Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane-derived vesicles (OMV secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including beta-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP-mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

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

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Ofir Bahar

2014-01-01

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

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii

International Nuclear Information System (INIS)

Park, Jeong Soon; Lee, Woo Cheol; Choi, Saehae; Yeo, Kwon Joo; Song, Jung Hyun; Han, Young-Hyun; Lee, Je Chul; Kim, Seung Il; Jeon, Young Ho; Cheong, Chaejoon; Kim, Hye-Yeon

2011-01-01

The crystallization of the OmpA periplasmic domain from A. baumannii is described. Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2 1 , with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å 3 Da −1

Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

Science.gov (United States)

Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

2009-11-01

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.

Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli

International Nuclear Information System (INIS)

Gu, Jiang; Ji, Xiaowei; Qi, Jianxun; Ma, Ying; Mao, Xuhu; Zou, Quanming

2010-01-01

In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a β-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. The crystals belonged to space group I4 1 32, with unit-cell parameter a = 158.99 Å. The Matthews coefficient and solvent content were calculated to be 2.55 Å 3 Da −1 and 51.77%, respectively, for two molecules in the asymmetric unit

Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

Directory of Open Access Journals (Sweden)

José Andrés Morgado-Díaz

2005-07-01

Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina.

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Michael W Stuck

Full Text Available The neural retinal leucine zipper (Nrl knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl(-/- retina, rods are converted into functional cone-like cells. The Nrl(-/- retina is characterized by large undulations of the outer nuclear layer (ONL commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl(-/- retina. We report that rosettes first appear at postnatal day (P8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM are not uniformly formed in the Nrl(-/- retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl(-/- retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM.

Proteomic study via a non-gel based approach of meningococcal outer membrane vesicle vaccine obtained from strain CU385: a road map for discovering new antigens.

Science.gov (United States)

Gil, Jeovanis; Betancourt, L Zaro H; Sardiñas, Gretel; Yero, Daniel; Niebla, Olivia; Delgado, Maité; García, Darien; Pajón, Rolando; Sánchez, Aniel; González, Luis J; Padrón, Gabriel; Campa, Concepción; Sotolongo, Franklin; Barberó, Ramón; Guillén, Gerardo; Herrera, Luis; Besada, Vladimir

2009-05-01

This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

Comparative Detection of Alkaline Protease Production in Exiguobacterium acetylicum

International Nuclear Information System (INIS)

Gomaa, O.M.; EI Shafey, H.M.

2009-01-01

Alkaline protease is one of the most important enzymes in industry, medicine, and research. In the present work, a comparative detection for alkaline protease activity was established for instant detection of enzyme activity. Eight different alkalophilic bacterial isolates were compared based on the clear zone they produced on skim milk agar. One strain gave an absolute clear zone in 16 hours and was used for alkaline protease detection. The result of Phenotypic identification using Biology Microlog 3 identified the isolate as Exiguobacterium acetylicum. The isolate under study showed slightly different characteristics from a known Exiguobacterium acetylicum strain. The isolate tolerated alkaline conditions up to ph 11, while good growth was evident at ph 7, the maximum alkaline protease activity was observed at ph 9 which reached up to 109.01 U/ml. The alkaline activity assay using alkaline protease enzyme assay were coordinating with those obtained by conductivity; there was a relevant decrease in conductivity at the maximum increase in enzyme activity, which proved the cell membrane conductivity has a close relation to alkaline protease production. This isolate has tolerated gamma radiation, the increase in dose (up to 4 Gy) gave wider clear zones in terms of diameter and this was relevant to the conductivity measurements

Biogenesis and Membrane Targeting of Lipoproteins.

Science.gov (United States)

Narita, Shin-Ichiro; Tokuda, Hajime

2010-09-01

Bacterial lipoproteins represent a unique class of membrane proteins, which are anchored to membranes through triacyl chains attached to the amino-terminal cysteine. They are involved in various functions localized in cell envelope. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane, with others being in the inner membrane. All lipoproteins are synthesized in the cytoplasm with an N-terminal signal peptide, translocated across the inner membrane by the Sec translocon to the periplasmic surface of the inner membrane, and converted to mature lipoproteins through sequential reactions catalyzed by three lipoprotein-processing enzymes: Lgt, LspA, and Lnt. The sorting of lipoproteins to the outer membrane requires a system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the sorting by mediating the detachment of lipoproteins from the inner membrane. Formation of the LolA-lipoprotein complex is coupled to this LolCDE-dependent release reaction. LolA accommodates the amino-terminal acyl chain of lipoproteins in its hydrophobic cavity, thereby generating a hydrophilic complex that can traverse the periplasmic space by diffusion. Lipoproteins are then transferred to LolB on the outer membrane and anchored to the inner leaflet of the outer membrane by the action of LolB. In contrast, since LolCDE does not recognize lipoproteins possessing Asp at position +2, these lipoproteins remain anchored to the inner membrane. Genes for Lol proteins are widely conserved among gram-negative bacteria, and Lol-mediated outer membrane targeting of lipoproteins is considered to be the general lipoprotein localization mechanism.

Lipopolysaccharide Membranes and Membrane Proteins of Pseudomonas aeruginosa Studied by Computer Simulation

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Straatsma, TP

2006-12-01

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is also a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid

Antibiotic trapping by plasmid-encoded cmy-2-lactamase combined with reduced outer membrane permeability as a mechanism of carbapenem resistance in escherichia coli

NARCIS (Netherlands)

W.H.F. Goessens (Wil); A.K. van der Bij (Akke); R. van Boxtel (Ria); J.D.D. Pitout (J. D D); P. van Ulsen (Peter); D.C. Melles (Damian); J. Tommassen (Jan)

2013-01-01

textabstractA liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane

MOLECULAR MODELING STUDY OF THE CONTRIBUTIONS OF SIDE AMINO ACID RESIDUES OF POLYMYXIN B3 TO ITS BINDING WITH E.COLI OUTER MEMBRANE LIPOPOLYSACCHARIDE

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Lisnyak Yu. V.

2014-12-01

Full Text Available Last decades, antimicrobial peptides (AMPs are the subject of intense investigations aimed to develop effective drugs against extremely resistant nosocomial bacterial pathogens (especially Gram-negative bacteria. In particular, there has been greatly renewed interest to polymyxins, the representatives of AMPs which are specific and highly potent against Gram-negative bacteria, but have potential nephrotoxic side effect. A prerequisite of purposeful enhancement of therapeutic properties of polymyxins is a detailed knowledge of the molecular mechanisms of their interactions with cell targets. Lipopolysaccharide (LPS, the main component of the outer leaflet of outer membrane of gram-negative bacteria, is a primary cell target of polymyxins. The aim of the paper was to study the peculiarities of molecular interactions of polymyxin В3 with lipopolysaccharide of the outer membrane of gram-negative bacterium. Materials and methods The complexes of polymyxin В3 (PmВ3 and its alaninederivatives with E. coli outer membrane lipopolysaccharide were built and studied by molecular modeling methods (minimization, simulated annealing, docking. Atom coordinates of polymyxin В3 and LPS structures were taken from nuclear magnetic resonance and X-ray crystallography experiments, respectively. The AMBER03 force field was used with a 1.05 nm force cutoff. Longrange electrostatic interactions were treated by the Particle Mesh Ewald method. Results and discussion Alanine scanning of PmВ3 molecule has been carried out and the role of its side amino acid residues in the formation of complex with lipopolysaccharide has been investigated. It has been shown that substitutions of polymyxin’s Dab residues in positions 1, 3, 5, 8 and 9 for alanine markedly reduce the binding energy of PmB3-LPS complex, where as the similar substitutions of residues in positions 2, 6, 7 and 10 leave the binding energy virtually unchanged. Structural aspects of antimicrobial action of

Identification of an archaeal presenilin-like intramembrane protease.

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Torres-Arancivia, Celia; Ross, Carolyn M; Chavez, Jose; Assur, Zahra; Dolios, Georgia; Mancia, Filippo; Ubarretxena-Belandia, Iban

2010-09-29

The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

Undressing of Waddlia chondrophila to enrich its outer membrane proteins to develop a new species-specific ELISA

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J. Lienard

2014-01-01

Full Text Available Waddlia chondrophila, an obligate intracellular bacterium of the Chlamydiales order, is considered as an agent of bovine abortion and a likely cause of miscarriage in humans. Its role in respiratory diseases was questioned after the detection of its DNA in clinical samples taken from patients suffering from pneumonia or bronchiolitis. To better define the role of Waddlia in both miscarriage and pneumonia, a tool allowing large-scale serological investigations of Waddlia seropositivity is needed. Therefore, enriched outer membrane proteins of W. chondrophila were used as antigens to develop a specific ELISA. After thorough analytical optimization, the ELISA was validated by comparison with micro-immunofluorescence and it showed a sensitivity above 85% with 100% specificity. The ELISA was subsequently applied to human sera to specify the role of W. chondrophila in pneumonia. Overall, 3.6% of children showed antibody reactivity against W. chondrophila but no significant difference was observed between children with and without pneumonia. Proteomic analyses were then performed using mass spectrometry, highlighting members of the outer membrane protein family as the dominant proteins. The major Waddlia putative immunogenic proteins were identified by immunoblot using positive and negative human sera. The new ELISA represents an efficient tool with high throughput applications. Although no association with pneumonia and Waddlia seropositivity was observed, this ELISA could be used to specify the role of W. chondrophila in miscarriage and in other diseases.

Negative Charge Neutralization in the Loops and Turns of Outer Membrane Phospholipase A Impacts Folding Hysteresis at Neutral pH.

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McDonald, Sarah K; Fleming, Karen G

2016-11-08

Hysteresis in equilibrium protein folding titrations is an experimental barrier that must be overcome to extract meaningful thermodynamic quantities. Traditional approaches to solving this problem involve testing a spectrum of solution conditions to find ones that achieve path independence. Through this procedure, a specific pH of 3.8 was required to achieve path independence for the water-to-bilayer equilibrium folding of outer membrane protein OmpLA. We hypothesized that the neutralization of negatively charged side chains (Asp and Glu) at pH 3.8 could be the physical basis for path-independent folding at this pH. To test this idea, we engineered variants of OmpLA with Asp → Asn and Glu → Gln mutations to neutralize the negative charges within various regions of the protein and tested for reversible folding at neutral pH. Although not fully resolved, our results show that these mutations in the periplasmic turns and extracellular loops are responsible for 60% of the hysteresis in wild-type folding. Overall, our study suggests that negative charges impact the folding hysteresis in outer membrane proteins and their neutralization may aid in protein engineering applications.

Involvement and necessity of the Cpx regulon in the event of aberrant β-barrel outer membrane protein assembly

Science.gov (United States)

Gerken, Henri; Leiser, Owen P.; Bennion, Drew; Misra, Rajeev

2010-01-01

Summary The Cpx and σE regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the σE pathway monitors the biogenesis of β-barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of β-barrel OMP mis-assembly, by utilizing mutants expressing either a defective β-barrel OMP assembly machinery (Bam) or assembly defective β-barrel OMPs. Analysis of specific mRNAs showed that ΔcpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the σE pathway. The synthetic conditional lethal phenotype of ΔcpxR in mutant Bam or β-barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant β-barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly-defective β-barrel OMP species. Together, these results showed that both the Cpx and σE regulons are required to reduce envelope stress caused by aberrant β-barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression. PMID:20487295

Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

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Nicolas Faller

Full Text Available Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

International Nuclear Information System (INIS)

Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S.

2005-01-01

Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V M ) of 3.3 Å 3 Da −1 , corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin

Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?

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Paramasivam Nagarajan

2012-09-01

Full Text Available Abstract Background In Gram-negative bacteria, the outer membrane is composed of an asymmetric lipid bilayer of phopspholipids and lipopolysaccharides, and the transmembrane proteins that reside in this membrane are almost exclusively β-barrel proteins. These proteins are inserted into the membrane by a highly conserved and essential machinery, the BAM complex. It recognizes its substrates, unfolded outer membrane proteins (OMPs, through a C-terminal motif that has been speculated to be species-specific, based on theoretical and experimental results from only two species, Escherichia coli and Neisseria meningitidis, where it was shown on the basis of individual sequences and motifs that OMPs from the one cannot easily be over expressed in the other, unless the C-terminal motif was adapted. In order to determine whether this species specificity is a general phenomenon, we undertook a large-scale bioinformatics study on all predicted OMPs from 437 fully sequenced proteobacterial strains. Results We were able to verify the incompatibility reported between Escherichia coli and Neisseria meningitidis, using clustering techniques based on the pairwise Hellinger distance between sequence spaces for the C-terminal motifs of individual organisms. We noticed that the amino acid position reported to be responsible for this incompatibility between Escherichia coli and Neisseria meningitidis does not play a major role for determining species specificity of OMP recognition by the BAM complex. Instead, we found that the signal is more diffuse, and that for most organism pairs, the difference between the signals is hard to detect. Notable exceptions are the Neisseriales, and Helicobacter spp. For both of these organism groups, we describe the specific sequence requirements that are at the basis of the observed difference. Conclusions Based on the finding that the differences between the recognition motifs of almost all organisms are small, we assume that

The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

Science.gov (United States)

König, Tim; Tröder, Simon E; Bakka, Kavya; Korwitz, Anne; Richter-Dennerlein, Ricarda; Lampe, Philipp A; Patron, Maria; Mühlmeister, Mareike; Guerrero-Castillo, Sergio; Brandt, Ulrich; Decker, Thorsten; Lauria, Ines; Paggio, Angela; Rizzuto, Rosario; Rugarli, Elena I; De Stefani, Diego; Langer, Thomas

2016-10-06

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca 2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca 2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca 2+ homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Antibiotic Trapping by Plasmid-Encoded CMY-2 beta-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

NARCIS (Netherlands)

Goessens, W.H.F.; van der Bij, A.K.; van Boxtel, R.; Pitout, J.D.D.; van Ulsen, J.P.; Melles, D.C.; Tommassen, J.

2013-01-01

A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein

Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

International Nuclear Information System (INIS)

Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

1986-01-01

The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes 14 C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg 2+ . Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-γ-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated

Identification of an archaeal presenilin-like intramembrane protease.

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Celia Torres-Arancivia

Full Text Available BACKGROUND: The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs. The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. METHODOLOGY AND PRINCIPAL FINDINGS: We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. CONCLUSIONS AND SIGNIFICANCE: Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

Functional characterization of the mammalian iAAA protease subunit, YME1L

OpenAIRE

Majczak, Joanna

2008-01-01

The iAAA protease is an ATP-dependent proteolytic complex in the mitochondrial inner membrane and belongs to the highly conserved family of AAA proteins. In the yeast Saccharomyces cerevisiae, the iAAA protease is a homo-oligomeric complex composed of Yme1p subunits which are active in the intermembrane space and mediate protein quality control. Yeast cells lacking Yme1p are characterized by pleiotropic phenotypes including a respiratory deficiency at elevated temperature and an aberrant mito...

An FtsH protease is recruited to the mitochondrion of Plasmodium falciparum.

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Aiman Tanveer

Full Text Available The two organelles, apicoplast and mitochondrion, of the malaria parasite Plasmodium falciparum have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (PfFtsH1 that exhibits ATP- and Zn(2+-dependent protease activity. PfFtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged PfFtsH1, and immunofluorescence assay with anti-PfFtsH1 Ab demonstrated that the protein localises to P. falciparum mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in PfFtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of PfFtsH1 in Escherichia coli converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the Plasmodium factor on the bacterial homolog, indicative of functional conservation with EcFtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in P. falciparum.

In-vivo identification of direct electron transfer from Shewanella oneidensis MR-1 to electrodes via outer-membrane OmcA-MtrCAB protein complexes

Energy Technology Data Exchange (ETDEWEB)

Okamoto, Akihiro [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Nakamura, Ryuhei, E-mail: nakamura@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Hashimoto, Kazuhito, E-mail: hashimoto@light.t.u-tokyo.ac.jp [Department of Applied Chemistry, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); ERATO/JST, HASHIMOTO Light Energy Conversion Project (Japan)

2011-06-30

Graphical abstract: . Display Omitted Highlights: > Monolayer biofilm of Shewanella cells was prepared on an ITO electrode. > Extracellular electron transfer (EET) process was examined with series of mutants. > Direct ET was confirmed with outer-membrane-bound OmcA-MtrCAB complex. > The EET process was not prominently influenced by capsular polysaccharide. - Abstract: The direct electron-transfer (DET) property of Shewanella bacteria has not been resolved in detail due to the complexity of in vivo electrochemistry in whole-cell systems. Here, we report the in vivo assignment of the redox signal indicative of the DET property in biofilms of Shewanella oneidensis MR-1 by cyclic voltammetry (CV) with a series of mutants and a chemical marking technique. The CV measurements of monolayer biofilms formed by deletion mutants of c-type cytochromes ({Delta}mtrA, {Delta}mtrB, {Delta}mtrC/{Delta}omcA, and {Delta}cymA), and pilin ({Delta}pilD), capsular polysaccharide ({Delta}SO3177) and menaquinone ({Delta}menD) biosynthetic proteins demonstrated that the electrochemical redox signal with a midpoint potential at 50 mV (vs. SHE) was due to an outer-membrane-bound OmcA-MtrCAB protein complex of decaheme cytochromes, and did not involve either inner-membrane-bound CymA protein or secreted menaquinone. Using the specific binding affinity of nitric monoxide for the heme groups of c-type cytochromes, we further confirmed this conclusion. The heterogeneous standard rate constant for the DET process was estimated to be 300 {+-} 10 s{sup -1}, which was two orders of magnitude higher than that previously reported for the electron shuttling process via riboflavin. Experiments using a mutant unable to produce capsular polysaccharide ({Delta}SO3177) revealed that the DET property of the OmcA-MtrCAB complex was not influenced by insulating and hydrophilic extracellular polysaccharide. Accordingly, under physiological conditions, S. oneidensis MR-1 utilizes a high density of outer-membrane

Outer hair cell piezoelectricity: frequency response enhancement and resonance behavior.

Science.gov (United States)

Weitzel, Erik K; Tasker, Ron; Brownell, William E

2003-09-01

Stretching or compressing an outer hair cell alters its membrane potential and, conversely, changing the electrical potential alters its length. This bi-directional energy conversion takes place in the cell's lateral wall and resembles the direct and converse piezoelectric effects both qualitatively and quantitatively. A piezoelectric model of the lateral wall has been developed that is based on the electrical and material parameters of the lateral wall. An equivalent circuit for the outer hair cell that includes piezoelectricity shows a greater admittance at high frequencies than one containing only membrane resistance and capacitance. The model also predicts resonance at ultrasonic frequencies that is inversely proportional to cell length. These features suggest all mammals use outer hair cell piezoelectricity to support the high-frequency receptor potentials that drive electromotility. It is also possible that members of some mammalian orders use outer hair cell piezoelectric resonance in detecting species-specific vocalizations.

Coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains.

Science.gov (United States)

Earnest, James T; Hantak, Michael P; Park, Jung-Eun; Gallagher, Tom

2015-06-01

Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases. Enveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus

TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

Science.gov (United States)

Yun, Bingling; Zhang, Yao; Liu, Yongzhen; Guan, Xiaolu; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Honglei; Gao, Li; Li, Kai; Gao, Yulong; Wang, Xiaomei

2016-12-15

The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope

Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

Science.gov (United States)

Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

2016-11-01

Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks.

Science.gov (United States)

Elliott, Michael H; Nash, Zack A; Takemori, Nobuaki; Fliesler, Steven J; McClellan, Mark E; Naash, Muna I

2008-01-01

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

The Mitochondrial m-AAA Protease Prevents Demyelination and Hair Greying.

Science.gov (United States)

Wang, Shuaiyu; Jacquemyn, Julie; Murru, Sara; Martinelli, Paola; Barth, Esther; Langer, Thomas; Niessen, Carien M; Rugarli, Elena I

2016-12-01

The m-AAA protease preserves proteostasis of the inner mitochondrial membrane. It ensures a functional respiratory chain, by controlling the turnover of respiratory complex subunits and allowing mitochondrial translation, but other functions in mitochondria are conceivable. Mutations in genes encoding subunits of the m-AAA protease have been linked to various neurodegenerative diseases in humans, such as hereditary spastic paraplegia and spinocerebellar ataxia. While essential functions of the m-AAA protease for neuronal survival have been established, its role in adult glial cells remains enigmatic. Here, we show that deletion of the highly expressed subunit AFG3L2 in mature mouse oligodendrocytes provokes early-on mitochondrial fragmentation and swelling, as previously shown in neurons, but causes only late-onset motor defects and myelin abnormalities. In contrast, total ablation of the m-AAA protease, by deleting both Afg3l2 and its paralogue Afg3l1, triggers progressive motor dysfunction and demyelination, owing to rapid oligodendrocyte cell death. Surprisingly, the mice showed premature hair greying, caused by progressive loss of melanoblasts that share a common developmental origin with Schwann cells and are targeted in our experiments. Thus, while both neurons and glial cells are dependant on the m-AAA protease for survival in vivo, complete ablation of the complex is necessary to trigger death of oligodendrocytes, hinting to cell-autonomous thresholds of vulnerability to m-AAA protease deficiency.

Molecular recognition in myxobacterial outer membrane exchange: functional, social and evolutionary implications.

Science.gov (United States)

Wall, Daniel

2014-01-01

Through cooperative interactions, bacteria can build multicellular communities. To ensure that productive interactions occur, bacteria must recognize their neighbours and respond accordingly. Molecular recognition between cells is thus a fundamental behaviour, and in bacteria important discoveries have been made. This MicroReview focuses on a recently described recognition system in myxobacteria that is governed by a polymorphic cell surface receptor called TraA. TraA regulates outer membrane exchange (OME), whereby myxobacterial cells transiently fuse their OMs to efficiently transfer proteins and lipids between cells. Unlike other transport systems, OME is rather indiscriminate in what OM goods are transferred. In contrast, the recognition of partnering cells is discriminatory and only occurs between cells that bear identical or closely related TraA proteins. Therefore TraA functions in kin recognition and, in turn, OME helps regulate social interactions between myxobacteria. Here, I discuss and speculate on the social and evolutionary implications of OME and suggest it helps to guide their transition from free-living cells into coherent and functional populations. © 2013 John Wiley & Sons Ltd.

Improved production process for native outer membrane vesicle vaccine against Neisseria meningitidis.

Directory of Open Access Journals (Sweden)

Bas van de Waterbeemd

Full Text Available An improved detergent-free process has been developed to produce vaccine based on native outer membrane vesicles (NOMV against Neisseria meningitidis serogroup B. Performance was evaluated with the NonaMen vaccine concept, which provides broad coverage based on nine distinct PorA antigens. Scalable aseptic equipment was implemented, replacing undesirable steps like ultracentrifugation, inactivation with phenol, and the use of preservatives. The resulting process is more consistent and gives a higher yield than published reference processes, enabling NOMV production at commercial scale. Product quality met preliminary specifications for 9 consecutive batches, and an ongoing study confirmed real-time stability up to 12 months after production. As the NOMV had low endotoxic activity and induced high bactericidal titres in mice, they are expected to be safe and effective in humans. The production process is not limited to NonaMen and may be applicable for other N. meningitidis serogroups and other gram-negative pathogens. The current results therefore facilitate the late-stage development and clinical evaluation of NOMV vaccines.

Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

International Nuclear Information System (INIS)

Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M.

2015-01-01

Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales

Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

Energy Technology Data Exchange (ETDEWEB)

Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M., E-mail: fmarassi@sbmri.org [Sanford-Burnham Medical Research Institute (United States)

2015-04-15

Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales.

Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB

Energy Technology Data Exchange (ETDEWEB)

Tanabe, Mikio; Iverson, Tina M.; (Vanderbilt)

2010-01-28

The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P6{sub 3}. The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P6{sub 3} crystal forms contained one PorB monomer in the asymmetric unit. Of the three, the P6{sub 3} crystal form had the best diffraction quality, yielding data extending to 2.3 {angstrom} resolution.

m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration.

Science.gov (United States)

Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

2018-03-01

The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.

Role of Tim50 in the transfer of precursor proteins from the outer to the inner membrane of mitochondria.

Science.gov (United States)

Mokranjac, Dejana; Sichting, Martin; Popov-Celeketić, Dusan; Mapa, Koyeli; Gevorkyan-Airapetov, Lada; Zohary, Keren; Hell, Kai; Azem, Abdussalam; Neupert, Walter

2009-03-01

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.

Iodo-gen-catalysed iodination for identification of surface-exposed outer membrane proteins of Escherichia coli K12

International Nuclear Information System (INIS)

Ferreira, L.C.S.; Almeida, D.F. de

1987-01-01

Surface proteins of Escherichia coli K12 were identified by radiolabelling using 1,3,4,6 - tatrachloro, 3-alpha, 6-alpha - diphenylgycoluryl (Iodo-Gen) and 131 I. Labelled proteins were localized in the outer membrane of the cells. Using this technique it has been possible to observe technique it has been possible to observe that the eletrophoretic pattern of surface proteins changes according to the growth phases in culture. Radiolabelling of E.coli cells inculbated at 42 0 C showed that the syntheses of two surface proteins were temperature-inducible. At least one such protein may be involved in the process of cell division in E.coli K12. (author) [pt

Iodo-gen-catalysed iodination for identification of surface-exposed outer membrane proteins of Escherichia coli K12

Energy Technology Data Exchange (ETDEWEB)

Ferreira, L C.S.; Almeida, D.F. de

1987-12-01

Surface proteins of Escherichia coli K12 were identified by radiolabelling using 1,3,4,6 - tatrachloro, 3-alpha, 6-alpha - diphenylgycoluryl (Iodo-Gen) and /sup 131/I. Labelled proteins were localized in the outer membrane of the cells. Using this technique it has been possible to observe technique it has been possible to observe that the eletrophoretic pattern of surface proteins changes according to the growth phases in culture. Radiolabelling of E.coli cells inculbated at 42/sup 0/C showed that the syntheses of two surface proteins were temperature-inducible. At least one such protein may be involved in the process of cell division in E.coli K12.

Outer membrane protein changes during bacteroid development are independent of nitrogen fixation and differ between indeterminate and determinate nodulating host plants of Rhizobium leguminosarum

NARCIS (Netherlands)

Roest, H.P.; Goosen-de Roo, L.; Wijffelman, C.A.; Maagd, de R.A.; Lugtenberg, B.J.J.

1995-01-01

The outer membrane of bacteroids contains largely decreased levels of protein antigen groups II and III in comparison with that of free-living rhizobia (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J, J. Lugtenberg, J.Bacteriol, 171:1136-1142, 1989). Since we intend to study the molecular

In silico local structure approach: a case study on outer membrane proteins.

Science.gov (United States)

Martin, Juliette; de Brevern, Alexandre G; Camproux, Anne-Claude

2008-04-01

The detection of Outer Membrane Proteins (OMP) in whole genomes is an actual question, their sequence characteristics have thus been intensively studied. This class of protein displays a common beta-barrel architecture, formed by adjacent antiparallel strands. However, due to the lack of available structures, few structural studies have been made on this class of proteins. Here we propose a novel OMP local structure investigation, based on a structural alphabet approach, i.e., the decomposition of 3D structures using a library of four-residue protein fragments. The optimal decomposition of structures using hidden Markov model results in a specific structural alphabet of 20 fragments, six of them dedicated to the decomposition of beta-strands. This optimal alphabet, called SA20-OMP, is analyzed in details, in terms of local structures and transitions between fragments. It highlights a particular and strong organization of beta-strands as series of regular canonical structural fragments. The comparison with alphabets learned on globular structures indicates that the internal organization of OMP structures is more constrained than in globular structures. The analysis of OMP structures using SA20-OMP reveals some recurrent structural patterns. The preferred location of fragments in the distinct regions of the membrane is investigated. The study of pairwise specificity of fragments reveals that some contacts between structural fragments in beta-sheets are clearly favored whereas others are avoided. This contact specificity is stronger in OMP than in globular structures. Moreover, SA20-OMP also captured sequential information. This can be integrated in a scoring function for structural model ranking with very promising results. (c) 2007 Wiley-Liss, Inc.

Loss of Drosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degeneration.

Science.gov (United States)

Qi, Y; Liu, H; Daniels, M P; Zhang, G; Xu, H

2016-02-01

Mitochondrial AAA (ATPases Associated with diverse cellular Activities) proteases i-AAA (intermembrane space-AAA) and m-AAA (matrix-AAA) are closely related and have major roles in inner membrane protein homeostasis. Mutations of m-AAA proteases are associated with neuromuscular disorders in humans. However, the role of i-AAA in metazoans is poorly understood. We generated a deletion affecting Drosophila i-AAA, dYME1L (dYME1L(del)). Mutant flies exhibited premature aging, progressive locomotor deficiency and neurodegeneration that resemble some key features of m-AAA diseases. dYME1L(del) flies displayed elevated mitochondrial unfolded protein stress and irregular cristae. Aged dYME1L(del) flies had reduced complex I (NADH/ubiquinone oxidoreductase) activity, increased level of reactive oxygen species (ROS), severely disorganized mitochondrial membranes and increased apoptosis. Furthermore, inhibiting apoptosis by targeting dOmi (Drosophila Htra2/Omi) or DIAP1, or reducing ROS accumulation suppressed retinal degeneration. Our results suggest that i-AAA is essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA leads to the accumulation of oxidative damage and progressive deterioration of membrane integrity, which might contribute to apoptosis upon the release of proapoptotic molecules such as dOmi. Containing ROS level could be a potential strategy to manage mitochondrial AAA protease deficiency.

Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

Science.gov (United States)

Liew, Lynette Sin Yee; Lee, Michelle Yueqi; Wong, Ying Lei; Cheng, Jinting; Li, Qingxin; Kang, CongBao

2016-05-01

Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease. Copyright © 2016 Elsevier Inc. All rights reserved.

Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants

OpenAIRE

Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

2016-01-01

Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri...

Incorporation of squalene into rod outer segments

International Nuclear Information System (INIS)

Keller, R.K.; Fliesler, S.J.

1990-01-01

We have reported previously that squalene is the major radiolabeled nonsaponifiable lipid product derived from [ 3 H]acetate in short term incubations of frog retinas. In the present study, we demonstrate that newly synthesized squalene is incorporated into rod outer segments under similar in vitro conditions. We show further that squalene is an endogenous constituent of frog rod outer segment membranes; its concentration is approximately 9.5 nmol/mumol of phospholipid or about 9% of the level of cholesterol. Pulse-chase experiments with radiolabeled precursors revealed no metabolism of outer segment squalene to sterols in up to 20 h of chase. Taken together with our previous absolute rate studies, these results suggest that most, if not all, of the squalene synthesized by the frog retina is transported to rod outer segments. Synthesis of protein is not required for squalene transport since puromycin had no effect on squalene incorporation into outer segments. Conversely, inhibition of isoprenoid synthesis with mevinolin had no effect on the incorporation of opsin into the outer segment. These latter results support the conclusion that the de novo synthesis and subsequent intracellular trafficking of opsin and isoprenoid lipids destined for the outer segment occur via independent mechanisms

Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host

OpenAIRE

Koussis, K.; Goulielmaki, E.; Chalari, A.; Withers-Martinez, C.; Siden-Kiamos, I.; Matuschewski, K.; Loukeris, T.

2017-01-01

Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane?bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways througho...

Focus on the Outer Membrane Factor OprM, the Forgotten Player from Efflux Pumps Assemblies

Directory of Open Access Journals (Sweden)

Gilles Phan

2015-11-01

Full Text Available Antibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains. It has become a major public concern, urging the need to discover new strategies to combat them. Among the different ways used by bacteria to resist antibiotics, the active efflux is one of the main mechanisms. In Gram-negative bacteria the efflux pumps are comprised of three components forming a long edifice crossing the complete cell wall from the inside to the outside of the cell. Blocking these pumps would permit the restoration of the effectiveness of the current antibiotherapy which is why it is important to increase our knowledge on the different proteins involved in these complexes. A tremendous number of experiments have been performed on the inner membrane protein AcrB from Escherichia coli and, to a lesser extent, the protein partners forming the AcrAB-TolC pump, but less information is available concerning the efflux pumps from other virulent Gram-negative bacteria. The present review will focus on the OprM outer membrane protein from the MexAB-OprM pump of Pseudomonas aeruginosa, highlighting similarities and differences compare to the archetypal AcrAB-TolC in terms of structure, function, and assembly properties.

Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins.

Science.gov (United States)

Sundara Baalaji, N; Mathew, M K; Krishnaswamy, S

2006-10-01

The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.

Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

International Nuclear Information System (INIS)

Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

1982-01-01

The presence of two distinct proteolytic activities in the rat uterus was confirmed with 14 C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition

Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology Estudo comparativo de técnicas de isolamento de membrana externa de Yersinia pestis e seu uso na epidemiologia da peste

Directory of Open Access Journals (Sweden)

Frederico Guilherme Coutinho Abath

1990-04-01

Full Text Available In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.No presente estudo três técnicas para isolamento de frações enriquecidas em membrana externa de Y. pestis foram avaliadas. As técnicas utilizadas foram: centrifugação em gradiente de densidade em sacarose e solubilização diferencial com Sarkosyl ou Triton X-100. A análise por eletroforese em gel de poliacrilamida na presença de dodecil sulfato de sódio (SDS-PAGE das membranas externas extraídas pelos diferentes métodos evidenciou perfis protéicos semelhantes. A determinação das atividades de NADH-desidrogenase e succinato-desidrogenase (enzimas de membrana interna indicou que todas as preparações estudadas eram adequadas a estudos analíticos. Obteve-se evidências preliminares sobre o possível uso de perfis protéicos de membrana externa na identificação de variantes geográficos entre isolados selvagens de Y. pestis.

The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

Science.gov (United States)

Chen, Hongyu; Kandel, Prem P; Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

2017-11-01

MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a β-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

Photoreceptor Outer Segment on Internal Limiting Membrane after Macular Hole Surgery: Implications for Pathogenesis.

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Grinton, Michael E; Sandinha, Maria T; Steel, David H W

2015-01-01

This report presents a case, which highlights key principles in the pathophysiology of macular holes. It has been hypothesized that anteroposterior (AP) and tangential vitreous traction on the fovea are the primary underlying factors causing macular holes [Nischal and Pearson; in Kanski and Bowling: Clinical Ophthalmology: A Systemic Approach, 2011, pp 629-631]. Spectral domain optical coherence tomography (OCT) has subsequently corroborated this theory in part but shown that AP vitreofoveal traction is the more common scenario [Steel and Lotery: Eye 2013;27:1-21]. This study was conducted as a single case report. A 63-year old female presented to her optician with blurred and distorted vision in her left eye. OCT showed a macular hole with a minimum linear diameter of 370 µm, with persistent broad vitreofoveal attachment on both sides of the hole edges. The patient underwent combined left phacoemulsification and pars plana vitrectomy, internal limiting membrane (ILM) peel and gas injection. The ILM was examined by electron microscopy and showed the presence of a cone outer segment on the retinal side. Post-operative OCT at 11 weeks showed a closed hole with recovery of the foveal contour and good vision. Our case shows the presence of a photoreceptor outer segment on the retinal side of the ILM and reinforces the importance of tangential traction in the development of some macula holes. The case highlights the theory of transmission of inner retinal forces to the photoreceptors via Müller cells and how a full thickness macular hole defect can occur in the absence of AP vitreomacular traction.

Genetic Manipulation of Outer Membrane Permeability: Generating Porous Heterogeneous Catalyst Analogs in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Patel, TN; Park, AHA; Bantat, S

2014-12-01

The limited permeability of the E. coli outer membrane can significantly hinder whole-cell biocatalyst performance. In this study, the SARS coronavirus small envelope protein (SCVE) was expressed in E. coli cells previously engineered for periplasmic expression of carbonic anhydrase (CA) activity. This maneuver increased small molecule uptake by the cells, resulting in increased apparent CA activity of the biocatalysts. The enhancements in activity were quantified using methods developed for traditional heterogeneous catalysis. The expression of the SCVE protein was found to significantly reduce the Thiele moduli (phi), as well as increase the effectiveness factors (eta), effective diffusivities (D-e), and permeabilities (P) of the biocatalysts. These catalytic improvements translated into superior performance of the biocatalysts for the precipitation of calcium carbonate from solution which is an attractive strategy for long-term sequestration of captured carbon dioxide. Overall, these results demonstrate that synthetic biology approaches can be used to enhance heterogeneous catalysts incorporated into microbial whole-cell scaffolds.

Dual effect of local anesthetics on the function of excitable rod outer segment disk membrane

Energy Technology Data Exchange (ETDEWEB)

Mashimo, T.; Abe, K.; Yoshiya, I.

1986-04-01

The effects of local anesthetics and a divalent cation, Ca2+, on the function of rhodopsin were estimated from the measurements of light-induced proton uptake. The light-induced proton uptake by rhodopsin in the rod outer segment disk membrane was enhanced at lower pH (4) but depressed at higher pHs (6 to 8) by the tertiary amine local anesthetics lidocaine, bupivacaine, tetracaine, and dibucaine. The order of local anesthetic-induced depression of the proton uptake followed that of their clinical anesthetic potencies. The depression of the proton uptake versus the concentration of the uncharged form of local anesthetic nearly describes the same curve for small and large dose of added anesthetic. Furthermore, a neutral local anesthetic, benzocaine, depressed the proton uptake at all pHs between 4 and 7. These results indicate that the depression of the proton uptake is due to the effect of only the uncharged form. It is hypothesized that the uncharged form of local anesthetics interacts hydrophobically with the rhodopsin in the disk membrane. The dual effect of local anesthetics on the proton uptake, on the other hand, suggests that the activation of the function of rhodopsin may be caused by the charged form. There was no significant change in the light-induced proton uptake by rhodopsin when 1 mM of Ca2+ was introduced into the disk membrane at varying pHs in the absence or presence of local anesthetics. This fact indicates that Ca2+ ion does not influence the diprotonating process of metarhodopsin; neither does it interfere with the local anesthetic-induced changes in the rhodopsin molecule.

OpnS, an outer membrane porin of Xenorhabdus nematophila, confers a competitive advantage for growth in the insect host.

Science.gov (United States)

van der Hoeven, Ransome; Forst, Steven

2009-09-01

The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded beta-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the DeltaopnS strain. Coinjection of the wild-type and DeltaopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or DeltaopnS strain were colonized by the wild-type strain. In addition, the DeltaopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The DeltaopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.

Evasion of IFN-γ signaling by Francisella novicida is dependent upon Francisella outer membrane protein C.

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Kalyan C Nallaparaju

2011-03-01

Full Text Available Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918 of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida FTN_0444 (homolog of FTT0918 fopC mutant to study the virulence-associated mechanism(s of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO mice, including MHC I, MHC II, and µmT (B cell deficient, but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity facilitating the activity and localization of acid phosphatases and other F. novicida cell components.

The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

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Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

2003-01-01

Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

Photoreceptor change and visual outcome after idiopathic epiretinal membrane removal with or without additional internal limiting membrane peeling.

Science.gov (United States)

Ahn, Seong Joon; Ahn, Jeeyun; Woo, Se Joon; Park, Kyu Hyung

2014-01-01

To compare the postoperative photoreceptor status and visual outcome after epiretinal membrane removal with or without additional internal limiting membrane (ILM) peeling. Medical records of 40 eyes from 37 patients undergoing epiretinal membrane removal with residual ILM peeling (additional ILM peeling group) and 69 eyes from 65 patients undergoing epiretinal membrane removal without additional ILM peeling (no additional peeling group) were reviewed. The length of defects in cone outer segment tips, inner segment/outer segment junction, and external limiting membrane line were measured using spectral domain optical coherence tomography images of the fovea before and at 1, 3, 6, and 12 months after the surgery. Cone outer segment tips and inner segment/outer segment junction line defects were most severe at postoperative 1 month and gradually restored at 12 months postoperatively. The cone outer segment tips line defect in the additional ILM peeling group was significantly greater than that in the no additional peeling group at postoperative 1 month (P = 0.006), and best-corrected visual acuity was significantly worse in the former group at the same month (P = 0.001). There was no significant difference in the defect size and best-corrected visual acuity at subsequent visits and recurrence rates between the two groups. Patients who received epiretinal membrane surgery without additional ILM peeling showed better visual and anatomical outcome than those with additional ILM peeling at postoperative 1 month. However, surgical outcomes were comparable between the two groups, thereafter. In terms of visual outcome and photoreceptor integrity, additional ILM peeling may not be an essential procedure.

Earthworm Protease

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Rong Pan

2010-01-01

Full Text Available The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibriniolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP. The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate proenzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

Earthworm Protease

International Nuclear Information System (INIS)

Pan, R.; Zhang, Z.; He, R.

2010-01-01

The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibrinolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate pro enzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

A Western-blot assay for the detection of antibodies against pathogenic Leptospira serogroups with recombinant outer membrane protein LipL32

OpenAIRE

Hong-yuan DUAN; Zhi-guo LIU; Shao-fu QIU; Bin HE; Hai ZHAO; Li-hua SONG; Hong ZHU; Qing DUAN

2011-01-01

Objective To provide a possible antigen for rapid serodiagnosis of leptospirosis,the present study focused on the activity of immune-reaction and cross-reaction between outer membrane protein LipL32 and multi-serogroup anti-pathogenic Leptospira antibodies.Methods Based on the given sequence of LipL32 gene of Leptospira icterohaemorrhagiae strain 56601,the primer pair was designed and the DNA fragment was amplified by PCR.The amplified product was inserted into vector pET-28a-(c) to construct...

Structure analysis of OmpC, one of the major proteins in the outer membrane of E. coli, by high resolution electron microscopy

International Nuclear Information System (INIS)

Chang, C.F.

1983-07-01

This dissertation is concerned with the structure analysis of a pore-forming membrane protein, OmpC, which is one of the major proteins in the outer membrane of Escherichia coli. In order to obtain structural information it was necessary to develop a suitable technique for preparing two-dimensional crystalline arrays of this membrane protein in an unfixed, unstained and hydrated condition. Electron micrographs were recorded at exposures of less than 5 electrons/A 2 in order to avoid severe radiation damage. The resulting images were crystallographically averaged, in order to overcome the statistical limitations associated with the low electron exposures. The resulting images, which extend to a resolution of approx. 13.5 A, lend themselves to a natural interpretation that is consistent with the mass density of protein, water and lipid, prior data from 2-D and 3-D structure studies of negatively stained specimens at approx. = 20 A resolution, and published spectroscopic data on the peptide chain secondary structure

Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity.

Science.gov (United States)

Dickey, Seth W; Baker, Rosanna P; Cho, Sangwoo; Urban, Siniša

2013-12-05

Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in more than a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (K(d) ~190 μM/0.1 mol%). Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in k(cat) values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately, a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but are strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications. Copyright © 2013 Elsevier Inc. All rights reserved.

Co-evolution of insect proteases and plant protease inhibitors.

Science.gov (United States)

Jongsma, Maarten A; Beekwilder, Jules

2011-08-01

Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase.

Science.gov (United States)

Moreno, S N; Mason, R P; Docampo, R

1984-12-10

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.

Modeling electrically active viscoelastic membranes.

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Sitikantha Roy

Full Text Available The membrane protein prestin is native to the cochlear outer hair cell that is crucial to the ear's amplification and frequency selectivity throughout the whole acoustic frequency range. The outer hair cell exhibits interrelated dimensional changes, force generation, and electric charge transfer. Cells transfected with prestin acquire unique active properties similar to those in the native cell that have also been useful in understanding the process. Here we propose a model describing the major electromechanical features of such active membranes. The model derived from thermodynamic principles is in the form of integral relationships between the history of voltage and membrane resultants as independent variables and the charge density and strains as dependent variables. The proposed model is applied to the analysis of an active force produced by the outer hair cell in response to a harmonic electric field. Our analysis reveals the mechanism of the outer hair cell active (isometric force having an almost constant amplitude and phase up to 80 kHz. We found that the frequency-invariance of the force is a result of interplay between the electrical filtering associated with prestin and power law viscoelasticity of the surrounding membrane. Paradoxically, the membrane viscoelasticity boosts the force balancing the electrical filtering effect. We also consider various modes of electromechanical coupling in membrane with prestin associated with mechanical perturbations in the cell. We consider pressure or strains applied step-wise or at a constant rate and compute the time course of the resulting electric charge. The results obtained here are important for the analysis of electromechanical properties of membranes, cells, and biological materials as well as for a better understanding of the mechanism of hearing and the role of the protein prestin in this mechanism.

Dissection of β-barrel Outer Membrane Protein Assembly Pathways through Characterizing BamA POTRA 1 Mutants of Escherichia coli

Science.gov (United States)

Bennion, Drew; Charlson, Emily S.; Coon, Eric; Misra, Rajeev

2010-01-01

Summary BamA of Escherichia coli is an essential component of the hetero-oligomeric machinery that mediates β-barrel outer membrane protein (OMP) assembly. The C- and N-termini of BamA fold into trans-membrane β-barrel and five soluble POTRA domains, respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β-barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site-specific cross-linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β-barrel OMPs except for TolC, which folds into a unique soluble α-helical barrel and an OM-anchored β-barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalyzed by the trans-membrane β-barrel domain of Bam A. PMID:20598079

Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries.

Science.gov (United States)

Hoang, Hanh H; Nickerson, Nicholas N; Lee, Vincent T; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P; Lory, Stephen

2011-01-01

In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect

StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

Science.gov (United States)

Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas

2014-01-01

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. PMID:24422629

Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

International Nuclear Information System (INIS)

Snyder, S.H.; Verma, A.; Trifiletti, R.R.

1987-01-01

The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for [ 3 H]diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with [ 3 H]flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines

A novel protease activity assay using a protease-responsive chaperone protein

International Nuclear Information System (INIS)

Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

2009-01-01

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

A novel protease activity assay using a protease-responsive chaperone protein

Energy Technology Data Exchange (ETDEWEB)

Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

2009-06-05

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin

DEFF Research Database (Denmark)

Friis, Stine; Sales, Katiuchia Uzzun; Schafer, Jeffrey Martin

2014-01-01

cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase......The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin...... form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating...

Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector.

Science.gov (United States)

Hoelzle, L E; Hoelzle, K; Wittenbrink, M M

2003-10-01

The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae.

Science.gov (United States)

Qiu, J; Hendrixson, D R; Baker, E N; Murphy, T F; St Geme, J W; Plaut, A G

1998-10-13

Haemophilus influenzae is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against H. influenzae colonization and disease. In the present study, we examined the effect of human milk on the H. influenzae IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the H. influenzae P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of H. influenzae by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.

A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Tan, Jingquan; Rouse, Sarah L.; Li, Dianfan; Pye, Valerie E.; Vogeley, Lutz; Brinth, Alette R.; El Arnaout, Toufic; Whitney, John C.; Howell, P. Lynne; Sansom, Mark S. P.; Caffrey, Martin

2014-01-01

Crystal structures of the β-barrel porin AlgE reveal a mechanism whereby alginate is exported from P. aeruginosa for biofilm formation. The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE

A conformational landscape for alginate secretion across the outer membrane of Pseudomonas aeruginosa

Energy Technology Data Exchange (ETDEWEB)

Tan, Jingquan [Trinity College, Dublin (Ireland); Rouse, Sarah L. [University of Oxford, South Parks Road, Oxford (United Kingdom); Li, Dianfan; Pye, Valerie E.; Vogeley, Lutz; Brinth, Alette R.; El Arnaout, Toufic [Trinity College, Dublin (Ireland); Whitney, John C.; Howell, P. Lynne [The Hospital for Sick Children, Toronto, Ontario (Canada); University of Toronto, Toronto, Ontario (Canada); Sansom, Mark S. P. [University of Oxford, South Parks Road, Oxford (United Kingdom); Caffrey, Martin, E-mail: martin.caffrey@tcd.ie [Trinity College, Dublin (Ireland)

2014-08-01

Crystal structures of the β-barrel porin AlgE reveal a mechanism whereby alginate is exported from P. aeruginosa for biofilm formation. The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.

Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

International Nuclear Information System (INIS)

Witt, P.L.; Bownds, M.D.

1987-01-01

Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

Science.gov (United States)

Parrell, Daniel; Zhang, Yang; Olenic, Sandra; Kroos, Lee

2017-10-01

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His 6 -MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His 6 -MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are

Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.

Science.gov (United States)

Díaz, M E; Rocha, G F; Kise, F; Rosso, A M; Guevara, M G; Parisi, M G

2018-05-08

Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 μmol l -1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 μmol l -1 . Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would

A distance measurement between specific sites on the cytoplasmic surface of bovine rhodopsin in rod outer segment disk membranes.

Science.gov (United States)

Albert, A D; Watts, A; Spooner, P; Groebner, G; Young, J; Yeagle, P L

1997-08-14

Structural information on mammalian integral membrane proteins is scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation with rhodopsin kinase. Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label. Magic angle spinning 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.

Purification and Characterization of Fibrinolytic Proteases from Mushroom Volvariela volvaceae Extract.

Directory of Open Access Journals (Sweden)

Willy Praira

2010-11-01

Full Text Available Purification and Characterization of Fibrinolytic Proteases from Mushroom Volvariela volvaceae Extract. Ediblestraw mushroom (V. volvaceae has been known used for improvement of blood circulation due to its fibrinolyticcontent. The objective of the study is to purify and characterize fibrinolytic protease from straw mushroom extract.Purification were performed through several steps, i.e. precipitation using ammonium sulphate 75%, dialyzed membran(cut-off 10 kDa, and ion-exchange chromatography using DEAE Sepharose. The active fraction of DEAE-Sepharosecontains two purified protein bands with molecular weight of 12.9 and 15.8 kDa. The active fraction has specificactivity of 0.383 U/mg with 2.7 fold higher purification compared to its crude extract. Both crude and purified enzymeshad optimum activity at temperature of 50 ºC and pH 7 in 10 minutes of incubation. Fibrin zymographic profiledemonstrated that the enzyme hydrolyzed fibrin, as well as casein, indicating their potent fibrinolytic activity. Theenzyme was strongly inhibited by phenilmethylsulphonyl fluoride and N-p-tosil-L-lysinchloromethyl keton. Thissuggested that it was a serine protease. In summary, these results showed that crude and purified protease of strawmushroom (V. volvaceae has fibrinolytic activities that can be applied for alternative thrombolytic therapy.

Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

OpenAIRE

Magdalena Opalińska; Katarzyna Parys; Hanna Jańska

2017-01-01

Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we...

OpnS, an Outer Membrane Porin of Xenorhabdus nematophila, Confers a Competitive Advantage for Growth in the Insect Host▿ †

Science.gov (United States)

van der Hoeven, Ransome; Forst, Steven

2009-01-01

The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded β-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ΔopnS strain. Coinjection of the wild-type and ΔopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ΔopnS strain were colonized by the wild-type strain. In addition, the ΔopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ΔopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment. PMID:19465651

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

Energy Technology Data Exchange (ETDEWEB)

Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

2017-09-01

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

GlyGly-CTERM and rhombosortase: a C-terminal protein processing signal in a many-to-one pairing with a rhomboid family intramembrane serine protease.

Directory of Open Access Journals (Sweden)

Daniel H Haft

Full Text Available The rhomboid family of serine proteases occurs in all domains of life. Its members contain at least six hydrophobic membrane-spanning helices, with an active site serine located deep within the hydrophobic interior of the plasma membrane. The model member GlpG from Escherichia coli is heavily studied through engineered mutant forms, varied model substrates, and multiple X-ray crystal studies, yet its relationship to endogenous substrates is not well understood. Here we describe an apparent membrane anchoring C-terminal homology domain that appears in numerous genera including Shewanella, Vibrio, Acinetobacter, and Ralstonia, but excluding Escherichia and Haemophilus. Individual genomes encode up to thirteen members, usually homologous to each other only in this C-terminal region. The domain's tripartite architecture consists of motif, transmembrane helix, and cluster of basic residues at the protein C-terminus, as also seen with the LPXTG recognition sequence for sortase A and the PEP-CTERM recognition sequence for exosortase. Partial Phylogenetic Profiling identifies a distinctive rhomboid-like protease subfamily almost perfectly co-distributed with this recognition sequence. This protease subfamily and its putative target domain are hereby renamed rhombosortase and GlyGly-CTERM, respectively. The protease and target are encoded by consecutive genes in most genomes with just a single target, but far apart otherwise. The signature motif of the Rhombo-CTERM domain, often SGGS, only partially resembles known cleavage sites of rhomboid protease family model substrates. Some protein families that have several members with C-terminal GlyGly-CTERM domains also have additional members with LPXTG or PEP-CTERM domains instead, suggesting there may be common themes to the post-translational processing of these proteins by three different membrane protein superfamilies.

Molecular biology of Neisseria meningitidis class 5 and H. 8 outer membrane proteins

Energy Technology Data Exchange (ETDEWEB)

Kawula, T.H.

1987-01-01

One of the surface structures responsible for inter- and intrastrain antigenic variability in meningococci is the heat-modifiable class 5 (C.5) protein. Neisseria meningitidis strain FAM18 (a meningococcal disease isolate) expressed two different C.5 proteins (C.5a and C.5b) identifiable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We generated two monoclonal antibodies (MAbs), each specific for one of the identified C.5 proteins. The MAbs, which were bactericidal for variants expressing the appropriate C.5 protein, were used to study C.5 expression changes in FAM18. The H.8 protein is an antigenically conserved outer membrane protein expressed almost exclusively by the pathogenic Neisseria. We have cloned and sequenced an H.8 gene from N. meningitidis FAM18. The predicted H.8 amino acid sequence indicated that the most probable signal peptide processing site matched the consensus prokaryotic lipoprotein processing/modification sequence. We then showed that the H.8 protein could be labeled with {sup 14}C-palmitic acid, confirming that H.8 was a lipoprotein. Processing of the H.8 protein was inhibited by globomycin in E. coli indicating that H.8 was modified by the described lipoprotein processing/modifying pathway described in both gram negative and gram positive genera.

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

Science.gov (United States)

Reuther, C; Ganjam, G K; Dolga, A M; Culmsee, C

2014-11-01

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography.

Science.gov (United States)

Kernaghan, Gavin; Mayerhofer, Michael

2017-01-01

This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.

Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria.

Directory of Open Access Journals (Sweden)

Louis S Ates

2015-05-01

Full Text Available Mycobacteria possess different type VII secretion (T7S systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of Msp

Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

KAUST Repository

Ates, Louis S.

2015-05-04

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow

Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria

KAUST Repository

Ates, Louis S.; Ummels, Roy; Commandeur, Susanna; van der Weerd, Robert; Sparrius, Marion; Weerdenburg, Eveline; Alber, Marina; Kalscheuer, Rainer; Piersma, Sander R.; Abdallah, Abdallah; Abd El Ghany, Moataz; Abdel-Haleem, Alyaa M.; Pain, Arnab; Jimé nez, Connie R.; Bitter, Wilbert; Houben, Edith N.G.

2015-01-01

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow

Nucleic Acid Aptamers Against Proteases

DEFF Research Database (Denmark)

Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

2011-01-01

, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection......Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, proteinaptamer recognition, protease inhibition, and advantages of aptamers...

Outer membrane protein A (OmpA: a new player in shigella flexneri protrusion formation and inter-cellular spreading.

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Cecilia Ambrosi

Full Text Available Outer membrane protein A (OmpA is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92 and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the

Transcriptional responses of Leptospira interrogans to host innate immunity: significant changes in metabolism, oxygen tolerance, and outer membrane.

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Feng Xue

Full Text Available BACKGROUND: Leptospira interrogans is the major causative agent of leptospirosis. Phagocytosis plays important roles in the innate immune responses to L. interrogans infection, and L. interrogans can evade the killing of phagocytes. However, little is known about the adaptation of L. interrogans during this process. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the interaction of pathogenic Leptospira and innate immunity, we employed microarray and comparative genomics analyzing the responses of L. interrogans to macrophage-derived cells. During this process, L. interrogans altered expressions of many genes involved in carbohydrate and lipid metabolism, energy production, signal transduction, transcription and translation, oxygen tolerance, and outer membrane proteins. Among them, the catalase gene expression was significantly up-regulated, suggesting it may contribute to resisting the oxidative pressure of the macrophages. The expressions of several major outer membrane protein (OMP genes (e.g., ompL1, lipL32, lipL41, lipL48 and ompL47 were dramatically down-regulated (10-50 folds, consistent with previous observations that the major OMPs are differentially regulated in vivo. The persistent down-regulations of these major OMPs were validated by immunoblotting. Furthermore, to gain initial insight into the gene regulation mechanisms in L. interrogans, we re-defined the transcription factors (TFs in the genome and identified the major OmpR TF gene (LB333 that is concurrently regulated with the major OMP genes, suggesting a potential role of LB333 in OMPs regulation. CONCLUSIONS/SIGNIFICANCE: This is the first report on global responses of pathogenic Leptospira to innate immunity, which revealed that the down-regulation of the major OMPs may be an immune evasion strategy of L. interrogans, and a putative TF may be involved in governing these down-regulations. Alterations of the leptospiral OMPs up interaction with host antigen

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Science.gov (United States)

Chatel-Chaix, Laurent; Baril, Martin; Lamarre, Daniel

2010-01-01

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection. PMID:21994705

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Directory of Open Access Journals (Sweden)

Laurent Chatel-Chaix

2010-08-01

Full Text Available Hepatitis C virus (HCV infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

Molecular analysis of interactions between dendrimers and asymmetric membranes at different transport stages.

Science.gov (United States)

He, XiaoCong; Qu, ZhiGuo; Xu, Feng; Lin, Min; Wang, JiuLing; Shi, XingHua; Lu, TianJian

2014-01-07

Studying dendrimer-biomembrane interactions is important for understanding drug and gene delivery. In this study, coarse-grained molecular dynamics simulations were performed to investigate the behaviors of polyamidoamine (PAMAM) dendrimers (G4 and G5) as they interacted with asymmetric membranes from different sides of the bilayer, thus mimicking different dendrimer transport stages. The G4 dendrimer could insert into the membrane during an equilibrated state, and the G5 dendrimer could induce pore formation in the membrane when the dendrimers interacted with the outer side (outer interactions) of an asymmetric membrane [with 10% dipalmitoyl phosphatidylserine (DPPS) in the inner leaflet of the membrane]. During the interaction with the inner side of the asymmetric membrane (inner interactions), the G4 and G5 dendrimers only adsorbed onto the membrane. As the membrane asymmetry increased (e.g., increased DPPS percentage in the inner leaflet of the membrane), the G4 and G5 dendrimers penetrated deeper into the membrane during the outer interactions and the G4 and G5 dendrimers were adsorbed more tightly onto the membrane for the inner interactions. When the DPPS content reached 50%, the G4 dendrimer could completely penetrate through the membrane from the outer side to the inner side. Our study provides molecular understanding and reference information about different dendrimer transport stages during drug and gene delivery.

The Outer Membrane Protein OmpW Forms an Eight-Stranded beta-Barrel with a Hydrophobic Channel

International Nuclear Information System (INIS)

Hong, H.; Patel, D.; Tamm, L.; van den Berg, B.

2006-01-01

Escherichia coli OmpW belongs to a family of small outer membrane (OM) proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. In order to gain insight into the function of these proteins we have determined the crystal structure of Escherichia coli OmpW to 2.7 Angstroms resolution. The structure shows that OmpW forms an eight-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound LDAO detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of LDAO. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial OM

Mitofilin complexes: conserved organizers of mitochondrial membrane architecture.

Science.gov (United States)

Zerbes, Ralf M; van der Klei, Ida J; Veenhuis, Marten; Pfanner, Nikolaus; van der Laan, Martin; Bohnert, Maria

2012-11-01

Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.

Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

Science.gov (United States)

Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

2015-01-01

Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

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Luca Musante

2015-01-01

Full Text Available Diabetic nephropathy (DN is one of the major complications of diabetes mellitus (DM, leads to chronic kidney disease (CKD, and, ultimately, is the main cause for end-stage kidney disease (ESKD. Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

Energy Technology Data Exchange (ETDEWEB)

Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

1987-08-01

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

International Nuclear Information System (INIS)

Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

1987-01-01

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon - cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [ 3 H]methyl-casein to acid-soluble products in the presence of ATP and Mg 2+ . ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

A genetic screen in Myxococcus xanthus identifies mutants that uncouple outer membrane exchange from a downstream cellular response.

Science.gov (United States)

Dey, Arup; Wall, Daniel

2014-12-01

Upon physical contact with sibling cells, myxobacteria transiently fuse their outer membranes (OMs) and exchange OM proteins and lipids. From previous work, TraA and TraB were identified to be essential factors for OM exchange (OME) in donor and recipient cells. To define the genetic complexity of OME, we carried out a comprehensive forward genetic screen. The screen was based on the observation that Myxococcus xanthus nonmotile cells, by a Tra-dependent mechanism, block swarm expansion of motile cells when mixed. Thus, mutants defective in OME or a downstream responsive pathway were readily identified as escape flares from mixed inocula seeded on agar. This screen was surprisingly powerful, as we found >50 mutants defective in OME. Importantly, all of the mutations mapped to the traAB operon, suggesting that there may be few, if any, proteins besides TraA and TraB directly required for OME. We also found a second and phenotypically different class of mutants that exhibited wild-type OME but were defective in a responsive pathway. This pathway is postulated to control inner membrane homeostasis by covalently attaching amino acids to phospholipids. The identified proteins are homologous to the Staphylococcus aureus MprF protein, which is involved in membrane adaptation and antibiotic resistance. Interestingly, we also found that a small number of nonmotile cells were sufficient to block the swarming behavior of a large gliding-proficient population. This result suggests that an OME-derived signal could be amplified from a few nonmotile producers to act on many responder cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Detection of nearest neighbors to specific fluorescently tagged ligands in rod outer segment and lymphocyte plasma membranes by photosensitization of 5-iodonaphthyl 1-azide

International Nuclear Information System (INIS)

Raviv, Y.; Bercovici, T.; Gitler, C.; Salomon, Y.

1989-01-01

Lima bean agglutinin-fluorescein 5-isothiocyanate conjugate (FluNCS-lima bean lectin) interacts with specific receptor molecules on membranes both from the rod outer segment (ROS) of the frog retina and from S49 mouse lymphoma cells. When [125I]-5-iodonaphthyl 1-azide (125I-INA), which freely and randomly partitions into the lipid bilayer, is added to membranes and the suspension is irradiated at 480 nm, the FluNCS-conjugated lectin photosensitizes the [125I]INA but only at discrete sites. This results in the selective labeling of specific proteins: an 88-kDa protein on ROS membranes and a 56-kDa protein on S49 plasma membranes. Labeling is dependent upon the interaction of the FluNCS-lectin with glycosylated receptor sites, since N-acetylgalactosamine, but not methyl alpha-mannoside, blocked labeling of the 56-kDa protein on S49 membranes. In contrast, a random labeling pattern of membrane proteins was observed upon irradiation at 480 nm using other fluorescein conjugates, such as FluNCS-bovine serum albumin (FluNCS-BSA) or FluNCS-soybean trypsin inhibitor (FluNCS-STI), which interact with cell membranes in a nonselective manner, or with N-(fluorescein-5-thiocarbamoyl)-n-undecyclamine (FluNCS-NHC11), which is freely miscible in the membrane lipid. Random labeling was also obtained by direct photoexcitation of [125I]INA at 314 nm, with no distinct labeling of the 88- and 56-kDa proteins in the respective membranes. These results suggest that protein ligands can be used to guide sensitizers to discrete receptor sites and lead to their selective labeling by photosensitized activation of [125I]INA

The plant i-AAA protease controls the turnover of an essential mitochondrial protein import component.

Science.gov (United States)

Opalińska, Magdalena; Parys, Katarzyna; Murcha, Monika W; Jańska, Hanna

2018-01-29

Mitochondria are multifunctional organelles that play a central role in energy metabolism. Owing to the life-essential functions of these organelles, mitochondrial content, quality and dynamics are tightly controlled. Across the species, highly conserved ATP-dependent proteases prevent malfunction of mitochondria through versatile activities. This study focuses on a molecular function of the plant mitochondrial inner membrane-embedded AAA protease (denoted i -AAA) FTSH4, providing its first bona fide substrate. Here, we report that the abundance of the Tim17-2 protein, an essential component of the TIM17:23 translocase (Tim17-2 together with Tim50 and Tim23), is directly controlled by the proteolytic activity of FTSH4. Plants that are lacking functional FTSH4 protease are characterized by significantly enhanced capacity of preprotein import through the TIM17:23-dependent pathway. Taken together, with the observation that FTSH4 prevents accumulation of Tim17-2, our data point towards the role of this i -AAA protease in the regulation of mitochondrial biogenesis in plants. © 2018. Published by The Company of Biologists Ltd.

A novel outer-membrane anion channel (porin) as part of a putatively two-component transport system for 4-toluenesulphonate in Comamonas testosteroni T-2

OpenAIRE

Mampel, Jörg; Maier, Elke; Tralau, Tewes; Ruff, Jürgen; Benz, Roland; Cook, Alasdair M.

2004-01-01

Inducible mineralization of TSA (4-toluenesulphonate) by Comamonas testosteroni T-2 is initiated by a secondary transport system, followed by oxygenation and oxidation by TsaMBCD to 4-sulphobenzoate under the regulation of TsaR and TsaQ. Evidence is presented for a novel, presumably two-component transport system (TsaST). It is proposed that TsaT, an outer-membrane porin, formed an anion-selective channel that works in co-operation with the putative secondary transporter, TsaS, located in the...

Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA).

Science.gov (United States)

Kihara, A; Akiyama, Y; Ito, K

1997-05-27

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

Legionella pneumophila-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages.

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Anna Lena Jung

2016-04-01

Full Text Available The formation and release of outer membrane vesicles (OMVs is a phenomenon of Gram-negative bacteria. This includes Legionella pneumophila (L. pneumophila, a causative agent of severe pneumonia. Upon its transmission into the lung, L. pneumophila primarily infects and replicates within macrophages. Here, we analyzed the influence of L. pneumophila OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of Legionella OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of L. pneumophila in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of Legionella-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a's targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for Legionella replication. In conclusion, L. pneumophila OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate L. pneumophila replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of L. pneumophila in the host.

B cell activation by outer membrane vesicles--a novel virulence mechanism.

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Maria Laura A Perez Vidakovics

2010-01-01

Full Text Available Secretion of outer membrane vesicles (OMV is an intriguing phenomenon of Gram-negative bacteria and has been suggested to play a role as virulence factors. The respiratory pathogens Moraxella catarrhalis reside in tonsils adjacent to B cells, and we have previously shown that M. catarrhalis induce a T cell independent B cell response by the immunoglobulin (Ig D-binding superantigen MID. Here we demonstrate that Moraxella are endocytosed and killed by human tonsillar B cells, whereas OMV have the potential to interact and activate B cells leading to bacterial rescue. The B cell response induced by OMV begins with IgD B cell receptor (BCR clustering and Ca(2+ mobilization followed by BCR internalization. In addition to IgD BCR, TLR9 and TLR2 were found to colocalize in lipid raft motifs after exposure to OMV. Two components of the OMV, i.e., MID and unmethylated CpG-DNA motifs, were found to be critical for B cell activation. OMV containing MID bound to and activated tonsillar CD19(+ IgD(+ lymphocytes resulting in IL-6 and IgM production in addition to increased surface marker density (HLA-DR, CD45, CD64, and CD86, whereas MID-deficient OMV failed to induce B cell activation. DNA associated with OMV induced full B cell activation by signaling through TLR9. Importantly, this concept was verified in vivo, as OMV equipped with MID and DNA were found in a 9-year old patient suffering from Moraxella sinusitis. In conclusion, Moraxella avoid direct interaction with host B cells by redirecting the adaptive humoral immune response using its superantigen-bearing OMV as decoys.

Lateral interactions in the photoreceptor membrane: a NMR study

International Nuclear Information System (INIS)

Mollevanger, L.C.P.J.

1987-01-01

The photoreceptor membrane has an exceptionally high content of polyunsaturated fatty acyl chains combined with a high amount of phosphatidyl ethanolamine. It is situated in a cell organelle, the rod outer segment, with a high biological activity in which controlable trans-membrane currents of different ions play an important role. These characteristics make it a very interesting biological membrane to search for the existence of non-bilayer structures. Therefore in this thesis a detailed study of the polymorphic phase behaviour of the rod outer segment photoreceptor lipids was undertaken, concerning modulation of the polymorphic phase behaviour of photoreceptor membrane lipids by divalent cations and temperature, polymorphism of the individual phospholipid classes phosphatidylethanolamine and phosphatidylserine and effects of cholesterol, bilayer stabilization by (rhod)opsin. Morphologically intact rod outer segment possesses a large magnetic anisotropy. This property is used to obtain 31 P-NMR of oriented photoreceptor membranes which allows spectral analysis and identification of individual phospholipid classes, and allows to study lateral lipid diffusion in intact disk membranes. The power of high resolution solid state 13 C-NMR to study the conformation of the chromophore in rhodopsin is demonstrated. (Auth.)

Mitofilin complexes : conserved organizers of mitochondrial membrane architecture

NARCIS (Netherlands)

Zerbes, Ralf M.; van der Klei, Ida J.; Veenhuis, Marten; Pfanner, Nikolaus; van der Laan, Martin; Bohnert, Maria

2012-01-01

Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner

Potentiometric determination of trypsin using a polymeric membrane polycation-sensitive electrode based on current-controlled reagent delivery.

Science.gov (United States)

Chen, Yan; Ding, Jiawang; Qin, Wei

2012-12-01

A potentiometric biosensor for the determination of trypsin is described based on current-controlled reagent delivery. A polymeric membrane protamine-sensitive electrode with dinonylnaphthalene sulfonate as cation exchanger is used for in situ generation of protamine. Diffusion of protamine across the polymeric membrane can be controlled precisely by applying an external current. The hydrolysis catalyzed with trypsin in sample solution decreases the concentration of free protamine released at the sample-membrane interface and facilitates the stripping of protamine out of the membrane surface via the ion-exchange process with sodium ions from the sample solution, thus decreasing the membrane potential, by which the protease can be sensed potentiometrically. The influences of anodic current amplitude, current pulse duration and protamine concentration in the inner filling solution on the membrane potential response have been studied. Under optimum conditions, the proposed protamine-sensitive electrode is useful for continuous and reversible detection of trypsin over the concentration range of 0.5-5UmL(-1) with a detection limit of 0.3UmL(-1). The proposed detection strategy provides a rapid and reagentless way for the detection of protease activities and offers great potential in the homogeneous immunoassays using proteases as labels. Copyright © 2012 Elsevier B.V. All rights reserved.

Outer Membrane Vesicle Vaccines from Biosafe Surrogates Prevent Acute Lethal Glanders in Mice

Directory of Open Access Journals (Sweden)

Michael H. Norris

2018-01-01

Full Text Available Burkholderia mallei is a host-adapted Gram-negative mammalian pathogen that causes the severe disease glanders. Glanders can manifest as a rapid acute progression or a chronic debilitating syndrome primarily affecting solipeds and humans in close association with infected animals. In USA, B. mallei is classified as one of the most important bacterial biothreat agents. Presently, there is no licensed glanders vaccine available for humans or animals. In this work, outer membrane vesicles (OMVs were isolated from three attenuated biosafe bacterial strains, Burkholderia pseudomallei Bp82, B. thailandensis E555, and B. thailandensis TxDOH and used to vaccinate mice. B. thailandensis OMVs induced significantly higher antibody responses that were investigated. B. mallei specific serum antibody responses were of higher magnitude in mice vaccinated with B. thailandensis OMVs compared to levels in mice vaccinated with B. pseudomallei OMVs. OMVs derived from biosafe strains protected mice from acute lethal glanders with vesicles from the two B. thailandensis strains affording significant protection (>90% up to 35 days post-infection with some up to 60 days. Organ loads from 35-day survivors indicated bacteria colonization of the lungs, liver, and spleen while those from 60 days had high CFUs in the spleens. The highest antibody producing vaccine (B. thailandensis E555 OMVs also protected C57BL/6 mice from acute inhalational glanders with evidence of full protection.

Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction.

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Beliaev, A S; Saffarini, D A

1998-12-01

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.

Mechanisms and cellular functions of intramembrane proteases.

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Urban, Siniša

2013-12-01

The turn of the millennium coincided with the branding of a fundamentally different class of enzyme - proteases that reside immersed inside the membrane. This new field was the convergence of completely separate lines of research focused on cholesterol homeostasis, Alzheimer's disease, and developmental genetics. None intended their ultimate path, but soon became a richly-integrated fabric for an entirely new field: regulated intramembrane proteolysis. Our aim in this Special Issue is to focus on the ancient and nearly ubiquitous enzymes that catalyze this unexpected yet important reaction. The pace of progress has been dramatic, resulting in a rapidly-expanding universe of known cellular functions, and a paradigm shift in the biochemical understanding of these once heretical enzymes. More recently, the first therapeutic successes have been attained by targeting an intramembrane protease. We consider these advances and identify oncoming opportunities in four parts: growing spectra of cellular roles, insights into biochemical mechanisms, therapeutic strategies, and newly-emerging topics. Recent studies also expose challenges for the future, including non-linear relationships between substrate identification and physiological functions, and the need for potent and specific, not broad-class, inhibitors. © 2013.

Bio-Inspired Polymer Membrane Surface Cleaning

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Agnes Schulze

2017-03-01

Full Text Available To generate polyethersulfone membranes with a biocatalytically active surface, pancreatin was covalently immobilized. Pancreatin is a mixture of digestive enzymes such as protease, lipase, and amylase. The resulting membranes exhibit self-cleaning properties after “switching on” the respective enzyme by adjusting pH and temperature. Thus, the membrane surface can actively degrade a fouling layer on its surface and regain initial permeability. Fouling tests with solutions of protein, oil, and mixtures of both, were performed, and the membrane’s ability to self-clean the fouled surface was characterized. Membrane characterization was conducted by investigation of the immobilized enzyme concentration, enzyme activity, water permeation flux, fouling tests, porosimetry, X-ray photoelectron spectroscopy, and scanning electron microscopy.

Sandwich-structured hollow fiber membranes for osmotic power generation

KAUST Repository

Fu, Feng Jiang; Zhang, Sui; Chung, Neal Tai-Shung

2015-01-01

In this work, a novel sandwich-structured hollow fiber membrane has been developed via a specially designed spinneret and optimized spinning conditions. With this specially designed spinneret, the outer layer, which is the most crucial part of the sandwich-structured membrane, is maintained the same as the traditional dual-layer membrane. The inner substrate layer is separated into two layers: (1) an ultra-thin middle layer comprising a high molecular weight polyvinylpyrrolidone (PVP) additive to enhance integration with the outer polybenzimidazole (PBI) selective layer, and (2) an inner-layer to provide strong mechanical strength for the membrane. Experimental results show that a high water permeability and good mechanical strength could be achieved without the expensive post treatment process to remove PVP which was necessary for the dual-layer pressure retarded osmosis (PRO) membranes. By optimizing the composition, the membrane shows a maximum power density of 6.23W/m2 at a hydraulic pressure of 22.0bar when 1M NaCl and 10mM NaCl are used as the draw and feed solutions, respectively. To our best knowledge, this is the best phase inversion hollow fiber membrane with an outer selective PBI layer for osmotic power generation. In addition, this is the first work that shows how to fabricate sandwich-structured hollow fiber membranes for various applications. © 2015 Elsevier B.V.

Sandwich-structured hollow fiber membranes for osmotic power generation

KAUST Repository

Fu, Feng Jiang

2015-11-01

In this work, a novel sandwich-structured hollow fiber membrane has been developed via a specially designed spinneret and optimized spinning conditions. With this specially designed spinneret, the outer layer, which is the most crucial part of the sandwich-structured membrane, is maintained the same as the traditional dual-layer membrane. The inner substrate layer is separated into two layers: (1) an ultra-thin middle layer comprising a high molecular weight polyvinylpyrrolidone (PVP) additive to enhance integration with the outer polybenzimidazole (PBI) selective layer, and (2) an inner-layer to provide strong mechanical strength for the membrane. Experimental results show that a high water permeability and good mechanical strength could be achieved without the expensive post treatment process to remove PVP which was necessary for the dual-layer pressure retarded osmosis (PRO) membranes. By optimizing the composition, the membrane shows a maximum power density of 6.23W/m2 at a hydraulic pressure of 22.0bar when 1M NaCl and 10mM NaCl are used as the draw and feed solutions, respectively. To our best knowledge, this is the best phase inversion hollow fiber membrane with an outer selective PBI layer for osmotic power generation. In addition, this is the first work that shows how to fabricate sandwich-structured hollow fiber membranes for various applications. © 2015 Elsevier B.V.

NMR analysis of the dynamic exchange of the NS2B cofactor between open and closed conformations of the West Nile virus NS2B-NS3 protease.

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Xun-Cheng Su

Full Text Available BACKGROUND: The two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation in the absence of inhibitor and lining the substrate binding site (closed conformation in the presence of an inhibitor. METHODS: In this work, nuclear magnetic resonance (NMR spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution. FINDINGS: In solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors. CONCLUSION: Partial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.

Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection.

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Matthew J Kesic

Full Text Available Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs human airway trypsin-like protease (HAT and transmembrane protease, serine 2 (TMPRSS2, whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI. Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

The Role of Helicobacter pylori Outer Membrane Proteins in Adherence and Pathogenesis

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Oleastro, Mónica; Ménard, Armelle

2013-01-01

Helicobacter pylori is one of the most successful human pathogens, which colonizes the mucus layer of the gastric epithelium of more than 50% of the world’s population. This curved, microaerophilic, Gram-negative bacterium induces a chronic active gastritis, often asymptomatic, in all infected individuals. In some cases, this gastritis evolves to more severe diseases such as peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. H. pylori has developed a unique set of factors, actively supporting its successful survival and persistence in its natural hostile ecological niche, the human stomach, throughout the individual’s life, unless treated. In the human stomach, the vast majority of H. pylori cells are motile in the mucus layer lining, but a small percentage adheres to the epithelial cell surfaces. Adherence to the gastric epithelium is important for the ability of H. pylori to cause disease because this intimate attachment facilitates: (1) colonization and persistence, by preventing the bacteria from being eliminated from the stomach, by mucus turnover and gastric peristalsis; (2) evasion from the human immune system and (3) efficient delivery of proteins into the gastric cell, such as the CagA oncoprotein. Therefore, bacteria with better adherence properties colonize the host at higher densities. H. pylori is one of the most genetically diverse bacterial species known and is equipped with an extraordinarily large set of outer membrane proteins, whose role in the infection and persistence process will be discussed in this review, as well as the different receptor structures that have been so far described for mucosal adherence. PMID:24833057

The Role of Helicobacter pylori Outer Membrane Proteins in Adherence and Pathogenesis

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Armelle Ménard

2013-08-01

Full Text Available Helicobacter pylori is one of the most successful human pathogens, which colonizes the mucus layer of the gastric epithelium of more than 50% of the world’s population. This curved, microaerophilic, Gram-negative bacterium induces a chronic active gastritis, often asymptomatic, in all infected individuals. In some cases, this gastritis evolves to more severe diseases such as peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. H. pylori has developed a unique set of factors, actively supporting its successful survival and persistence in its natural hostile ecological niche, the human stomach, throughout the individual’s life, unless treated. In the human stomach, the vast majority of H. pylori cells are motile in the mucus layer lining, but a small percentage adheres to the epithelial cell surfaces. Adherence to the gastric epithelium is important for the ability of H. pylori to cause disease because this intimate attachment facilitates: (1 colonization and persistence, by preventing the bacteria from being eliminated from the stomach, by mucus turnover and gastric peristalsis; (2 evasion from the human immune system and (3 efficient delivery of proteins into the gastric cell, such as the CagA oncoprotein. Therefore, bacteria with better adherence properties colonize the host at higher densities. H. pylori is one of the most genetically diverse bacterial species known and is equipped with an extraordinarily large set of outer membrane proteins, whose role in the infection and persistence process will be discussed in this review, as well as the different receptor structures that have been so far described for mucosal adherence.

Colistin resistance associated with outer membrane protein change in Klebsiella pneumoniae and Enterobacter asburiae.

Science.gov (United States)

Kádár, Béla; Kocsis, Béla; Tóth, Ákos; Kristóf, Katalin; Felső, Péter; Kocsis, Béla; Böddi, Katalin; Szabó, Dóra

2017-06-01

In this study, outer membrane proteins (OMPs) of colistin-resistant Klebsiella pneumoniae and Enterobacter asburiae were analyzed. One colistin-susceptible and three colistin-resistant K. pneumoniae sequence type 258 strains as well as one colistin-susceptible E. asburiae and its colistin-heteroresistant counterpart strain were involved in the study. OMP analysis of each strain was performed by microchip method. Matrix-assisted laser desorption ionization time of flight/mass spectrometry (MALDI-TOF/MS) investigation was carried out after separation of OMPs by two-dimensional gel electrophoresis and in-gel digestion. The MALDI-TOF/MS analysis of OMPs in the colistin-susceptible K. pneumoniae found 16 kDa proteins belonging to the LysM domain/BON superfamily, as well as DNA starvation proteins, whereas OmpX and OmpW were detected in the colistin-resistant counterpart strains. OmpC and OmpW were detected in the colistin-susceptible E. asburiae, whereas OmpA and OmpX were identified in the colistin-resistant counterpart. This study demonstrated that OMP differences were between colistin-susceptible and -resistant counterpart strains. The altered Gram-negative cell wall may contribute to acquired colistin resistance in Enterobacteriaceae.

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

International Nuclear Information System (INIS)

Yao, Yong; Dutta, Samit Kumar; Park, Sang Ho; Rai, Ratan; Fujimoto, L. Miya; Bobkov, Andrey A.; Opella, Stanley J.; Marassi, Francesca M.

2017-01-01

The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with 13 C or 1 H detection, have very narrow line widths (0.40–0.60 ppm for 13 C, 0.11–0.15 ppm for 1 H, and 0.46–0.64 ppm for 15 N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The 1 H-detected solid-state NMR 1 H/ 15 N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR 1 H/ 15 N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

Science.gov (United States)

Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

1999-03-01

The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

Immunochemical and biological characterization of outer membrane proteins of Porphyromonas endodontalis.

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Ogawa, T; Kuribayashi, S; Shimauchi, H; Toda, T; Hamada, S

1992-01-01

Outer membrane proteins (OMP) of Porphyromonas endodontalis HG 370 (ATCC 35406) were prepared from the cell envelope fraction of the organisms. The cell envelope that had been obtained by sonication of the whole cells was extracted in 2% lithium dodecyl sulfate and then successively chromatographed with Sephacryl S-200 HR and DEAE-Sepharose Fast Flow. Two OMP fractions, OMP-I and OMP-II, were obtained, and their immunochemical properties and induction of specific antibodies were examined. The OMP-I preparation consisted of a major protein with an apparent molecular mass of 31 kDa and other moderate to minor proteins of 40.3, 51.4, 67, and 71.6 kDa, while the OMP-II preparation contained 14-, 15.5-, 27-, and 44-kDa proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. OMP-I was found to form hydrophilic diffusion pores by incorporation into artificial liposomes composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that OMP-I exhibited significant porin activity. However, the liposomes containing heat-denatured OMP-I were scarcely active. Spontaneous and antigen-specific immunoglobulin M (IgM)-, IgG-, and IgA-secreting spot-forming cells (SFC) enzymatically dissociated into single-cell suspensions from chronically inflamed periapical tissues and were enumerated by enzyme-linked immunospot assay. In patients with radicular cysts or dental granulomas, the major isotype of spontaneous SFC was IgG. In radicular cysts, the OMP-II-specific IgG SFC represented 0.13% of the total IgG SFC, while the antigen-specific IgA or IgM SFC was not observed. It was also found that none of these mononuclear cells produced antibodies specific for OMP-I or lipopolysaccharide of P. endodontalis. Images PMID:1328059

BSp66 protease is widespread in the acrosomal region of sperm from several mammalian species

International Nuclear Information System (INIS)

Cesari, A.; Katunar, M.R.; Monclus, M.A.; Vincenti, A.; Fornes, M.W.

2004-01-01

Fertilization in mammals comprises a sequence of events leading to the fusion of sperm and oocyte membranes. Although proteases are known to be involved in this process, their role in fertilization is controversial. There is extensive work on the characterization of proteolytic systems, including serine proteases, which demonstrates that acrosomal proteases can be distinguished among the sperm of different mammalian species on the basis of the gelatin-hydrolyzing activity on SDS-PAGE by the quantity and variety of the enzymes. In this report, we investigated the occurrence and activity of the serine protease BSp66, previously characterized in bovine spermatozoa, in various mammalian sperm. A protein with a molecular mass of 66 kDa cross-reacted with heterologous antibodies against bovine BSp66 when sperm extracts of several mammalian species were analyzed by Western blot. In agreement, proteolytic activity corresponding to the molecular mass of BSp66 was detected by gelatin zymography in all the species analyzed. This protein was located on the acrosomal region of sperm cells by immunofluorescence methods. We concluded that BSp66 is widespread in mammalian sperm, with a conserved location in the acrosomal region

Application of zwitterionic detergent to the solubilization of Klebsiella pneumoniae outer membrane proteins for two-dimensional gel electrophoresis.

Science.gov (United States)

Bednarz-Misa, I; Serek, P; Dudek, B; Pawlak, A; Bugla-Płoskońska, G; Gamian, A

2014-12-01

Klebsiella pneumoniae is a frequent cause of nosocomial respiratory, urinary and gastrointestinal tract infections and septicemia with the multidrug-resistant K. pneumoniae being a major public health concern. Outer membrane proteins (OMPs) are important virulence factors responsible for the appropriate adaptation to the host environment. They constitute of the antigens being the first in contact with infected organism. However, K. pneumoniae strains are heavily capsulated and it is important to establish the OMPs isolation procedure prior to proteomics extensive studies. In this study we used Zwittergent Z 3-14® as a detergent to isolate the OMPs from K. pneumoniae cells and resolve them using two-dimensional electrophoresis (2-DE). As a result we identified 134 protein spots. The OMPs identified in this study are possible candidates for the development of a protein-based vaccine against K. pneumoniae infections. Copyright © 2014 Elsevier B.V. All rights reserved.

Insecticide resistance and intracellular proteases.

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Wilkins, Richard M

2017-12-01

Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Nanodisc-solubilized membrane protein library reflects the membrane proteome.

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Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

2013-05-01

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

Ferric-Pyoverdine Recognition by Fpv Outer Membrane Proteins of Pseudomonas protegens Pf-5

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Hartney, Sierra L.; Mazurier, Sylvie; Girard, Maëva K.; Mehnaz, Samina; Davis, Edward W.; Gross, Harald; Lemanceau, Philippe

2013-01-01

The soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp. PMID:23222724

FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence.

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Xiaojun Wu

Full Text Available Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence and FTT0602c (fmvB, which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential

Majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

International Nuclear Information System (INIS)

Wilcox, C.A.; Olson, E.N.

1987-01-01

The BC 2 Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, the authors examined the subcellular localization of the major fatty acylated proteins in BC 4 Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [ 3 H]palmitate and [ 3 H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins

Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa

Science.gov (United States)

Castagliuolo, Ignazio; Riegler, Martin F.; Valenick, Leyla; LaMont, J. Thomas; Pothoulakis, Charalabos

1999-01-01

Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibits C. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardii on toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of S. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii on C. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease. PMID:9864230

Vacuum Outer-Gap Structure in Pulsar Outer Magnetospheres

International Nuclear Information System (INIS)

Gui-Fang, Lin; Li, Zhang

2009-01-01

We study the vacuum outer-gap structure in the outer magnetosphere of rotation-powered pulsars by considering the limit of trans-field height through a pair production process. In this case, the trans-field height is limited by the photon-photon pair production process and the outer boundary of the outer gap can be extended outside the light cylinder. By solving self-consistently the Poisson equation for electrical potential and the Boltzmann equations of electrons/positrons and γ-rays in a vacuum outer gap for the parameters of Vela pulsar, we obtain an approximate geometry of the outer gap, i.e. the trans-field height is limited by the pair-production process and increases with the radial distance to the star and the width of the outer gap starts at the inner boundary (near the null charge surface) and ends at the outer boundary which locates inside or outside the light cylinder depending on the inclination angle. (geophysics, astronomy, and astrophysics)

The Effects of Urethane on Rat Outer Hair Cells

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Mingyu Fu

2016-01-01

Full Text Available The cochlea converts sound vibration into electrical impulses and amplifies the low-level sound signal. Urethane, a widely used anesthetic in animal research, has been shown to reduce the neural responses to auditory stimuli. However, the effects of urethane on cochlea, especially on the function of outer hair cells, remain largely unknown. In the present study, we compared the cochlear microphonic responses between awake and urethane-anesthetized rats. The results revealed that the amplitude of the cochlear microphonic was decreased by urethane, resulting in an increase in the threshold at all of the sound frequencies examined. To deduce the possible mechanism underlying the urethane-induced decrease in cochlear sensitivity, we examined the electrical response properties of isolated outer hair cells using whole-cell patch-clamp recording. We found that urethane hyperpolarizes the outer hair cell membrane potential in a dose-dependent manner and elicits larger outward current. This urethane-induced outward current was blocked by strychnine, an antagonist of the α9 subunit of the nicotinic acetylcholine receptor. Meanwhile, the function of the outer hair cell motor protein, prestin, was not affected. These results suggest that urethane anesthesia is expected to decrease the responses of outer hair cells, whereas the frequency selectivity of cochlea remains unchanged.

Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study.

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Petousis-Harris, Helen; Paynter, Janine; Morgan, Jane; Saxton, Peter; McArdle, Barbara; Goodyear-Smith, Felicity; Black, Steven

2017-09-30

Gonorrhoea is a major global public health problem that is exacerbated by drug resistance. Effective vaccine development has been unsuccessful, but surveillance data suggest that outer membrane vesicle meningococcal group B vaccines affect the incidence of gonorrhoea. We assessed vaccine effectiveness of the outer membrane vesicle meningococcal B vaccine (MeNZB) against gonorrhoea in young adults aged 15-30 years in New Zealand. We did a retrospective case-control study of patients at sexual health clinics aged 15-30 years who were born between Jan 1, 1984, and Dec 31, 1998, eligible to receive MeNZB, and diagnosed with gonorrhoea or chlamydia, or both. Demographic data, sexual health clinic data, and National Immunisation Register data were linked via patients' unique personal identifier. For primary analysis, cases were confirmed by laboratory isolation or detection of Neisseria gonorrhoeae only from a clinical specimen, and controls were individuals with a positive chlamydia test only. We estimated odds ratios (ORs) comparing disease outcomes in vaccinated versus unvaccinated participants via multivariable logistic regression. Vaccine effectiveness was calculated as 100×(1-OR). 11 of 24 clinics nationally provided records. There were 14 730 cases and controls for analyses: 1241 incidences of gonorrhoea, 12 487 incidences of chlamydia, and 1002 incidences of co-infection. Vaccinated individuals were significantly less likely to be cases than controls (511 [41%] vs 6424 [51%]; adjusted OR 0·69 [95% CI 0·61-0·79]; pvaccine effectiveness of MeNZB against gonorrhoea after adjustment for ethnicity, deprivation, geographical area, and sex was 31% (95% CI 21-39). Exposure to MeNZB was associated with reduced rates of gonorrhoea diagnosis, the first time a vaccine has shown any protection against gonorrhoea. These results provide a proof of principle that can inform prospective vaccine development not only for gonorrhoea but also for meningococcal vaccines. GSK

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

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Zückert, Wolfram R.

2014-01-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Mental retardation-related protease, motopsin (prss12), binds to the BRICHOS domain of the integral membrane protein 2a.

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Mitsui, Shinichi; Osako, Yoji; Yuri, Kazunari

2014-01-01

Motopsin (prss12), a mosaic serine protease secreted by neuronal cells, is believed to be important for cognitive function, as the loss of its function causes severe nonsyndromic mental retardation. To understand the molecular role of motopsin, we identified the integral membrane protein 2a (Itm2a) as a motopsin-interacting protein using a yeast two-hybrid system. A pull-down assay showed that the BRICHOS domain of Itm2a was essential for this interaction. Motopsin and Itm2a co-localized in COS cells and in cultured neurons when transiently expressed in these cells. Both proteins were co-immunoprecipitated from lysates of these transfected COS cells. Itm2a was strongly detected in a brain lysate prepared between postnatal day 0 and 10, during which period motopsin protein was also enriched in the brain. Immunohistochemistry detected Itm2a as patchy spots along endothelial cells of brain capillaries (which also expressed myosin II regulatory light chain [RLC]), and on glial fibrillary acidic protein (GFAP)-positive processes in the developing cerebral cortex. The data raise the possibility that secreted motopsin interacts with endothelial cells in the developing brain. © 2013 International Federation for Cell Biology.

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

Energy Technology Data Exchange (ETDEWEB)

Yao, Yong; Dutta, Samit Kumar [Sanford Burnham Prebys Medical Discovery Institute (United States); Park, Sang Ho; Rai, Ratan [University of California San Diego, Department of Chemistry and Biochemistry (United States); Fujimoto, L. Miya; Bobkov, Andrey A. [Sanford Burnham Prebys Medical Discovery Institute (United States); Opella, Stanley J. [University of California San Diego, Department of Chemistry and Biochemistry (United States); Marassi, Francesca M., E-mail: fmarassi@sbp.edu [Sanford Burnham Prebys Medical Discovery Institute (United States)

2017-03-15

The outer membrane protein Ail (Adhesion invasion locus) is one of the most abundant proteins on the cell surface of Yersinia pestis during human infection. Its functions are expressed through interactions with a variety of human host proteins, and are essential for microbial virulence. Structures of Ail have been determined by X-ray diffraction and solution NMR spectroscopy, but those samples contained detergents that interfere with functionality, thus, precluding analysis of the structural basis for Ail’s biological activity. Here, we demonstrate that high-resolution solid-state NMR spectra can be obtained from samples of Ail in detergent-free phospholipid liposomes, prepared with a lipid to protein molar ratio of 100. The spectra, obtained with {sup 13}C or {sup 1}H detection, have very narrow line widths (0.40–0.60 ppm for {sup 13}C, 0.11–0.15 ppm for {sup 1}H, and 0.46–0.64 ppm for {sup 15}N) that are consistent with a high level of sample homogeneity. The spectra enable resonance assignments to be obtained for N, CO, CA and CB atomic sites from 75 out of 156 residues in the sequence of Ail, including 80% of the transmembrane region. The {sup 1}H-detected solid-state NMR {sup 1}H/{sup 15}N correlation spectra obtained for Ail in liposomes compare very favorably with the solution NMR {sup 1}H/{sup 15}N TROSY spectra obtained for Ail in nanodiscs prepared with a similar lipid to protein molar ratio. These results set the stage for studies of the molecular basis of the functional interactions of Ail with its protein partners from human host cells, as well as the development of drugs targeting Ail.

Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

Science.gov (United States)

Bai, Jaewoo; Kim, Seul I; Ryu, Sangryeol

2014-01-01

Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes. PMID:24935973

Proteolytic crosstalk in multi-protease networks

Science.gov (United States)

Ogle, Curtis T.; Mather, William H.

2016-04-01

Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis.

Science.gov (United States)

Zimbler, Daniel L; Eddy, Justin L; Schroeder, Jay A; Lathem, Wyndham W

2016-01-01

Pneumonic plague represents the most severe form of disease caused by Yersinia pestis due to its ease of transmission, rapid progression, and high mortality rate. The Y. pestis outer membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed by Y. pestis in a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2 activities of Prdx6. In addition, we found that infection with wild-type Y. pestis reduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with Δpla Y. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 function in vitro and reduce Prdx6 levels in vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The structure of the COPII transport-vesicle coat assembled on membranes.

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Zanetti, Giulia; Prinz, Simone; Daum, Sebastian; Meister, Annette; Schekman, Randy; Bacia, Kirsten; Briggs, John A G

2013-09-17

Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers. DOI:http://dx.doi.org/10.7554/eLife.00951.001.

Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea).

Science.gov (United States)

Sharma, Ranu; Suresh, C G

2015-01-01

Proteases are a family of enzymes present in almost all living organisms. In plants they are involved in many biological processes requiring stress response in situations such as water deficiency, pathogen attack, maintaining protein content of the cell, programmed cell death, senescence, reproduction and many more. Similarly, protease inhibitors (PIs) are involved in various important functions like suppression of invasion by pathogenic nematodes, inhibition of spores-germination and mycelium growth of Alternaria alternata and response to wounding and fungal attack. As much as we know, no genome-wide study of proteases together with proteinaceous PIs is reported in any of the sequenced genomes till now. Phylogenetic studies and domain analysis of proteases were carried out to understand the molecular evolution as well as gene and protein features. Structural analysis was carried out to explore the binding mode and affinity of PIs for cognate proteases and prolyl oligopeptidase protease with inhibitor ligand. In the study reported here, a significant number of proteases and PIs were identified in chickpea genome. The gene expression profiles of proteases and PIs in five different plant tissues revealed a differential expression pattern in more than one plant tissue. Molecular dynamics studies revealed the formation of stable complex owing to increased number of protein-ligand and inter and intramolecular protein-protein hydrogen bonds. The genome-wide identification, characterization, evolutionary understanding, gene expression, and structural analysis of proteases and PIs provide a framework for future analysis when defining their roles in stress response and developing a more stress tolerant variety of chickpea. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana.

Science.gov (United States)

Kelly, Amélie A; Kalisch, Barbara; Hölzl, Georg; Schulze, Sandra; Thiele, Juliane; Melzer, Michael; Roston, Rebecca L; Benning, Christoph; Dörmann, Peter

2016-09-20

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites.

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Okamura, Masashi; Yokoyama, Naoaki; Takabatake, Noriyuki; Okubo, Kazuhiro; Ikehara, Yuzuru; Igarashi, Ikuo

2007-02-01

In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC showed that the trypsin-treated bovine RBC, but not the chymotrypsin-treated ones, significantly reduced the growth of B. bovis and B. bigemina as compared to the control. In contrast, the growth of B. equi and B. caballi was not affected by any of these proteases. Thus, the bovine, but not the equine, Babesia parasites require the trypsin-sensitive membrane (sialoglyco) proteins to infect the RBC.

Membrane-bound transcription factors: regulated release by RIP or RUP.

Science.gov (United States)

Hoppe, T; Rape, M; Jentsch, S

2001-06-01

Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA–HasR from Serratia marcescens

International Nuclear Information System (INIS)

Huché, Frédéric; Delepelaire, Philippe; Wandersman, Cécile; Welte, Wolfram

2005-01-01

The expression, purification, and crystallization in space group P2 1 2 1 2 1 of the complex HasA-HasR from S. marcescens are reported. Diffraction data have been collected and processed to 6.8 Å. Serratia marcescens is able to acquire iron using its haem-acquisition system (‘has’), which contains an outer membrane receptor HasR and a soluble haemophore HasA. After secretion, HasA binds free haem in the extracellular medium or extracts it from haemoproteins and delivers it to the receptor. Here, the crystallization of a HasA–HasR complex is reported. HasA and HasR have been overexpressed in Escherichia coli and the complex formed and crystallized. Small platelets and bunches of needles of dimensions 0.01 × 0.1 × 1 mm were obtained. A native data set has been collected to 6.8 Å

Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA–HasR from Serratia marcescens

Energy Technology Data Exchange (ETDEWEB)

Huché, Frédéric, E-mail: huche@pasteur.fr [Fachbereich Biologie, Universität Konstanz, 78457 Konstanz (Germany); Unité des Membranes Bactériennes, CNRS URA 2172, Département de Microbiologie Fondamentale et Médicale, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris CEDEX 15 (France); Delepelaire, Philippe; Wandersman, Cécile [Unité des Membranes Bactériennes, CNRS URA 2172, Département de Microbiologie Fondamentale et Médicale, Institut Pasteur, 25-28 Rue du Dr Roux, 75724 Paris CEDEX 15 (France); Welte, Wolfram [Fachbereich Biologie, Universität Konstanz, 78457 Konstanz (Germany)

2006-01-01

The expression, purification, and crystallization in space group P2{sub 1}2{sub 1}2{sub 1} of the complex HasA-HasR from S. marcescens are reported. Diffraction data have been collected and processed to 6.8 Å. Serratia marcescens is able to acquire iron using its haem-acquisition system (‘has’), which contains an outer membrane receptor HasR and a soluble haemophore HasA. After secretion, HasA binds free haem in the extracellular medium or extracts it from haemoproteins and delivers it to the receptor. Here, the crystallization of a HasA–HasR complex is reported. HasA and HasR have been overexpressed in Escherichia coli and the complex formed and crystallized. Small platelets and bunches of needles of dimensions 0.01 × 0.1 × 1 mm were obtained. A native data set has been collected to 6.8 Å.

Diversity of both the cultivable protease-producing bacteria and bacterial extracellular proteases in the coastal sediments of King George Island, Antarctica.

Directory of Open Access Journals (Sweden)

Ming-Yang Zhou

Full Text Available Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 10(5 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%, Flavobacterium (21.0% and Lacinutrix (16.2%. Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea.

Planar ceramic membrane assembly and oxidation reactor system

Science.gov (United States)

Carolan, Michael Francis; Dyer, legal representative, Kathryn Beverly; Wilson, Merrill Anderson; Ohm, Ted R.; Kneidel, Kurt E.; Peterson, David; Chen, Christopher M.; Rackers, Keith Gerard; Dyer, deceased, Paul Nigel

2007-10-09

Planar ceramic membrane assembly comprising a dense layer of mixed-conducting multi-component metal oxide material, wherein the dense layer has a first side and a second side, a porous layer of mixed-conducting multi-component metal oxide material in contact with the first side of the dense layer, and a ceramic channeled support layer in contact with the second side of the dense layer. The planar ceramic membrane assembly can be used in a ceramic wafer assembly comprising a planar ceramic channeled support layer having a first side and a second side; a first dense layer of mixed-conducting multi-component metal oxide material having an inner side and an outer side, wherein the inner side is in contact with the first side of the ceramic channeled support layer; a first outer support layer comprising porous mixed-conducting multi-component metal oxide material and having an inner side and an outer side, wherein the inner side is in contact with the outer side of the first dense layer; a second dense layer of mixed-conducting multi-component metal oxide material having an inner side and an outer side, wherein the inner side is in contact with the second side of the ceramic channeled layer; and a second outer support layer comprising porous mixed-conducting multi-component metal oxide material and having an inner side and an outer side, wherein the inner side is in contact with the outer side of the second dense layer.

Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces.

Science.gov (United States)

Ionescu, Michael; Zaini, Paulo A; Baccari, Clelia; Tran, Sophia; da Silva, Aline M; Lindow, Steven E

2014-09-16

Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an "exploratory" lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents.

Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

DEFF Research Database (Denmark)

Kousted, Tina Mostrup; Skjoedt, K; Petersen, S V

2013-01-01

abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between...

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

Directory of Open Access Journals (Sweden)

Garabaya Cecilia

2006-03-01

Full Text Available Abstract Background We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity. Results Following our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the α-chain of fibrinogen as well as pro

Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli – An Anti-thrombotic Mechanism in the Kidney

Directory of Open Access Journals (Sweden)

Ramesh Tati

2017-02-01

Full Text Available Adequate cleavage of von Willebrand factor (VWF prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3, cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

Disruption the Outer Membrane of Enteropathogenic and Enterotoxigenic Escherichia coli using Proanthocyanidins

Science.gov (United States)

American cranberry (Vaccinium macrocarpon) proanthocyanidins (PACs) have been reported as a natural antibacterial agent to suppress the growth of pathogenic Escherichia coli. The objective of this study was to investigate the efficacy of cranberry-derived proanthocyanidins on destabilizing the outer...

Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

Science.gov (United States)

Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

2009-04-01

Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

Usher syndrome type 1-associated cadherins shape the photoreceptor outer segment.

Science.gov (United States)

Schietroma, Cataldo; Parain, Karine; Estivalet, Amrit; Aghaie, Asadollah; Boutet de Monvel, Jacques; Picaud, Serge; Sahel, José-Alain; Perron, Muriel; El-Amraoui, Aziz; Petit, Christine

2017-06-05

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis , these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23 , encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15-containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. © 2017 Schietroma et al.

A preliminary study of aquaporin 1 immunolocalization in chronic subdural hematoma membranes.

Science.gov (United States)

Basaldella, Luca; Perin, Alessandro; Orvieto, Enrico; Marton, Elisabetta; Itskevich, David; Dei Tos, Angelo Paolo; Longatti, Pierluigi

2010-07-01

Aquaporin 1 (AQP1) is a molecular water channel expressed in many anatomical locations, particularly in epithelial barriers specialized in water transport. The aim of this study was to investigate AQP1 expression in chronic subdural hematoma (CSDH) membranes. In this preliminary study, 11 patients with CSDH underwent burr hole craniectomy and drainage. Membrane specimens were stained with a monoclonal antibody targeting AQP1 for immunohistochemical analysis. The endothelial cells of the sinusoid capillaries of the outer membranes exhibited an elevated immunoreactivity to AQP1 antibody compared to the staining intensity of specimens from the inner membrane and normal dura. These findings suggest that the outer membrane might be the source of the increased fluid accumulation responsible for chronic hematoma enlargement.

Processing Proteases

DEFF Research Database (Denmark)

Ødum, Anders Sebastian Rosenkrans

-terminal of the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is one...... of few known proteases to have substrate specificity for the C-terminal side of the scissile bond. LysN exhibits specificity for lysine, and has primarily been used to complement trypsin in to proteomic studies. A working hypothesis during this study was the potential of LysN as a processing protease...

Rotating bubble membrane radiator

Science.gov (United States)

Webb, Brent J.; Coomes, Edmund P.

1988-12-06

A heat radiator useful for expelling waste heat from a power generating system aboard a space vehicle is disclosed. Liquid to be cooled is passed to the interior of a rotating bubble membrane radiator, where it is sprayed into the interior of the bubble. Liquid impacting upon the interior surface of the bubble is cooled and the heat radiated from the outer surface of the membrane. Cooled liquid is collected by the action of centrifical force about the equator of the rotating membrane and returned to the power system. Details regarding a complete space power system employing the radiator are given.

Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

Science.gov (United States)

Zückert, Wolfram R

2014-08-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

Science.gov (United States)

Huang, Qiwei; Li, Qingxin; Joy, Joma; Chen, Angela Shuyi; Ruiz-Carrillo, David; Hill, Jeffrey; Lescar, Julien; Kang, Congbao

2013-12-01

Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV. Copyright © 2013 Elsevier Inc. All rights reserved.

A biotechnology perspective of fungal proteases

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Paula Monteiro de Souza

2015-06-01

Full Text Available Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens.

Science.gov (United States)

Pors, Susanne E; Pedersen, Ida J; Skjerning, Ragnhild Bager; Thøfner, Ida C N; Persson, Gry; Bojesen, Anders M

2016-11-15

Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have previously shown promising results in protection against infections and we hypothesized that OMVs could serve as an immunogen to protect egg-laying hens against G. anatis. To investigate the immunogenic potential of G. anatis OMVs, two in vivo studies in egg-laying hens were made. The trials assessedthe degree of protection provided by immunization with G. anatis OMV against challenge and the IgY responses in serum after immunization and challenge, respectively. A total of 64 egg-laying hens were included in the trials. OMVs for immunization were produced and purified from a high-producing G. anatis ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re-isolation remained unchanged. Furthermore, a high OMV-specific IgY response was induced by immunization and subsequent challenge of the hens. The results strongly indicate that immunization with G. anatis OMVs provides significant protection against G. anatis challenge and induces specific antibody responses with high titers of OMV-specific IgY in serum. The results therefore show great promise for OMV based vaccines aiming at providing protecting against G. anatis in egg-laying hens. Copyright © 2016 Elsevier B.V. All rights reserved.

Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

Energy Technology Data Exchange (ETDEWEB)

Kocab, S.; Erdem, B. [Middle East Technical University, Ankara (Turkey). Dept. of Biological Sciences

2002-08-01

In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70{sup o}C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes. (author)

[Study on immunogenicity of group A and group C meningococcal conjugate vaccine with coupling group B meningococcal outer membrane protein].

Science.gov (United States)

Ma, Fu-Bao; Tao, Hong; Wang, Hong-Jun

2009-10-01

To evaluate the Immunogenicity of Group A and Group C Meningococcal conjugate Vaccine with coupling Group B Meningococcal Outer Membrane Protein (Men B-OMP). 458 healthy children aged 3-5 months, 6-23 months, 2-6 years and 7-24 years were given the Groups A and C conjugate Vaccine with MenB-OMP or other vaccine as control group to measure the pre-and post-vaccination Men A and C and B by Serum Bactericidal Assay (SBA) in the double-blind randomized controlled trial. 97.65%-100% were 4 times or greater increase in SBA titer for the healthy children given the Groups A and C conjugate Vaccine with MenB-OMP, The geometric mean titer of SBA were 1:194-1:420, which significantly higber than controls. The Group A and C conjugate Vaccine with MenB-OMP was safe and well immunogenic.

Site-SpecificCu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N), Using Copper Free Click Chemistry

DEFF Research Database (Denmark)

Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H

2018-01-01

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migrati...

Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

Directory of Open Access Journals (Sweden)

Magdalena Opalińska

2017-11-01

Full Text Available Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4’s physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4 and Pam18-2 and known (Tim17-2 substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach.

Science.gov (United States)

Opalińska, Magdalena; Parys, Katarzyna; Jańska, Hanna

2017-11-18

Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i -AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4's in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4's physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

Directory of Open Access Journals (Sweden)

Thomas A Prohaska

Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

Science.gov (United States)

Maddur, Ashoka A; Swanson, Richard; Izaguirre, Gonzalo; Gettins, Peter G W; Olson, Steven T

2013-11-01

Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to (i) serpin reactive center loop insertion into sheet A with full protease translocation but incomplete protease distortion followed by, (ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism.

Relationship of the CreBC two-component regulatory system and inner membrane protein CreD with swimming motility in Stenotrophomonas maltophilia.

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Hsin-Hui Huang

Full Text Available The CreBC two-component system (TCS is a conserved regulatory system found in Escherichia coli, Aeromonas spp., Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. In this study, we determined how CreBC TCS regulates secreted protease activities and swimming motility using creB, creC, and creBC in-frame deletion mutants (KJΔCreB, KJΔCreC, and KJΔBC of S. maltophilia KJ. Compared to wild-type KJ, KJΔCreB had a comparable secreted protease activity; however, the secreted protease activities were obviously reduced in KJΔCreC and KJΔBC, suggesting that CreC works together with another unidentified response regulator (not CreB to regulate secreted protease activity. Single gene inactivation of creB or creC resulted in mutants with an enhanced swimming motility, and this phenotype was exacerbated in a double mutant KJΔBC. To elucidate the underlying mechanism responsible for the ΔcreBC-mediated swimming enhancement, flagella morphology observation, RNA-seq based transcriptome assay, qRT-PCR, and membrane integrity and potential assessment were performed. Flagella morphological observation ruled out the possibility that swimming enhancement was due to altered flagella morphology. CreBC inactivation upregulated the expression of creD and flagella-associated genes encoding the basal body- and motor-associated proteins. Furthermore, KJΔBC had an increased membrane susceptibility to Triton X-100 and CreD upregulation in KJΔBC partially alleviated the compromise of membrane integrity. The impact of creBC TCS on bacterial membrane potential was assessed by carbonyl cyanide m-chlorophenyl hydrazine (CCCP50 concentration at which 50% of bacterial swimming is inhibited. CCCP50 of wild-type KJ increased when creBC was deleted, indicating an association between the higher membrane potential of KJΔBC cells and enhanced motility. Upregulation of the basal body- and motor-associated genes of flagella in KJΔBC cells may explain the increased

Advances in protease engineering for laundry detergents.

Science.gov (United States)

Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

2015-12-25

Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

Usher syndrome type 1–associated cadherins shape the photoreceptor outer segment

Science.gov (United States)

Parain, Karine; Aghaie, Asadollah; Picaud, Serge

2017-01-01

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. PMID:28495838

Interaction of MreB-derived antimicrobial peptides with membranes.

Science.gov (United States)

Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

Cloning and Characterization of an Outer Membrane Protein of Vibrio vulnificus Required for Heme Utilization: Regulation of Expression and Determination of the Gene Sequence

Science.gov (United States)

Litwin, Christine M.; Byrne, Burke L.

1998-01-01

Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V. vulnificus. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V. cholerae HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the V. vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the V. vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V. vulnificus pathogenesis. PMID:9632577

In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.

Directory of Open Access Journals (Sweden)

Jeong Hee Kim

Full Text Available This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s. The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

International Nuclear Information System (INIS)

Watanabe, Yoshihisa; Okui, Akira; Mitsui, Shinichi; Kawarabuki, Kentaro; Yamaguchi, Tatsuyuki; Uemura, Hidetoshi; Yamaguchi, Nozomi

2004-01-01

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression

Protein secretion and membrane insertion systems in gram-negative bacteria.

Science.gov (United States)

Saier, Milton H

2006-01-01

In contrast to other organisms, gram-negative bacteria have evolved numerous systems for protein export. Eight types are known that mediate export across or insertion into the cytoplasmic membrane, while eight specifically mediate export across or insertion into the outer membrane. Three of the former secretory pathway (SP) systems, type I SP (ISP, ABC), IIISP (Fla/Path) and IVSP (Conj/Vir), can export proteins across both membranes in a single energy-coupled step. A fourth generalized mechanism for exporting proteins across the two-membrane envelope in two distinct steps (which we here refer to as type II secretory pathways [IISP]) utilizes either the general secretory pathway (GSP or Sec) or the twin-arginine targeting translocase for translocation across the inner membrane, and either the main terminal branch or one of several protein-specific export systems for translocation across the outer membrane. We here survey the various well-characterized protein translocation systems found in living organisms and then focus on the systems present in gram-negative bacteria. Comparisons between these systems suggest specific biogenic, mechanistic and evolutionary similarities as well as major differences.

A Meningococcal Outer Membrane Vesicle Vaccine Incorporating Genetically Attenuated Endotoxin Dissociates Inflammation From Immunogenicity

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David J. Dowling

2016-12-01

Full Text Available Background. Group B Neisseria meningitidis, an endotoxin-producing gram-negative bacterium, causes the highest incidence of group B meningococcus (MenB disease in the first year of life. The Bexsero vaccine is indicated in Europe from 8 weeks of age. Endotoxin components of outer membrane vesicles (OMVs or soluble lipopolysaccharide (LPS represent a potential source of inflammation and residual reactogenicity. The purpose of this study was to compare novel candidate MenB vaccine formulations with licensed vaccines, including Bexsero, using age-specific in vitro culture systems.Methods. OMVs from wild type and inactivated lpxL1 gene mutant N. meningitidis strains were characterized in human neonatal and adult in vitro whole blood assays and dendritic cell arrays. OMVs were benchmarked against licensed vaccines, including Bexsero and whole cell pertussis formulations, with respect to Th-polarizing cytokine and PGE2 production, as well as cell surface activation markers (HLA-DR, CD86, CCR7. OMV immunogenicity was assessed in mice.Results. ΔlpxLI native OMVs demonstrated significantly less cytokine induction in human blood and DCs than Bexsero and most of the other pediatric vaccines (e.g., PedvaxHib, EasyFive, Bacillus Calmette–Guérin (BCG tested. Despite a much lower inflammatory profile in vitro than Bexsero, ΔlpxLI native OMVs still had moderate DC maturing ability and induced robust anti-N. meningitidis antibody responses after murine immunization.Conclusions. A meningococcal vaccine comprised of attenuated LPS-based OMVs with a limited inflammatory profile in vitro induces robust antigen-specific immunogenicity in vivo.

Comparative Outer Membrane Protein Analysis of High and Low-Invasive Strains of Cronobacter malonaticus

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Maha A. Aldubyan

2017-11-01

Full Text Available Cronobacter are an important group of foodborne pathogens that has been linked to life-threatening infections in both infants and adults. The major infections associated with Cronobacter species are neonatal meningitis, necrotizing enterocolitis, and septicaemia. There are seven species in the Cronobacter genus, of which only three are of clinical importance; Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter turicensis. To date most studies have focussed on C. sakazakii as it is the major species associated with neonatal infections. However, recently C. malonaticus, in particular sequence type 7 (ST7, has been noted as being prevalent in adult infections and therefore warranting further investigation. In this study, eight strains of C. malonaticus ST7, that had been isolated from a wide range of sources and varied in their in vitro virulence, were chosen for proteomic analysis of their outer membrane proteins (OMPs. One-dimensional gel analysis revealed a ~29 kDa size band that was only present in the highly invasive strains. Subsequent mass spectrometric analysis identified several peptides that matched the flagellin protein. The presence of flagellin protein was confirmed in 2D gel spot. Mass spectrometry analysis of total OMPs revealed that the four highly invasive C. malonaticus strains expressed the main flagellum proteins that were absent from the four low invasive strains. These were the flagellar hook protein FlgE, flagellar hook-associated protein 1, flagellar hook-associated protein, flagellin, and flagellar hook-filament junction protein FlgL. This data indicates that C. malonaticus flagellar proteins may have an important role in the organism's invasion properties.

Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles.

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Horrevoets, A J; Hackeng, T M; Verheij, H M; Dijkman, R; de Haas, G H

1989-02-07

The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.

Understanding serine proteases implications on Leishmania spp lifecycle.

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Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

2018-01-01

Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.

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Kauffman, Karlina J; Yu, Shenliang; Jin, Jiaxin; Mugo, Brian; Nguyen, Nathan; O'Brien, Aidan; Nag, Shanta; Lystad, Alf Håkon; Melia, Thomas J

2018-04-10

During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.

The establishment of polarized membrane traffic in Xenopus laevis embryos.

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Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin

Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

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Nicole Hofmann

Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

Structure of the Integral Membrane Protein CAAX Protease Ste24p

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Pryor Jr., Edward E. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Horanyi, Peter S. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Clark, Kathleen M. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Fedoriw, Nadia [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Connelly, Sara M. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States); Koszelak-Rosenblum, Mary [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); Zhu, Guangyu [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); Malkowski, Michael G. [Membrane Protein Structural Biology Consortium (United States); Hauptman-Woodward Inst., Buffalo, NY (United States); State Univ. of New York, Buffalo, NY (United States); Wiener, Michael C. [Membrane Protein Structural Biology Consortium (United States); Univ. of Virginia, Charlottesville, VA (United States); Dumont, Mark E. [Membrane Protein Structural Biology Consortium (United States); Univ. of Rochester School of Medicine and Dentistry, Rochester, NY (United States)

2012-10-26

Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a -factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.

Membrane transport of anandamide through resealed human red blood cell membranes

DEFF Research Database (Denmark)

Bojesen, I.N.; Hansen, Harald S.

2005-01-01

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments...... at 0°C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 ± 0.023, and the rate constant...... of unidirectional flux from inside to outside is 0.361 ± 0.023 s. The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]) increases with the square root of [BSA] in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane...

A genomic survey of proteases in Aspergilli

NARCIS (Netherlands)

Budak, Sebnem Ozturkoglu; Zhou, M.; Brouwer, Carlo; Wiebenga, A.; Benoit, Isabelle; Di Falco, Marcos; Tsang, Adrian; de Vries, Ronald P; van den Brink, J.

2014-01-01

BACKGROUND: Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide

Aspartate tightens the anchoring of staphylococcal lipoproteins to the cytoplasmic membrane.

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Kumari, Nimerta; Götz, Friedrich; Nguyen, Minh-Thu

2017-12-01

In gram-negative bacteria, the ABC transporter LolCDE complex translocates outer membrane-specific lipoproteins (Lpp) from the inner membrane to the outer membrane. Lpp possessing aspartate (Asp) at position +2 are not translocated because it functions as a LolCDE avoidance signal. In gram-positive bacteria, lacking an outer membrane and the Lol system, Lpp are only anchored at the outer leaflet of the cytoplasmic membrane. However, the release of Lpp particularly in pathogenic or commensal species is crucial for immune modulation. Here, we provide evidence that in Staphylococcus aureus Asp at position +2 plays a role in withholding Lpp to the cytoplasmic membrane. Screening of published exoproteomic data of S. aureus revealed that Lpp mainly with Gly or Ser at position +2 were found in exoproteome, but there was no Lpp with Asp+2. The occurrence of Lpp with Asp+2 is infrequent in gram-positive bacteria. In S. aureus USA300 only seven of the 67 Lpp possess Asp+2; among them five Lpp represented Lpl lipoproteins involved in host cell invasion. Our study demonstrated that replacing the Asp+2 present in Lpl8 with a Ser enhances its release into the supernatant. However, there is no different release of Asp+2 and Ser+2 in mprF mutant that lacks the positive charge of lysyl-phosphatidylglycerol (Lys-PG). Moreover, substitution of Ser+2 by Asp in SitC (MntC) did not lead to a decreased release indicating that in staphylococci positions +3 and +4 might also be important for a tighter anchoring of Lpp. Here, we show that Asp in position +2 and adjacent amino acids contribute in tightening the anchoring of Lpp by interaction of the negative charged Asp with the positive charged Lys-PG. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Increase in average foveal thickness after internal limiting membrane peeling

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Kumagai K

2017-04-01

Full Text Available Kazuyuki Kumagai,1 Mariko Furukawa,1 Tetsuyuki Suetsugu,1 Nobuchika Ogino2 1Department of Ophthalmology, Kami-iida Daiichi General Hospital, 2Department of Ophthalmology, Nishigaki Eye Clinic, Aichi, Japan Purpose: To report the findings in three cases in which the average foveal thickness was increased after a thin epiretinal membrane (ERM was removed by vitrectomy with internal limiting membrane (ILM peeling.Methods: The foveal contour was normal preoperatively in all eyes. All cases underwent successful phacovitrectomy with ILM peeling for a thin ERM. The optical coherence tomography (OCT images were examined before and after the surgery. The changes in the average foveal (1 mm thickness and the foveal areas within 500 µm from the foveal center were measured. The postoperative changes in the inner and outer retinal areas determined from the cross-sectional OCT images were analyzed.Results: The average foveal thickness and the inner and outer foveal areas increased significantly after the surgery in each of the three cases. The percentage increase in the average foveal thickness relative to the baseline thickness was 26% in Case 1, 29% in Case 2, and 31% in Case 3. The percentage increase in the foveal inner retinal area was 71% in Case 1, 113% in Case 2, and 110% in Case 3, and the percentage increase in foveal outer retinal area was 8% in Case 1, 13% in Case 2, and 18% in Case 3.Conclusion: The increase in the average foveal thickness and the inner and outer foveal areas suggests that a centripetal movement of the inner and outer retinal layers toward the foveal center probably occurred due to the ILM peeling. Keywords: internal limiting membrane, optical coherence tomography, average foveal thickness, epiretinal membrane, vitrectomy

Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

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Renault, Thibaud T; Floros, Konstantinos V; Elkholi, Rana; Corrigan, Kelly-Ann; Kushnareva, Yulia; Wieder, Shira Y; Lindtner, Claudia; Serasinghe, Madhavika N; Asciolla, James J; Buettner, Christoph; Newmeyer, Donald D; Chipuk, Jerry E

2015-01-08

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Cytomegalovirus protease targeted prodrug development.

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Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

2013-04-01

Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

Protease-resistant prions selectively decrease Shadoo protein.

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Joel C Watts

2011-11-01

Full Text Available The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C into PrP(Sc, a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho, a protein that resembles the flexibly disordered N-terminal domain of PrP(C, were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc. Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc. Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc during prion disease.

Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

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Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.; Heffron, Fred

2011-06-01

We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548, PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.

Role of Proteases in Chronic Obstructive Pulmonary Disease

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Kailash C. Pandey

2017-08-01

Full Text Available Chronic obstructive pulmonary disease (COPD is generally associated with progressive destruction of airways and lung parenchyma. Various factors play an important role in the development and progression of COPD, like imbalance of proteases, environmental and genetic factors and oxidative stress. This review is specifically focused on the role of proteases and their imbalance in COPD. There are three classes (serine, mettalo, and cysteine of proteases involved in COPD. In serine proteases, neutrophil elastase, cathepsin G, and proteinase-3 are involved in destruction of alveolar tissue. Matrix-mettaloproteinase-9, 12, 13, plays an influential role in severity of COPD. Among cysteine proteases, caspase-3, caspases-8 and caspase-9 play an important role in controlling apoptosis. These proteases activities can be regulated by inhibitors like α-1-antitrypsin, neutrophil elastase inhibitor, and leukocyte protease inhibitor. Studies suggest that neutrophil elastase may be a therapeutic target for COPD, and specific inhibitor against this enzyme has potential role to control the disease. Current study suggests that Dipeptidyl Peptidase IV is a potential marker for COPD. Since the expression of proteases and its inhibitors play an important role in COPD pathogenesis, therefore, it is worth investigating the role of proteases and their regulation. Understanding the biochemical basis of COPD pathogenesis using advanced tools in protease biochemistry and aiming toward translational research from bench-to-bedside will have great impact to deal with this health problem.

tolerant alkaline protease from Bacillus coagulans PSB

African Journals Online (AJOL)

oyaide

2013-05-22

May 22, 2013 ... suggest the suitability of the enzyme for applications in peptide synthesis, detergent formulation and ... The cell free supernatant was recovered as crude enzyme preparation and used for further studies. Assay of protease activity. Protease activity was ... Effect of pH on growth and protease production.

Relationship of Triamine-Biocide Tolerance of Salmonella enterica Serovar Senftenberg to Antimicrobial Susceptibility, Serum Resistance and Outer Membrane Proteins.

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Futoma-Kołoch, Bożena; Dudek, Bartłomiej; Kapczyńska, Katarzyna; Krzyżewska, Eva; Wańczyk, Martyna; Korzekwa, Kamila; Rybka, Jacek; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-07-11

A new emerging phenomenon is the association between the incorrect use of biocides in the process of disinfection in farms and the emergence of cross-resistance in Salmonella populations. Adaptation of the microorganisms to the sub-inhibitory concentrations of the disinfectants is not clear, but may result in an increase of sensitivity or resistance to antibiotics, depending on the biocide used and the challenged Salmonella serovar. Exposure of five Salmonella enterica subsp. enterica serovar Senftenberg ( S. Senftenberg) strains to triamine-containing disinfectant did not result in variants with resistance to antibiotics, but has changed their susceptibility to normal human serum (NHS). Three biocide variants developed reduced sensitivity to NHS in comparison to the sensitive parental strains, while two isolates lost their resistance to serum. For S. Senftenberg, which exhibited the highest triamine tolerance (6 × MIC) and intrinsic sensitivity to 22.5% and 45% NHS, a downregulation of flagellin and enolase has been demonstrated, which might suggest a lower adhesion and virulence of the bacteria. This is the first report demonstrating the influence of biocide tolerance on NHS resistance. In conclusion, there was a potential in S. Senftenberg to adjust to the conditions, where the biocide containing triamine was present. However, the adaptation did not result in the increase of antibiotic resistance, but manifested in changes within outer membrane proteins' patterns. The strategy of bacterial membrane proteins' analysis provides an opportunity to adjust the ways of infection treatments, especially when it is connected to the life-threating bacteremia caused by Salmonella species.

Mosaic serine proteases in the mammalian central nervous system.

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Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

2008-01-01

We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

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Kim, Nak Hyun; Hwang, Byung Kook

2015-01-01

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Differences in outer membrane proteins of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis

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Batteiger, B.E.; Jones, R.B.

1985-01-01

The lymphogranuloma venereum (LGV) and trachoma biovars of Chlamydia trachomatis exhibit differences in biological properties both in vivo and in vitro. To identify analogous biochemical differences, the authors studied the molecular charges of chlamydial outer membrane proteins (OMPs) by means of isoelectric focusing and nonequilibrium pH gradient electrophoresis. Analysis of proteins of whole elementary bodies biosynthetically labeled with L-[35S]cysteine revealed that most chlamydial proteins were neutral or acidic. The major OMPs (MOMPs) of all strains tested were acidic and had apparent isoelectric points (pIs) that varied within narrow limits despite differences in molecular mass of up to 3,000 daltons (Da). However, a low-molecular-mass cysteine-rich OMP analogous to that previously described for Chlamydia psittaci varied consistently in molecular mass (12,500 versus 12,000 Da) and pI (5.4 versus 6.9) between LGV strains and trachoma strains, respectively. OMPs with a molecular mass of 60,000 Da in the trachoma biovar strains had pIs in the 7.3 to 7.7 range. However, analogous OMPs in the LGV strains existed as a doublet with a molecular mass of about 60,000 Da. These data indicate substantial differences in biochemical characteristics of analogous OMPs in the LGV and trachoma biovars. Such differences are the first structural differences described between LGV and trachoma strains which support their distinction into separate biovars and may be related to some of their biological differences

Vaccinomics Approach for Designing Potential Peptide Vaccine by Targeting Shigella spp. Serine Protease Autotransporter Subfamily Protein SigA

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Arafat Rahman Oany

2017-01-01

Full Text Available Shigellosis, a bacillary dysentery, is closely associated with diarrhoea in human and causes infection of 165 million people worldwide per year. Casein-degrading serine protease autotransporter of enterobacteriaceae (SPATE subfamily protein SigA, an outer membrane protein, exerts both cytopathic and enterotoxic effects especially cytopathic to human epithelial cell type-2 (HEp-2 and is shown to be highly immunogenic. In the present study, we have tried to impose the vaccinomics approach for designing a common peptide vaccine candidate against the immunogenic SigA of Shigella spp. At first, 44 SigA proteins from different variants of S. flexneri, S. dysenteriae, S. boydii, and S. sonnei were assessed to find the most antigenic protein. We retrieved 12 peptides based on the highest score for human leukocyte antigen (HLA supertypes analysed by NetCTL. Initially, these peptides were assessed for the affinity with MHC class I and class II alleles, and four potential core epitopes VTARAGLGY, FHTVTVNTL, HTTWTLTGY, and IELAGTLTL were selected. From these, FHTVTVNTL and IELAGTLTL peptides were shown to have 100% conservancy. Finally, IELAGTLTL was shown to have the highest population coverage (83.86% among the whole world population. In vivo study of the proposed epitope might contribute to the development of functional and unique widespread vaccine, which might be an operative alleyway to thwart dysentery from the world.

The membrane-topogenic vectorial behaviour of Nrf1 controls its post-translational modification and transactivation activity.

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Zhang, Yiguo; Hayes, John D

2013-01-01

The integral membrane-bound Nrf1 transcription factor fulfils important functions in maintaining cellular homeostasis and organ integrity, but how it is controlled vectorially is unknown. Herein, creative use of Gal4-based reporter assays with protease protection assays (GRAPPA), and double fluorescence protease protection (dFPP), reveals that the membrane-topogenic vectorial behaviour of Nrf1 dictates its post-translational modification and transactivation activity. Nrf1 is integrated within endoplasmic reticulum (ER) membranes through its NHB1-associated TM1 in cooperation with other semihydrophobic amphipathic regions. The transactivation domains (TADs) of Nrf1, including its Asn/Ser/Thr-rich (NST) glycodomain, are transiently translocated into the ER lumen, where it is glycosylated in the presence of glucose to become a 120-kDa isoform. Thereafter, the NST-adjoining TADs are partially repartitioned out of membranes into the cyto/nucleoplasmic side, where Nrf1 is subject to deglycosylation and/or proteolysis to generate 95-kDa and 85-kDa isoforms. Therefore, the vectorial process of Nrf1 controls its target gene expression.

Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

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Madhusudhan Budatha

Full Text Available Mice deficient for the fibulin-5 gene (Fbln5(-/- develop pelvic organ prolapse (POP due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/- mice, herein named V1 (25 kDa. V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/- mice. PRSS3 was (a localized in epithelial secretions, (b detected in media of vaginal organ culture from both Fbln5(-/- and wild type mice, and (c cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin and Elafin] was dysregulated in Fbln5(-/- epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

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Zhao Haiyan; Sequeira, Reuben D.; Galeva, Nadezhda A.; Tang Liang

2011-01-01

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.

Extracellular proteases of Trichoderma species. A review.

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Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

2005-01-01

Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

Outer- and middle-ear contributions to presbycusis in the Brown Norway rat.

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Gratton, Michael Anne; Bateman, Kristin; Cannuscio, Joseph F; Saunders, James C

2008-01-01

This paper examines the contribution of the outer and middle ears to the hearing loss associated with presbycusis in Brown Norway rats. Animals were formed into two groups; young adults (2-3 months old) and aged animals (approximately 34 months old). Auditory brainstem response (ABR) thresholds were obtained with the outer ear intact or surgically removed. Tympanic membrane (TM) velocity transfer functions were measured from the umbo with the outer ear removed. The length of the auditory meatus, TM surface area, and TM thickness were quantified. The ABR thresholds were 17-26 dB less sensitive in the aged animals between 8.0 and 40.0 kHz when the outer ear was intact. A significant and reliable reduction in the aged rat velocity transfer function of 5-8 dB occurred between 10.0 and 32.0 kHz, while the low frequency velocity response was only a few decibels greater in the younger animals. The ABR threshold differences between young adult and aged ears were compensated by removing the outer/middle ear effects of aging to reveal a purely sensorineural component of presbycusis. The outer and middle ear effects were calculated directly when the ABR and TM velocity data were obtained with the outer ear removed. The outer ear intact condition was modeled in order to compare the ABR data obtained with the outer ear intact with the TM velocity data obtained with the outer removed. With either procedure, removal of the age-related contributions of the outer and middle ear to the ABR threshold resulted in similar age-related ABR threshold shifts between the two age groups. The pure sensorineural threshold shift component of the ABR response was restricted to frequencies between 5.0 and 20.0 kHz and reached a maximum of approximately 15 dB. These results support the conclusion that there is an outer- and middle-ear contribution to the threshold loss defining presbycusis. (c) 2008 S. Karger AG, Basel.

The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

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Mark J White

2011-03-01

Full Text Available Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.

Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

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Olivier Barré

Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

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Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Isolation and Identification of Outer Membrane Proteins of Helicobacter Pylori of Iranian Patient by SDS-PAGE

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M. Doosty

1998-04-01

Full Text Available The function of Helicobacter pylori (H.pylori is confirmed as one of the factors which motivates gastric and duodenal ulcer and gastritis. Various methods are used to diagnose the infection. Serological tests are the easiest and most harmless for the patients. Probably, H.pylori strains in Iran are different from the strains in other countries. Hence, it seems neccessary to design a specific serological test to recognize and identify different strains of bacterial antigenic proteins of Iranian patients."nSince the most manifest and specific to these bacterial antigens are the "Outer Membrane Protein" (OMP, therefore, the first necessary step is to separate and purify H.pylori OMP and then to identify antigenic proteins."nIn this study, we received bacteria colony that belonged to 15 patients with gastric or duodenal ulcer, which had been growed in blood agar or brucella broth. After processing such as washing, freezing and defreezing, sonicating, centrifugation with high speed (10,000 g and treatment with sarcosyl, the sarcosyl insoluble fraction was extracted. Sodium Dodecyl Sulfate - Poly Acrylamide Gel Electrophoresis (SDS-PAGE was preformed. From all 15 OMP specimens, we isolated protein bands."nThe first two bands with higher MW, were major bands and the two lighter bands were the minor bands. Approximate MW of these 4 proteins are equal to 67000, 61000, 30000 and 17000 dalton

Plasma membrane organization and dynamics is probe and cell line dependent.

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Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue

Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

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Jing Zhang

2017-12-01

Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

Curcumin derivatives as HIV-1 protease inhibitors

Energy Technology Data Exchange (ETDEWEB)

Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

1993-12-31

Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

Mitochondrial shaping cuts.

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Escobar-Henriques, Mafalda; Langer, Thomas

2006-01-01

A broad range of cellular processes are regulated by proteolytic events. Proteolysis has now also been established to control mitochondrial morphology which results from the balanced action of fusion and fission. Two out of three known core components of the mitochondrial fusion machinery are under proteolytic control. The GTPase Fzo1 in the outer membrane of mitochondria is degraded along two independent proteolytic pathways. One controls mitochondrial fusion in vegetatively growing cells, the other one acts upon mating factor-induced cell cycle arrest. Fusion also depends on proteolytic processing of the GTPase Mgm1 by the rhomboid protease Pcp1 in the inner membrane of mitochondria. Functional links of AAA proteases or other proteolytic components to mitochondrial dynamics are just emerging. This review summarises the current understanding of regulatory roles of proteolytic processes for mitochondrial plasticity.

Permeability testing of biomaterial membranes

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Dreesmann, L; Hajosch, R; Nuernberger, J Vaz; Schlosshauer, B [NMI Natural and Medical Sciences Institute at University Tuebingen, Markwiesenstr. 55, D-72770 Reutlingen (Germany); Ahlers, M [GELITA AG, Gammelsbacher Str. 2, D-69412 Eberbach (Germany)], E-mail: schlosshauer@nmi.de

2008-09-01

The permeability characteristics of biomaterials are critical parameters for a variety of implants. To analyse the permeability of membranes made from crosslinked ultrathin gelatin membranes and the transmigration of cells across the membranes, we combined three technical approaches: (1) a two-chamber-based permeability assay, (2) cell culturing with cytochemical analysis and (3) biochemical enzyme electrophoresis (zymography). Based on the diffusion of a coloured marker molecule in conjunction with photometric quantification, permeability data for a gelatin membrane were determined in the presence or absence of gelatin degrading fibroblasts. Cytochemical evaluation after cryosectioning of the membranes was used to ascertain whether fibroblasts had infiltrated the membrane inside. Zymography was used to investigate the potential release of proteases from fibroblasts, which are known to degrade collagen derivatives such as gelatin. Our data show that the diffusion equilibrium of a low molecular weight dye across the selected gelatin membrane is approached after about 6-8 h. Fibroblasts increase the permeability due to cavity formation in the membrane inside without penetrating the membrane for an extended time period (>21 days in vitro). Zymography indicates that cavity formation is most likely due to the secretion of matrix metalloproteinases. In summary, the combination of the depicted methods promises to facilitate a more rational development of biomaterials, because it provides a rapid means of determining permeability characteristics and bridges the gap between descriptive methodology and the mechanistic understanding of permeability alterations due to biological degradation.

Permeability testing of biomaterial membranes

International Nuclear Information System (INIS)

Dreesmann, L; Hajosch, R; Nuernberger, J Vaz; Schlosshauer, B; Ahlers, M

2008-01-01

The permeability characteristics of biomaterials are critical parameters for a variety of implants. To analyse the permeability of membranes made from crosslinked ultrathin gelatin membranes and the transmigration of cells across the membranes, we combined three technical approaches: (1) a two-chamber-based permeability assay, (2) cell culturing with cytochemical analysis and (3) biochemical enzyme electrophoresis (zymography). Based on the diffusion of a coloured marker molecule in conjunction with photometric quantification, permeability data for a gelatin membrane were determined in the presence or absence of gelatin degrading fibroblasts. Cytochemical evaluation after cryosectioning of the membranes was used to ascertain whether fibroblasts had infiltrated the membrane inside. Zymography was used to investigate the potential release of proteases from fibroblasts, which are known to degrade collagen derivatives such as gelatin. Our data show that the diffusion equilibrium of a low molecular weight dye across the selected gelatin membrane is approached after about 6-8 h. Fibroblasts increase the permeability due to cavity formation in the membrane inside without penetrating the membrane for an extended time period (>21 days in vitro). Zymography indicates that cavity formation is most likely due to the secretion of matrix metalloproteinases. In summary, the combination of the depicted methods promises to facilitate a more rational development of biomaterials, because it provides a rapid means of determining permeability characteristics and bridges the gap between descriptive methodology and the mechanistic understanding of permeability alterations due to biological degradation

Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope.

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Preetinder K Dhanoa

Full Text Available BACKGROUND: Tail-anchored (TA proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34 and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9. Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. CONCLUSIONS/SIGNIFICANCE: Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie

Indispensable Role of Proteases in Plant Innate Immunity.

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Balakireva, Anastasia V; Zamyatnin, Andrey A

2018-02-23

Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered.

Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

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Rogers, Jason V; Rose, Mark D

2014-12-02

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. Copyright © 2015 Rogers and Rose.

Gut proteases target Yersinia invasin in vivo

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Freund Sandra

2011-04-01

Full Text Available Abstract Background Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches. Findings Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases. Conclusions We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

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Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

2016-08-01

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

Artificial Cochlear Sensory Epithelium with Functions of Outer Hair Cells Mimicked Using Feedback Electrical Stimuli

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Tetsuro Tsuji

2018-05-01

Full Text Available We report a novel vibration control technique of an artificial auditory cochlear epithelium that mimics the function of outer hair cells in the organ of Corti. The proposed piezoelectric and trapezoidal membrane not only has the acoustic/electric conversion and frequency selectivity of the previous device developed mainly by one of the authors and colleagues, but also has a function to control local vibration according to sound stimuli. Vibration control is achieved by applying local electrical stimuli to patterned electrodes on an epithelium made using micro-electro-mechanical system technology. By choosing appropriate phase differences between sound and electrical stimuli, it is shown that it is possible to both amplify and dampen membrane vibration, realizing better control of the response of the artificial cochlea. To be more specific, amplification and damping are achieved when the phase difference between the membrane vibration by sound stimuli and electrical stimuli is zero and π , respectively. We also demonstrate that the developed control system responds automatically to a change in sound frequency. The proposed technique can be applied to mimic the nonlinear response of the outer hair cells in a cochlea, and to realize a high-quality human auditory system.