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Sample records for extracellular protease activity

  1. Extracellular Protease Activity of Enteropathogenic Escherechia coli on Mucin Substrate

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    SRI BUDIARTI

    2007-03-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC causes gastrointestinal infections in human. EPEC invasion was initiated by attachment and aggressive colonization on intestinal surface. Attachment of EPEC alter the intestine mucosal cells. Despite this, the pathogenic mechanism of EPEC infectior has not been fully understood. This research hypothesizes that extracellular proteolytic enzymes is necessary for EPEC colonization. The enzyme is secreted into gastrointestinal milieu and presumably destroy mucus layer cover the gastrointestinal tract. The objective of this study was to assay EPEC extracellular protease enzyme by using mucin substrate. The activity of EPEC extracellular proteolytic enzyme on 1% mucin substrate was investigated. Non-pathogenic E. coli was used as a negative control. Positive and tentative controls were Yersinia enterocolitica and Salmonella. Ten EPEC strains were assayed, seven of them were able to degrade mucin, and the highest activity was produced by K1.1 strain. Both positive and tentative controls also showed the ability to digest 0.20% mucin.

  2. Vibrio cholerae hemagglutinin(HA)/protease: an extracellular metalloprotease with multiple pathogenic activities

    Science.gov (United States)

    Benitez, Jorge A.; Silva, Anisia J.

    2016-01-01

    Vibrio cholerae of serogroup O1 and O139, the etiological agent of the diarrheal disease cholera, expresses the extracellular Zn-dependent metalloprotease hemagglutinin (HA)/protease also reported as vibriolysin. This enzyme is also produced by non-O1/O139 (non-cholera) strains that cause mild, sporadic illness (i.e. gastroenteritis, wound or ear infections). Orthologs of HA/protease are present in other members of the Vibrionaceae family pathogenic to humans and fish. HA/protease belongs to the M4 neutral peptidase family and displays significant amino acid sequence homology to Pseudomonas aeruginosa elastase (LasB) and Bacillus thermoproteolyticus thermolysin. It exhibits a broad range of potentially pathogenic activities in cell culture and animal models. These activities range from the covalent modification of other toxins, the degradation of the protective mucus barrier and disruption of intestinal tight junctions. Here we review (i) the structure and regulation of HA/protease expression, (ii) its interaction with other toxins and the intestinal mucosa and (iii) discuss the possible role(s) of HA/protease in the pathogenesis of cholera. PMID:26952544

  3. The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

    NARCIS (Netherlands)

    Braaksma, M.; Smilde, A.K.; Werf, M.J. van der; Punt, P.J.

    2009-01-01

    Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically

  4. Antibacterial Activity of Extracellular Protease Isolated From an Algicolous Fungus Xylaria psidii KT30 Against Gram-Positive Bacteria

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    Taufik Indarmawan

    2016-04-01

    Full Text Available Infectious diseases became more serious problem for public health in recent years. Although existing antibacterial drugs have been relatively effective, they do not rule out the emergence of resistance to the drug. Therefore, the intensive exploration of new bioactive compounds from natural, especially peptide compounds began in recent decades in order-handling infection. This study aimed to isolate, purify and test the potential application of Xylaria psidii KT30 extracellular protease as antibacterial agent against Gram-positive bacteria. X. psidii KT30, a marine fungus isolated from red seaweed Kappaphycus alvarezii showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Antibacterial compounds of this fungus were predicted as a group of proteases. Extracellular protease exhibited an optimum activity when potato dextrose broth was used as cultivation medium. Furthermore, the highest activity of these proteases was found on fungal extract after day 15 of cultivation with value of 2.33 ± 0.19 U/mL. The partial purification of proteases using G-75 column chromatography resulted in 2 groups of fractions and showed protease activity based on zymogram assay. The extracellular proteases obtained from those fractions have 3 patterns of molecular mass based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis which are 56.62, 89.12, 162.18 kDa.

  5. Antarctic bacterial isolates that produce cold-active extracellular proteases at low temperature but are active and stable at high temperature

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    Cecilia Martínez-Rosales

    2011-04-01

    Full Text Available We report the isolation and identification of bacteria that produce extracellular cold-active proteases, obtained from water samples collected near the Uruguayan Antarctic Base on King George Island, South Shetlands. The bacteria belonged to the genera Pseudomonas (growth between 4 and 30 °C and Flavobacterium (growth between 4 and 18 °C. In all cases, extracellular protease production was evident when reaching the stationary phase at 18 and 4 °C but was not detected at 30 °C. The zymogram revealed the secretion of one extracellular protease per isolate, each with different relative electrophoretic mobility. The extracellular proteases produced at 4 °C showed thermal activity and stability at 30 °C. Both activity and stability at a temperature higher that 10 °C have no physiological meaning because the isolates do not experience such temperatures in the Antarctic environment; however, the possible ecological value of cold-active and -stable extracellular proteases is discussed.

  6. Purification and characterization of an extracellular protease from Clonostachys rosea

    Institute of Scientific and Technical Information of China (English)

    LI Jun; HUANG Xiao-wei; ZHANG Ke-qin

    2004-01-01

    @@ An extracellular protease from Clonostachys rosea (syn. Gliocladium roseum) was purified to SDSPAGE homogeneity with 14-fold purification by ultrafiltration、 ammonium sulfate precipetation、hydrophobic interaction chromatography and anion exchange chromatography. The molecular weight of the protease was 32 kDa as estimated by SDS-PAGE. The N-terminal sequence of first 10 amino acids was A-T-Q-S-N-A-P-W-G-L. This enzyme exhibited pH and temperature optima of 9-10 and 60℃, respectively, and was stable over a wide range of pH 4-10 and temperature 4-50 ℃. It did not require Ca2+ for activity and thermal stability. Pre-incubation of enzyme with Zn2+ , Cu2+ , Hg2+,Fe3+ inhibited most of the enzyme activity, but Mn2+ increased enzyme activity up to 38%. It remained stable in the presence of Tween20, H2O2, EDTA. The inhibition profile of the enzymes by PMSF, suggested that this purified protease belongs to the serine protease family. The protease could immobilize nematodes (Panagrellus redivirus) in bioassays and hydrolyzed proteins of the purified cuticle.

  7. Isolation, purification and characterization of extracellular protease produced by marine-derived endophytic fungus Xylaria psidii KT30

    Institute of Scientific and Technical Information of China (English)

    Bugi Ratno Budiarto; Apon Zaenal Mustopa; Kustiariyah Tarman

    2015-01-01

    Objective:To isolate, purify and characterize extracellular protease produced by Xylaria psidii (X. psidii) KT30. Methods:In the present study, the extracellular protease secreted by X. psidii KT30 was isolated and purified by using three steps of protein purification, then the purified protease was characterized by applying qualitative and quantitative enzymatic assays. Results:Extracellular protease with molecular mass 71 kDa has been purified successfully by applying diethylaminoethanol-Sepharose followed by sephadex SG75 with its final specific protease activity of 0.091 IU/mg. Protease was the most active at temperature 60 °C and pH 7. The activity of enzyme was abolished mostly by phenylmethanesulfonyl fluoride, showing it is family of serine protease. Conclusions:Extracellular serine protease produced by X. psidii KT30 with good biochemical properties displayed some promising results for its further application in field of biotechnology or medicine.

  8. Isolation, purification and characterization of extracellular protease produced by marine-derived endophytic fungus Xylaria psidii KT30

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    Bugi Ratno Budiarto

    2015-01-01

    Full Text Available Objective: To isolate, purify and characterize extracellular protease produced by Xylaria psidii (X. psidii KT30. Methods: In the present study, the extracellular protease secreted by X. psidii KT30 was isolated and purified by using three steps of protein purification, then the purified protease was characterized by applying qualitative and quantitative enzymatic assays. Results: Extracellular protease with molecular mass 71 kDa has been purified successfully by applying diethylaminoethanol-Sepharose followed by sephadex SG75 with its final specific protease activity of 0.091 IU/mg. Protease was the most active at temperature 60 °C and pH 7. The activity of enzyme was abolished mostly by phenylmethanesulfonyl fluoride, showing it is family of serine protease. Conclusions: Extracellular serine protease produced by X. psidii KT30 with good biochemical properties displayed some promising results for its further application in field of biotechnology or medicine.

  9. Detection of extracellular proteases from microorganisms on agar plates

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    Alane Beatriz Vermelho

    1996-12-01

    Full Text Available We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

  10. A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4.

    Science.gov (United States)

    Sangawa, Takeshi; Nogi, Terukazu; Takagi, Junichi

    2008-10-01

    Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.

  11. Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA.

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    Martin Löwer

    Full Text Available Exported proteases of Helicobacter pylori (H. pylori are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI. Among these, we found the predicted serine protease HtrA (Hp1019, which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.

  12. Purification Crude Extracellular Protease of Saccharomycopsis fibuligera strain R64 Isolated from Tape’ Indonesian Fermented Food

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    L.B. Roostita

    2013-08-01

    Full Text Available The research aims to purify crude extracellular protease produced by Saccharomycopsis fibuligera strain R64 that isolated from Tape' Indonesian fermented food. Saccharomycopsis fibuligera strain R64 was tested on Malt Extract Agar (MEA that added 3% skim milk powder to discover the proteolytic activity. Extracellular protease purified with ammonium sulfate precipitation, dialysis and gel filtration chromatography with Sephadex G-100. Bradford method used to measure protein content, protease activity measured with Walter method and SDS-PAGE used to measure the molecule weight of protease. Results shown that isolated yeasts give 2-5 mm of clear zones on MEA that added with skim milk powder. Precipitations with 45% ammonium sulfate give the best protein content of 5.05 mg/mL and protease activity of 0.051 U/mL. After dialysis the protein content the protease activity were increased to 5.57 mg/mL and 0.0631 U/mL. Moreover, the gel filtration chromatography with sephadex G-100 yields 0.023 U/mL proteolytic activities, 2.1 mg/mL protein contents with 97.4 kD protease molecular weight.

  13. Partial Purification and Characterization of Extracellular Protease ...

    African Journals Online (AJOL)

    USER

    Purification of the enzyme by gel filtration chromatography on Sephadex G75 following ammonium sulphate precipitation gave 2.26 fold increase in purification with specific activity of 46.13 .... minutes in a water bath and were allowed to cool.

  14. Mutations affecting extracellular protease production in the filamentous fungus Aspergillus nidulans.

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    Katz, M E; Flynn, P K; vanKuyk, P A; Cheetham, B F

    1996-04-10

    The extracellular proteases of Aspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designated xprE, located on chromosome VI. The xprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations, xprF1, xprF2 and xprG1, which suppress xprE1, were characterised. Both xprF and xprG map to chromosome VII but the two genes are unlinked. The xprF1, xprF2 and xprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that the xprE1 and xprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.

  15. Production of extracellular proteases by Mucor circinelloides using D-glucose as carbon source / substrate

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    Andrade Vânia Sousa

    2002-01-01

    Full Text Available Recently, some Mucorales species have been reported as protease producers. The production of extracellular proteases by Mucor circinelloides using glucose as substrate was studied. Experiments were carried out with different D-glucose concentrations (40, 60 and 80 g/L. Biomass, pH and protease activity were determined. Although biomass production had reached best yields for the medium containing D-glucose in a concentration of 80 g/L, the enzymatic production was higher when the substrate concentration was reduced to 40 g/L. The yield factor for product on cell growth and the yield factor for product on carbon substrate were higher when the microorganism grew in medium containing 40 g/L glucose. The kinetics parameters suggest that this strain seems to be promising as an alternative microorganism for protease production.

  16. Optimized production of extracellular proteases by Bacillus subtilis from degraded abattoir waste

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    PALLAVI BADHE

    2016-04-01

    Full Text Available Proteases are ubiquitous in occurrence and are found in all living organisms. These are essential for cell growth and differentiation. The extracellular proteases are of a high commercial value and find multiple applications in various industrial sectors. The present study describes the screening of protease producing bacteria from a hitherto unexplored source i.e. degraded waste from abattoir. Three isolates were found namely yellow, white and orange coloured bacteria. Amongst them, white colored colony was found to be more suitable for protease production. The morphological, cultural, biochemical and 16S rRNA confirmed that the isolate was Bacillus subtilis. Physical and chemical parameters were optimized for maximum protease production and optimum temperature and pH was found to be 40oC at pH 14. Glucose as a carbon source and yeast extract as a nitrogen source further stimulated the production process giving maximum protease activity to be 20.74 U/ml and 20.67 U/ml. The applications of protease in detergent and solvent industry were tested and it was revealed that the purified enzyme can be used as an additive in detergent industry.

  17. Purification and characterization of a novel extracellular alkaline protease from Cellulomonas bogoriensis.

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    Li, Fan; Yang, Liyuan; Lv, Xue; Liu, Dongbo; Xia, Hongmei; Chen, Shan

    2016-05-01

    An extracellular alkaline protease produced by the alkali-tolerant Cellulomonas bogoriensis was purified by a combination of ammonium sulfate precipitation and cation exchange chromatography. The purity of the protease was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was confirmed to be 18.3 kDa. The enzyme showed optimum activity at 60 °C and pH 11. The stability of the protease was maintained at a wide temperature range of 4-60 °C and pH range of 3-12. Irreversible inhibition of the enzyme activity by phenylmethylsulfonyl fluoride and tosyl-l-phenylalanine chloromethyl ketone demonstrated that the purified enzyme is a chymotrypsin of the serine protease family. The Km and Vmax of the protease activity on casein were 19.2 mg/mL and 25000 μg/min/mg, respectively. The broad substrate specificity and remarkable stability in the presence of organic solvents, salt, and commercial detergents, as well as its excellent stain removal and dehairing capability, make the purified alkaline protease a promising candidate for industrial applications.

  18. PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR ALKALINE PROTEASE PRODUCED FROM AN ISOLATED BACILLUS SUBTILIS

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    Vijaya Bundela

    2013-03-01

    Full Text Available This paper describes the studies on the purification and partial characterization of serine alkaline protease produced through submerged fermentation process from a locally isolated Bacillus subtilis. This strain, grown in a highly alkaline medium (pH 10, produces an extracellular proteolytic enzyme. The alkaline protease was purified in a simple two-step procedure involving ammonium sulphate precipitation and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE analysis of the purified alkaline protease indicated an estimated molecular mass of 30KDa. It was more active in the range of 20-60ºC and had an optimum activity at 55ºC with optimum pH of 10.5. Characterization of the protease showed that it required certain cations such as Mg++, Mn++ and Ca++ for maximal activity. The serine nature of the alkaline protease was confirmed by PMSF inhibition. The temperature and pH stability of this Alkaline Protease from Bacillus Subtilismakes it potentially useful forindustrial applications.

  19. Extracellular acid protease from Aspergillus niger I1: purification and ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... Proteases with high activity and stability in acid pH range have .... at a flow rate of 25 ml/h after elution with the same buffer. Protein content (abs. at ..... Role of the hydrophobic amino acid residue in the bitterness of peptides.

  20. In vivo imaging of protease activity by Probody therapeutic activation.

    Science.gov (United States)

    Wong, Kenneth R; Menendez, Elizabeth; Craik, Charles S; Kavanaugh, W Michael; Vasiljeva, Olga

    2016-03-01

    Probody™ therapeutics are recombinant, proteolytically-activated antibody prodrugs, engineered to remain inert until activated locally by tumor-associated proteases. Probody therapeutics exploit the fundamental dysregulation of extracellular protease activity that exists in tumors relative to healthy tissue. Leveraging the ability of a Probody therapeutic to bind its target at the site of disease after proteolytic cleavage, we developed a novel method for profiling protease activity in living animals. Using NIR optical imaging, we demonstrated that a non-labeled anti-EGFR Probody therapeutic can become activated and compete for binding to tumor cells in vivo with a labeled anti-EGFR monoclonal antibody. Furthermore, by inhibiting matriptase activity in vivo with a blocking-matriptase antibody, we show that the ability of the Probody therapeutic to bind EGFR in vivo was dependent on protease activity. These results demonstrate that in vivo imaging of Probody therapeutic activation can be used for screening and characterization of protease activity in living animals, and provide a method that avoids some of the limitations of prior methods. This approach can improve our understanding of the activity of proteases in disease models and help to develop efficient strategies for cancer diagnosis and treatment.

  1. The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

    Science.gov (United States)

    Xie, Peng; Pan, Luqing; Xu, Wujie; Yue, Feng

    2013-09-01

    We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

  2. Cellular lysis in Bacillus subtilis; the affect of multiple extracellular protease deficiencies

    NARCIS (Netherlands)

    Stephenson, K; Bron, S; Harwood, CR

    1999-01-01

    Cellular lysis properties of strains of Bacillus subtilis deficient in the synthesis of extracellular proteases was investigated. In all cases, extracellular protease deficiency was found to increase the extent of cellular lysis of batch cultured strains following the transition to stationary phase,

  3. Cellular lysis in Bacillus subtilis; the affect of multiple extracellular protease deficiencies

    NARCIS (Netherlands)

    Stephenson, K; Bron, S; Harwood, CR

    Cellular lysis properties of strains of Bacillus subtilis deficient in the synthesis of extracellular proteases was investigated. In all cases, extracellular protease deficiency was found to increase the extent of cellular lysis of batch cultured strains following the transition to stationary phase,

  4. Semi-continuous in situ magnetic separation for enhanced extracellular protease productionmodeling and experimental validation

    DEFF Research Database (Denmark)

    Cerff, M.; Scholz, A.; Käppler, T.;

    2013-01-01

    In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS......) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations....... Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98...

  5. Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

    Science.gov (United States)

    Santos, Anderson F.; Valle, Roberta S.; Pacheco, Clarissa A.; Alvarez, Vanessa M.; Seldin, Lucy; Santos, André L.S.

    2013-01-01

    Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties. PMID:24688526

  6. Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

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    Anderson F. Santos

    2013-12-01

    Full Text Available Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9, a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i molecular masses ranging from 30 to 80 kDa, (ii better hydrolytic activities under neutral-alkaline pH range, (iii expression modulated according to the culture age, (iv susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v specific cleavage over the chymotrypsin substrate, and (vi enzymatic stability in the presence of salt (up to 20% NaCl and organic solvents (e.g., ether, isooctane and cyclohexane. The proteases described herein are promising for industrial practices due to its haloalkaline properties.

  7. Biochemical study of the extracellular aspartyl protease Eap1 from the phytopathogen fungus Sporisorium reilianum.

    Science.gov (United States)

    Mandujano-González, Virginia; Arana-Cuenca, Ainhoa; Anducho-Reyes, Miguel Ángel; Téllez-Jurado, Alejandro; González-Becerra, Aldo E; Mercado-Flores, Yuridia

    2013-12-01

    In this work, the extracellular protease Eap1 from Sporisorium reilianum was characterized in solid and liquid cultures using different culture media. The results showed that Eap1 was produced in all media and under all culture conditions, with the most activity in solid culture at an acidic pH of 3-5. Following purification, the 41 kDa protease demonstrated aspartyl protease activity. The enzyme was stable at a wide range of temperatures and pH values, but 45°C and pH 3 were optimal. The K(m) and V(max( values obtained were 0.69 mg/mL and 0.66 μmol/min, respectively, with albumin as the substrate. Eap1 degraded hemoglobin as well as proteins obtained from corn germ, roots, stems and slides at pH 3 and also had milk-clotting activity. Sequencing analysis showed that this protein has 100% similarity to the peptide sequence theoretically obtained from the sr11394 gene, which encodes an aspartyl protease secreted by S. reilianum.

  8. Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

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    Rachana Fulzele

    2011-12-01

    Full Text Available Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i motile ii rod shaped iii non spore forming iv catalase and amylase positive v able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7 exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp. The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

  9. Extracellular proteolysis of apolipoprotein E (apoE by secreted serine neuronal protease.

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    Irfan Y Tamboli

    Full Text Available Under normal conditions, brain apolipoprotein E (apoE is secreted and lipidated by astrocytes, then taken up by neurons via receptor mediated endocytosis. Free apoE is either degraded in intraneuronal lysosomal compartments or released. Here we identified a novel way by which apoE undergoes proteolysis in the extracellular space via a secreted neuronal protease. We show that apoE is cleaved in neuronal conditioned media by a secreted serine protease. This apoE cleavage was inhibited by PMSF and α1-antichymotrypsin, but not neuroserpin-1 or inhibitors of thrombin and cathepsin G, supporting its identity as a chymotrypsin like protease. In addition, apoE incubation with purified chymotrypsin produced a similar pattern of apoE fragments. Analysis of apoE fragments by mass spectrometry showed cleavages occurring at the C-terminal side of apoE tryptophan residues, further supporting our identification of cleavage by chymotrypsin like protease. Hippocampal neurons were more efficient in mediating this apoE cleavage than cortical neurons. Proteolysis of apoE4 generated higher levels of low molecular weight fragments compared to apoE3. Primary glial cultures released an inhibitor of this proteolytic activity. Together, these studies reveal novel mechanism by which apoE can be regulated and therefore could be useful in designing apoE directed AD therapeutic approaches.

  10. Variation in Extracellular Protease Production among Clinical Isolates of Staphylococcus aureus Due to Different Levels of Expression of the Protease Repressor sarA

    OpenAIRE

    Karlsson, Anna; Arvidson, Staffan

    2002-01-01

    Staphylococcus aureus produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92 S. aureus strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 s...

  11. Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes.

    Science.gov (United States)

    Palaniyandi, S A; Yang, S H; Suh, J-W

    2013-07-01

    To study the antifungal mechanism of proteases from Streptomyces phaeopurpureus strain ExPro138 towards Colletotrichum coccodes and to evaluate its utilization as biofungicide. We screened proteolytic Streptomyces strains from the yam rhizosphere with antifungal activity. Forty proteolytic Streptomyces were isolated, among which eleven isolates showed gelatinolytic activity and antagonistic activity on C. coccodes. Of the 11 isolates, protease preparation from an isolate designated ExPro138 showed antifungal activity. 16S rDNA sequence analysis of the strain showed 99% similarity with Streptomyces phaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culture filtrate revealed that the strain produced several extracellular proteases. The protease preparation inhibited spore germination, spore adhesion to polystyrene surface and appressorium formation. Microscopic study of the interaction between ExPro138 and C. coccodes revealed that ExPro138 was mycoparasitic on C. coccodes. The protease preparation also reduced anthracnose incidence on tomato fruits compared with untreated control. This study demonstrates possibility of utilizing antifungal proteases derived from antagonistic microbes as biofungicide. Microbial proteases having the ability to inhibit spore adhesion and appressorium formation could be used to suppress infection establishment by foliar fungal pathogens at the initial stages of the infection process. Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.

  12. Preparation of Antioxidant Peptides from Salmon Byproducts with Bacterial Extracellular Proteases

    Science.gov (United States)

    Wu, Ribang; Chen, Leilei; Liu, Dan; Huang, Jiafeng; Zhang, Jiang; Xiao, Xiao; Lei, Ming; Chen, Yuelin; He, Hailun

    2017-01-01

    Bacterial extracellular proteases from six strains of marine bacteria and seven strains of terrestrial bacteria were prepared through fermentation. Proteases were analyzed through substrate immersing zymography and used to hydrolyze the collagen and muscle proteins from a salmon skin byproduct, respectively. Collagen could be degraded much more easily than muscle protein, but it commonly showed weaker antioxidant capability. The hydrolysate of muscle proteins was prepared with crude enzymes from Pseudoalteromonas sp. SQN1 displayed the strongest activity of antioxidant in DPPH and hydroxyl radical scavenging assays (74.06% ± 1.14% and 69.71% ± 1.97%), but did not perform well in Fe2+ chelating assay. The antioxidant fractions were purified through ultrafiltration, cation exchange chromatography, and size exclusion chromatography gradually, and the final purified fraction U2-S2-I displayed strong activity of antioxidant in DPPH, hydroxyl radical scavenging assays (IC50 = 0.263 ± 0.018 mg/mL and 0.512 ± 0.055 mg/mL), and oxygen radical absorption capability assay (1.960 ± 0.381 mmol·TE/g). The final purified fraction U2-S2-I possessed the capability to protect plasmid DNA against the damage of hydroxyl radical and its effect was similar to that of the original hydrolysis product. It indicated that U2-S2-I might be the major active fraction of the hydrolysate. This study proved that bacterial extracellular proteases could be utilized in hydrolysis of a salmon byproduct. Compared with collagen, muscle proteins was an ideal material used as an enzymatic substrate to prepare antioxidant peptides. PMID:28085023

  13. Biotechnology of cold-active proteases.

    Science.gov (United States)

    Joshi, Swati; Satyanarayana, Tulasi

    2013-05-03

    The bulk of Earth's biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth's surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  14. Biotechnology of Cold-Active Proteases

    Directory of Open Access Journals (Sweden)

    Tulasi Satyanarayana

    2013-05-01

    Full Text Available The bulk of Earth’s biosphere is cold (<5 °C and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  15. Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

    OpenAIRE

    Cupp-Enyard, Carrie

    2009-01-01

    The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.

  16. Impact of protease activity of yeasts on wine fermentation and formation of volatile and non-volatile metabolites

    OpenAIRE

    Chomsri, Ni-orn

    2008-01-01

    Fifty yeast strains previously isolated from grapes and wines were investigated for extracellular protease activity. Saccharomyces yeasts showed no detectable protease activity, while most of the non-Saccharomyces yeasts exhibited proteolytic activity. Nine of the fifty yeast strains were cultivated in grape juice and were analysed for the production of extracellular proteases. The proteolytic activity of Metschnikowia pulcherrima (M 004) was as high as that of Rhodotolura sp. (R3). Very low ...

  17. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  18. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    Science.gov (United States)

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. The AprV5 subtilase is required for the optimal processing of all three extracellular serine proteases from Dichelobacter nodosus.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Han

    Full Text Available Dichelobacter nodosus is the principal causative agent of ovine footrot and its extracellular proteases are major virulence factors. Virulent isolates of D. nodosus secrete three subtilisin-like serine proteases: AprV2, AprV5 and BprV. These enzymes are each synthesized as precursor molecules that include a signal (pre- peptide, a pro-peptide and a C-terminal extension, which are processed to produce the mature active forms. The function of the C-terminal regions of these proteases and the mechanism of protease processing and secretion are unknown. AprV5 contributes to most of the protease activity secreted by D. nodosus. To understand the role of the C-terminal extension of AprV5, we constructed a series of C-terminal-deletion mutants in D. nodosus by allelic exchange. The proteases present in the resultant mutants and their complemented derivatives were examined by protease zymogram analysis, western blotting and mass spectrometry. The results showed that the C-terminal region of AprV5 is required for the normal expression of protease activity, deletion of this region led to a delay in the processing of these enzymes. D. nodosus is an unusual bacterium in that it produces three closely related extracellular serine proteases. We have now shown that one of these enzymes, AprV5, is responsible for its own maturation, and for the optimal cleavage of AprV2 and BprV, to their mature active forms. These studies have increased our understanding of how this important pathogen processes these virulence-associated extracellular proteases and secretes them into its external environment.

  20. Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

    Directory of Open Access Journals (Sweden)

    Evan L Pannkuk

    Full Text Available White nose syndrome (WNS is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1 was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE, broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

  1. Lipase and protease extraction from activated sludge

    DEFF Research Database (Denmark)

    Gessesse, Amare; Dueholm, Thomas; Petersen, Steffen B.

    2003-01-01

    of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination...... for the extraction of lipases and proteases from activated sludge. The sludge was continuously stirred in the presence of either buffer alone or in the presence of detergent and/or chelating agents. In all cases, a marked reduction in floc size was observed upon continuous stirring. However, no lipase activity...... and negligible protease activity was extracted in the presence of buffer alone, indicating that enzyme extraction was not due to shear force alone. The highest lipase activity was extracted using 0.1% Triton X-100 above which the activity was gradually decreasing. For proteases, the highest activity was obtained...

  2. A novel extracellular protease from Pseudomonas aeruginosa MCM B-327: enzyme production and its partial characterization.

    Science.gov (United States)

    Zambare, Vasudeo; Nilegaonkar, Smita; Kanekar, Pradnya

    2011-02-28

    The focus of this study was on production, purification and characterization of dehairing protease from Pseudomonas aeruginosa MCM B-327, isolated from vermicompost pit soil. Optimum protease activity, 395 U mL(-1), was observed in the medium containing soybean meal and tryptone, at pH 7 and 30 °C. The crude enzyme exhibited dehairing activity. As compared to chemical method, enzymatic method of dehairing showed reduction in COD, TDS and TSS by 34.28%, 37.32% and 51.58%, respectively. Zymogram of crude enzyme on native-PAGE presented two bands with protease activity of molecular weights of 56 and 67 kDa. Both proteases showed dehairing activity. Out of these, 56kDa protease (PA02) was purified 3.05-folds with 2.71% recovery. The enzyme was active in pH range 7-9 and temperature 20-50 °C with optimum pH of 8 and temperature 35°C. Moreover, the enzyme activity of PA02 protease was not strongly inhibited by specific inhibitor showing the novel nature of enzyme compared to serine, cysteine, aspartyl and metalloproteases. Kinetic studies indicated that substrate specificity of PA02 protease was towards various natural and synthetic proteolytic substrates but inactive against collagen and keratin. These findings suggest protease secreted by P. aeruginosa MCM B-327 may have application in dehairing for environment-friendly leather processing.

  3. Production of extracellular protease by a Brazilian strain of Beauveria bassiana reactivated on coffee berry borer, Hypothenemus hampei

    Directory of Open Access Journals (Sweden)

    Eliana Tiemi Ito

    2007-03-01

    Full Text Available Studies were carried out on extracellular protease production by Beauveria bassiana CG432 in liquid medium containing glucose and yeast extract. B. Bassiana presented active growth after lag period of 24 h., produced 80% of the total of the extracellular protease activity in 48 h which was maximum on the 5th culture day. The extracellular protease presented optimum activity at 60ºC, was stable up to 1M Cl-, maintained the stability during 15 day at 4ºC and -18ºC, but was not stable if frozen repeatedly.A cepa brasileira do fungo entomopatogênico Beauveria bassiana CG432 foi cultivada em meio de cultivo líquido contendo glucose e extrato de levedura a 28°C, 150 rpm, durante 9 dias utilizando 1% de inóculo constituído por 10(8conídios/mL pré-ativados em insetos vivos de broca-do-café. Avaliou-se diariamente o crescimento do fungo através da quantificação da biomassa e a atividade de proteases extracelulares no filtrado do cultivo pela determinação de peptídeos solúveis liberados pela hidrólise de soro albumina bovina a 37°C durante 30minutos. O fungo cresceu ativamente após reduzido período lag de 24 horas, produziu 80% da atividade total de proteases em 48 horas e atingiu produção máxima em 5 dias de cultivo, sem adição de substratos indutores no meio de cultivo. As proteases apresentaram atividade ótima a 60ºC, estabilidade até 1M de íons cloreto, mantiveram mais de 80% de atividade durante 15 dias a 4ºC e -18ºC, porém foram sensíveis a congelamentos repetitivos.

  4. Cysteine proteases: Modes of activation and future prospects as pharmacological targets

    Directory of Open Access Journals (Sweden)

    Sonia eVerma

    2016-04-01

    Full Text Available Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria and parasite to the higher organisms (mammals. Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases and metallo-proteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a pro-domain (regulatory and a mature domain (catalytic. The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases.

  5. Detection of Extracellular Enzyme Activities in Ganoderma neo-japonicum

    OpenAIRE

    Jo, Woo-Sik; Park, Ha-Na; Cho, Doo-Hyun; Yoo, Young-Bok; Park, Seung-Chun

    2011-01-01

    The ability of Ganoderma to produce extracellular enzymes, including β-glucosidase, cellulase, avicelase, pectinase, xylanase, protease, amylase, and ligninase was tested in chromogenic media. β-glucosidase showed the highest activity, among the eight tested enzymes. In particular, Ganoderma neo-japonicum showed significantly stronger activity for β-glucosidase than that of the other enzymes. Two Ganoderma lucidum isolates showed moderate activity for avicelase; however, Ganoderma neo-japonic...

  6. DEGUMMING OF RAW SILK FABRIC WITH HELP OF MARINE EXTRACELLULAR PROTEASE

    Directory of Open Access Journals (Sweden)

    Das Sumana

    2013-01-01

    Full Text Available Protease secreting microbe was isolated and characterized on the basis of their morphological, biochemical, physiological and 16S rDNA based molecular properties. The extracellular protease was quantified and characterized. Protease was used for different time (4, 8, 12 and 24 h at different temperature (RT and 37°C for optimization of the degumming process for raw silk fabric with enzyme dosage (0.2-1 unit/cm2 of fabric. Post-enzymatic treatment, the fabric quality and texture was compared with conventionally treated as well as untreated fabric in terms of degumming loss, tensile strength and yarn count and colour fastness to light/water. The isolate SM1 (Bacillus thuringensis was able to grow in Carbon Minimal Salt Medium (CMSM with jaggery or tamarind as the carbon source (0.3% w/v. Energy Dispersive X-Ray Fluorescense (EDXRF data showed intracellular accumulation of heavy metal by the isolate. Extracellular protease was able to degum silk fabric within 4 h at RT with enzyme concentration of 0.8unit/cm2 and the maximum degumming loss was 21.72%. Post enzymatic degumming, a shiny texture was observed under Environmental Scanning Electron Microscope (ESEM and the yarn volume also increased. Utilization of CMSM made the process cost effective during large scale application. Intracellular metal accumulation and growth in a wide range of temperature and pH made the isolate a potential candidate for bioremediation. Extracellular protease with significant degumming property could be used as an eco friendly approach as compared to the conventional chemical treatment.

  7. Identification and properties of two extracellular proteases from Brevundimonas diminuta Identificação e propriedades de duas proteases extracelulares de Brevundimonas diminuta

    Directory of Open Access Journals (Sweden)

    André Adriano Chaia

    2000-03-01

    Full Text Available Extracellular proteases from Brevundimonas diminuta (syn. Pseudomonas diminuta were studied in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE containing a copolymerized substrate. Two proteases were detected migrating at 67 kDa and 50 kDa: both of them hydrolysed preferentially gelatin, but casein was also degraded and a slight hydrolysis was observed with hemoglobin. No detectable extracellular proteolytic activity was found in bovine serum albumin-containing gels. The optima temperature and pH for proteolytic activity were between 40ºC and 50ºC in a pH ranging from 7.0 to 11.0, respectively. These enzymes were isolated by analytical high performance liquid chromatography (HPLC. Protease assays with the synthetic substrate Z-Phe-Arg-MCA and the inhibitors EGTA, EDTA and 1, 10 phenanthroline point out that these enzymes are metalloproteases.Proteases extracelulares de Brevundimonas diminuta (Pseudomonas diminuta foram identificadas e caracterizadas por eletroforese em gel de poliacrilamida com dodecilsulfato de sódio, contendo um substrato co-polimerizado. Duas proteases foram detectadas migrando em 67 kDa e 50 kDa: ambas hidrolisaram preferencialmente a gelatina, embora a caseína também tenha sido degradada e uma pequena hidrólise tenha sido observada com hemoglobina. Nenhuma atividade proteolítica extracelular foi detectada nos géis contendo soro albumina bovina. Condições ótimas de temperatura e pH para a atividade proteolítica foram observadas entre 40ºC e 50ºC e numa faixa de pH que variou de 7,0 a 11,0, respectivamente. Essas enzimas foram isoladas por cromatografia líquida de alta resolução. Os ensaios enzimáticos com o substrato sintético Z-Phe-Arg-MCA e com os inibidores EGTA, EDTA e 1, 10 fenantrolina indicam que essas enzimas são metaloproteases.

  8. Activation of ADAM 12 protease by copper

    DEFF Research Database (Denmark)

    Loechel, F; Wewer, Ulla M.

    2001-01-01

    Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency......: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism...

  9. Lipase and protease extraction from activated sludge

    DEFF Research Database (Denmark)

    Gessesse, Amare; Dueholm, Thomas; Petersen, Steffen B.

    2003-01-01

    In the process of wastewater treatment hydrolysis of polymeric substances is the first and rate-limiting step. A closer study of the enzymes catalysing these reactions is essential for a better understanding of the microbial activity in the wastewater treatment process. Therefore, development...... of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination...... and negligible protease activity was extracted in the presence of buffer alone, indicating that enzyme extraction was not due to shear force alone. The highest lipase activity was extracted using 0.1% Triton X-100 above which the activity was gradually decreasing. For proteases, the highest activity was obtained...

  10. Identification of a cell-bound extracellular protease overproduced by Sulfolobus solfataricus in peptide-rich media.

    Science.gov (United States)

    Cannio, Raffaele; Catara, Giuliana; Fiume, Imma; Balestrieri, Marco; Rossi, Mosè; Palmieri, Gianna

    2010-01-01

    A new protease, named SsMTP was identified from the archeon Sulfolobus solfataricus. The enzyme is associated to the cell-membrane and over-produced in response to the peptide-enriched media. SsMTP has a molecular mass of 120 kDa showing optimal activity at pH 2.0 in the temperature range 70 - 90 degrees C, and a half-life of 20 days at 80 degrees C. Primary structure analysis revealed that SsMTP represents a novel type of multi-domain thermopsin-like protease containing the catalytic domain followed by two distinct domains, PKD and Y_Y_Y, which are usually involved in a range of protein-protein interactions among the extracellular proteins.

  11. Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor.

    Science.gov (United States)

    Ju, Shouyong; Lin, Jian; Zheng, Jinshui; Wang, Shaoying; Zhou, Hongying; Sun, Ming

    2016-01-29

    Root knot nematodes (RKNs) are the world's most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs.

  12. Protease Inhibitors from Plants with Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Yoonkyung Park

    2009-06-01

    Full Text Available Antimicrobial proteins (peptides are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides. Plants produce a variety of proteins (peptides that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins. Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents.

  13. Production of extracellular alkaline protease from Bacillus subtilis RSKK96 with solid state fermentation

    Directory of Open Access Journals (Sweden)

    Nurullah Akcan

    2011-09-01

    Full Text Available The production of extracellular alkaline protease by producing Bacillus subtilis RSKK96 was studied with solid state fermentation (SSF. Different agro residues as substrate were studied for enzyme production. The highest enzyme production was expressed with lentil husk as units per mass of dry substrate (3937.0 U/mg. Production parameters were optimized as incubation time 120 h, extraction medium Triton-X100 1%, initial moisture content 30%, initial pH 9.0. The high level of alkaline protease was obtained in the medium containing arabinose followed by lactose, galactose, and fructose. Among various nitrogen sources, beef extract was found to be the best inducer of alkaline protease, while other nitrogen sources repressed enzyme production. Among metal salts FeSO4.7H2O and MgSO4.7H2O was found to increase protease production. The maximum enzyme production (5759.2 U/mg was observed with lentil husk in 1000 mL of fermentation medium volume.

  14. Optimal conditions for production of extracellular protease from newly isolated Bacillus cereus strain CA15

    Directory of Open Access Journals (Sweden)

    Fikret Uyar

    2011-02-01

    Full Text Available An alkaline protease producer Bacillus sp. strain CA15 was isolated from soil. The microorganism was found to be closely related to Bacillus cereus based on 16S ribosomal DNA sequencing. The culture conditions for higher protease production were optimized with respect to carbon and nitrogen sources, metal ions, pH and temperature. Maximum protease production was obtained in the medium supplemented with 1% skim milk, 1% starch and 0.6% MgSO4.7H2O, initial pH 8.0 at 35oC. The best enzyme production was obtained during the stationary phase in which the cell density reached to 1.8x108 cells/mL. The level of protease was found to be low in the presence of inorganic nitrogen sources. The protease production was diminished in the presence of sucrose and lactose. The extreme stability towards Triton X-100, Tween 20 and SDS was observed by Bacillus sp. CA15 alkaline protease. The enzyme activity was inhibited by PMSF suggested that presence of serine residues at the active sites.

  15. Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Carolina Piña-Vázquez

    2012-01-01

    Full Text Available Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina. The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa.

  16. Protease activities of rumen protozoa.

    Science.gov (United States)

    Forsberg, C W; Lovelock, L K; Krumholz, L; Buchanan-Smith, J G

    1984-01-01

    Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids. PMID:6364968

  17. Optimization Conditions of Extracellular Proteases Production from a Newly Isolated Streptomyces Pseudogrisiolus NRC-15

    Directory of Open Access Journals (Sweden)

    El-Sayed E. Mostafa

    2012-01-01

    Full Text Available Microbial protease represents the most important industrial enzymes, which have an active role in biotechnological processes. The objective of this study was to isolate new strain of Streptomyces that produce proteolytic enzymes with novel properties and the development of the low-cost medium. An alkaline protease producer strain NRC-15 was isolated from Egyptian soil sample. The cultural, morphological, physiological characters and chemotaxonomic evidence strongly indicated that the NRC-15 strain represents a novel species of the genus Streptomyces, hence the name Strptomyces pseudogrisiolus NRC-15. The culture conditions for higher protease production by NRC-15 were optimized with respect to carbon and nitrogen sources, metal ions, pH and temperature. Maximum protease production was obtained in the medium supplemented with 1% glucose, 1% yeast extract, 6% NaCl and 100 μmol/L of Tween 20, initial pH 9.0 at 50 °C for 96 h. The current results confirm that for this strain, a great ability to produce alkaline proteases, which supports the use of applications in industry.

  18. Tumor necrosis factor-α and receptor activator of nuclear factor-κB ligand augment human macrophage foam-cell destruction of extracellular matrix through protease-mediated processes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Larsen, Lise;

    2012-01-01

    component of extracellular matrix (ECM) in plaques, and to establish whether the pro-inflammatory molecules, tumor necrosis factor (TNF)-alpha, and receptor activator of nuclear factor-κB ligand (RANK-L) increase this degradation. CD14+ monocytes isolated from peripheral blood were differentiated......% and 72%, respectively. This is, to our knowledge, the first data describing a simple in vitro system in which macrophage foam cells degradation of matrix proteins can be monitored. This degradation can be enhanced by cytokines since TNF-alpha and RANK-L significantly increased the matrix degradation...

  19. Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

    Science.gov (United States)

    2010-01-01

    Background The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels. Results Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects. Conclusion Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might

  20. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    Science.gov (United States)

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

  1. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    Science.gov (United States)

    Park, Mi Kyung; Cho, Min Kyoung; Kang, Shin Ae; Park, Hye-Kyung; Kim, Dong-Hee; Yu, Hak Sun

    2014-01-01

    Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES) proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25) in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF) inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  2. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    Directory of Open Access Journals (Sweden)

    Mi Kyung Park

    Full Text Available Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25 in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  3. Structural determinants of MALT1 protease activity.

    Science.gov (United States)

    Wiesmann, Christian; Leder, Lukas; Blank, Jutta; Bernardi, Anna; Melkko, Samu; Decock, Arnaud; D'Arcy, Allan; Villard, Frederic; Erbel, Paulus; Hughes, Nicola; Freuler, Felix; Nikolay, Rainer; Alves, Juliano; Bornancin, Frederic; Renatus, Martin

    2012-05-25

    The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.

  4. Biased signaling by peptide agonists of protease activated receptor 2.

    Science.gov (United States)

    Jiang, Yuhong; Yau, Mei-Kwan; Kok, W Mei; Lim, Junxian; Wu, Kai-Chen; Liu, Ligong; Hill, Timothy A; Suen, Jacky Y; Fairlie, David P

    2017-02-07

    Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

  5. Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function.

    Science.gov (United States)

    Watanabe, Masanori; Techapun, Charin; Kuntiya, Ampin; Leksawasdi, Noppol; Seesuriyachan, Phisit; Chaiyaso, Thanongsak; Takenaka, Shinji; Maeda, Isamu; Koyama, Masahiro; Nakamura, Kozo

    2017-02-01

    A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the CC and CO stretching and OH group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extracellular protease.

  6. Purification and properties of detergent-compatible extracellular alkaline protease from Scopulariopsis spp.

    Science.gov (United States)

    Niyonzima, Francois Niyongabo; More, Sunil

    2014-10-03

    A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0-12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co(2+) and Mn(2+) at 10 mM but was unaffected by Ba(2+), Mg(2+), Cu(2+), Na(+), K(+), and Fe(2+). Ca(2+) and Fe(3+) moderately reduced the activity (∼18%); however, a reduction of about 40% was seen for Zn(2+) and Hg(2+). The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H2O2 and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (-20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel-enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity

  7. Detection of protease and protease activity using a single nanoscrescent SERS probe

    Science.gov (United States)

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2013-01-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  8. Extracellular Lipase and Protease Production from a Model Drinking Water Bacterial Community Is Functionally Robust to Absence of Individual Members.

    Directory of Open Access Journals (Sweden)

    Graham G Willsey

    Full Text Available Bacteria secrete enzymes into the extracellular space to hydrolyze macromolecules into constituents that can be imported for microbial nutrition. In bacterial communities, these enzymes and their resultant products can be modeled as community property. Our goal was to investigate the impact of individual community member absence on the resulting community production of exoenzymes (extracellular enzymes involved in lipid and protein hydrolysis. Our model community contained nine bacteria isolated from the potable water system of the International Space Station. Bacteria were grown in static conditions individually, all together, or in all combinations of eight species and exoproduct production was measured by colorimetric or fluorometric reagents to assess short chain and long chain lipases, choline-specific phospholipases C, and proteases. The exoenzyme production of each species grown alone varied widely, however, the enzyme activity levels of the mixed communities were functionally robust to absence of any single species, with the exception of phospholipase C production in one community. For phospholipase C, absence of Chryseobacterium gleum led to increased choline-specific phospholipase C production, correlated with increased growth of Burkholderia cepacia and Sphingomonas sanguinis. Because each individual species produced different enzyme activity levels in isolation, we calculated an expected activity value for each bacterial mixture using input levels or known final composition. This analysis suggested that robustness of each exoenzyme activity is not solely mediated by community composition, but possibly influenced by bacterial communication, which is known to regulate such pathways in many bacteria. We conclude that in this simplified model of a drinking water bacterial community, community structure imposes constraints on production and/or secretion of exoenzymes to generate a level appropriate to exploit a given nutrient environment.

  9. 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.

  10. Heighten the Study on Factor Seven Activating Protease

    Institute of Scientific and Technical Information of China (English)

    贺石林; 陈方平; 张广森; 文志斌

    2008-01-01

    @@ Recent studies have showed that factor seven activating protease (FSAP) is a novel serine protease in human plasma. Immunoreactivity for FSAP has been observed in vascular endothelial cells,epithelial cells and macrophages but FSAP-specific mRNA expression only exists in the former two cells. FSAP has three epidermal growth factor (EGF) domains,a kringle domain and a serine protease domain.

  11. Amylase,. beta. -glucanase and protease activities from a mutant of Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Yin, X.S.; Li, Y.X. (Wuxi Inst. of Light Industry, JS (China). Dept. of Fermentation Technology); Stark, J.R. (International Centre for Brewing and Distilling, Edinburgh (UK))

    1991-10-01

    Enzymes in the shake culture of a mutant of Bacillus subtilis developed mainly during the stationary phase of growth. Extracellular {alpha}-amylase and protease activities increased and then declined to relatively low levels after 48 h of incubation while the {beta}-glucanase activity remained high. The production of extracellular proteolytic activity commenced only when the low molecular weight nitrogen source in the medium was completely consumed. Enzymes were fractionated by ion-exchange chromatography and the molecular weights of {beta}-glucanase, {alpha}-amylase, protease I and protease II were estimated by gel filtration to be 1.3x10{sup 4}, 3.2x10{sup 4}, 6.3x10{sup 3} and 2.0x10{sup 4}, respectively. The {beta}-glucanase and {alpha}-amylase showed maximum activities at around pH 7, but the two proteases had different pH optima (pH 6-7 and 8.5, respectivelty). The presence of proteolytic activity in the enzyme preparation had a significant effect on the stability of the {beta}-glucanase and the {alpha}-amylase at 65deg C. (orig.).

  12. Presence of a lethal protease in the extracellular products of Vibrio splendidus-Vibrio lentus related strains.

    Science.gov (United States)

    Farto, R; Armada, S P; Montes, M; Perez, M J; Nieto, T P

    2006-12-01

    The presence of a lethal extracellular 39-kDa protease, a virulence determinant of a Listonella pelagia strain which produces vibriosis in turbot, was determined in the extracellular products (ECP) of 33 Vibrionaceae strains. Both immunological and enzymatic techniques distinguished this specific protease from other Vibrionaceae proteins. It was detected in 15% (5/33) of the ECPs assayed belonging to strains of the Vibrio splendidus-V. lentus related group isolated in Galician aquaculture systems (NW Spain). As these strains were associated with diseased octopus and cultured turbot, were able to colonize the internal organs of fish and produced a lethal ECP for fish, they are a potential risk for the health of reared aquatic organisms.

  13. Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii.

    Science.gov (United States)

    Serrano-Luna, José de Jesús; Cervantes-Sandoval, Isaac; Calderón, Jesús; Navarro-García, Fernando; Tsutsumi, Victor; Shibayama, Mineko

    2006-01-01

    Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.

  14. Immunoglobulin A1 protease activity in Gemella haemolysans

    DEFF Research Database (Denmark)

    Lomholt, JA; Kilian, Mogens

    2000-01-01

    The purpose of this study was to determine the occurrence and nature of immunoglobulin A1 (IgA1) protease activity in members of the genus Gemella and related taxa. Among a total of 22 Gemella strains belonging to the four species Gemella haemolysans, Gemella morbillorum, Gemella sanguinis......, and Gemella bergeriae and four reference strains of the species Helcococcus kunzii, Facklamia hominis, and Globicatella sanguinis, IgA1 protease activity was an exclusive character of all nine isolates of G. haemolysans. The IgA1 protease of G. haemolysans appears to be a metallo-type IgA1 protease...... that cleaves the Pro(227)-Thr(228) peptide bond in the hinge region of the alpha1 chain like that of several Streptococcus species. Phenotypic characterization of the isolates demonstrates that screening for IgA1 protease activity provides a valuable means for species differentiation in this group of bacteria....

  15. Effect of pH and temperature on stability and kinetics of novel extracellular serine alkaline protease (70 kDa).

    Science.gov (United States)

    Bhunia, Biswanath; Basak, Bikram; Mandal, Tamal; Bhattacharya, Pinaki; Dey, Apurba

    2013-03-01

    A novel extracellular serine protease (70 kDa by SDS-PAGE) was purified and characterized. This enzyme retained more than 93% of its initial activity after preincubation for 30 min at 37 °C in the presence of 25% (v/v) tested organic solvents and showed feather degradation activity. The purified enzyme was deactivated at various combinations of pH and temperature to examine the interactive effect of them on enzyme activity. The deactivation process was modeled as first-order kinetics and the deactivation rate constant (k(d)) was found to be minimum at pH 9 and 37 °C. The kinetic analysis of enzyme over a range of pH values indicated two pK values at 6.21 and at 10.92. The lower pK value was likely due to the catalytic histidine in the free enzyme and higher pK value likely reflected deprotonation of the proline moiety of the substrate but ionization of the active site serine is another possibility. Inhibition kinetic showed that enzyme is serine protease because enzyme was competitively inhibited by antipain and aprotinin as these compounds are known to be competitive inhibitors of serine protease. The organic solvent, thermal and pH tolerances of enzyme suggested that it may have potential for use as a biocatalyst in industry.

  16. Protease activity, localization and inhibition in the human hair follicle.

    Science.gov (United States)

    Bhogal, R K; Mouser, P E; Higgins, C A; Turner, G A

    2014-02-01

    In humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials. We measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin. Our data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen) and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (U.K., Brazil, China, first-generation Mexicans in the U.S.A., Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system. These studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen and climbazole. This technology may have potential to reduce excessive hair shedding. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  17. Allergens with Protease Activity from House Dust Mites

    Directory of Open Access Journals (Sweden)

    Manuel Reithofer

    2017-06-01

    Full Text Available Globally, house dust mites (HDM are one of the main sources of allergens causing Type I allergy, which has a high risk of progressing into a severe disabling disease manifestation such as allergic asthma. The strong protease activities of a number of these allergens are thought to be involved in several steps of the pathophysiology of this allergic disease. It has been a common notion that protease activity may be one of the properties that confers allergenicity to proteins. In this review we summarize and discuss the roles of the different HDM proteases in the development of Type I allergy.

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

    Science.gov (United States)

    Fyfe, Cameron D; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W; Cogdell, Richard J; Wall, Daniel M; Burchmore, Richard J S; Byron, Olwyn; Walker, Daniel

    2015-07-01

    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.

  19. Useful Extracellular Activity of Bacteria Isolated from Bhitarkanika Mangrove Ecosystem of Orissa Coast

    Directory of Open Access Journals (Sweden)

    Gupta, N.

    2007-01-01

    Full Text Available This paper describes the isolation of bacteria from Bhitarkanika mangrove ecosystem of Orissa and screening for their antifungal properties against five pathogenic fungi, extracellular enzyme activity like amylase, protease and lipase, and phosphate solubilization capacity. From 567 bacterial isolates obtained, 26 bacterial isolates have exhibited wide spectrum antifungal activity against all five fungi tested. Most of the bacterial isolates were found to be amylase and protease producers. In present study, 41 lipase producers and 33 phosphate solubilisers were also found. Bacterial isolates from plant origin exhibited all the four extracellular enzyme activity except lipase. The maximum % occurrence of phosphate solubilisers was found in soil and plant system of mangrove. This screening study opens an avenue to work with some of the potent strains for useful product formation at large scale.

  20. Different patterns of extracellular proteolytic activity in W303a and BY4742 Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Seredyński, Rafał; Wolna, Dorota; Kędzior, Mateusz; Gutowicz, Jan

    2017-01-01

    Protease secretion in Saccharomyces cerevisiae cultures is a complex process, important for the application of this organism in the food industry and biotechnology. Previous studies provide rather quantitative data, yielding no information about the number of enzymes involved in proteolysis and their individual biochemical properties. Here we demonstrate that W303a and BY4742 S. cerevisiae strains reveal different patterns of spontaneous and gelatin-induced extracellular proteolytic activity. We applied the gelatin zymography assay to track changes of the proteolytic profile in time, finding the protease secretion dependent on the growth phase and the presence of the protein inducer. Detected enzymes were characterized regarding their substrate specificity, pH tolerance, and susceptibility to inhibitors. In case of the W303a strain, only one type of gelatin-degrading secretory protease (presumably metalloproteinase) was observed. However, the BY4742 strain secreted different proteases of the various catalytic types, depending on the substrate availability. Our study brings the evidence that S. cerevisiae strains secrete several kinds of proteases depending on the presence and type of the substrate. Protein induction may cause not only quantitative but also qualitative changes in the extracellular proteolytic patterns.

  1. Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

    Institute of Scientific and Technical Information of China (English)

    Zeng Yinxin; Yu Yong; Chen Bo; Li Huirong

    2004-01-01

    The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.

  2. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A. (UMASS, MED)

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  3. 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.

  4. Secreted proteases. Regulation of their activity and their possible role in metastasis.

    Science.gov (United States)

    Goldberg, G I; Frisch, S M; He, C; Wilhelm, S M; Reich, R; Collier, I E

    1990-01-01

    Extracellular matrix metalloproteases are secreted by the resident cells of the tissue in a proenzyme form, and their extracellular activity is regulated at the level of gene expression, proenzyme activation, and interaction with inhibitors. To understand the molecular mechanisms that control the activity of ECM metalloproteases and their effect on the cellular phenotype, we have established cell lines in which the transcription of the protease genes is repressed. We also have undertaken a detailed study of the pathway of extracellular activation of interstitial procollagenase. Stable transfection of three human tumor cell lines--H-ras-transformed bronchial epithelial cells TBE-1, fibrosarcoma cells HT1080, and melanoma cells A2058--with the adenovirus E1A gene dramatically repressed the expression of the secreted proteases, type IV and interstitial collagenases, and urokinase-type plasminogen activator. Concomitantly, E1A-expressing cells showed reduced metastatic activity in vivo and reduced ability to traverse a reconstituted basement membrane in vitro. Monospecific anti-type IV collagenase antibody inhibited the invasive activity of parental tumor cell lines in the in vitro system, suggesting a possible causal relationship between the effect of E1A on the expression of secreted proteases and the reduced metastatic potential of the E1A-expressing transformants. We have also studied the mechanism of regulation of metalloprotease activity at the level of extracellular activation by investigating the cascade of proteolytic events that results in the activation of interstitial procollagenase. Cocultivation of the major cellular components of skin, dermal fibroblasts, and epidermal keratinocytes induces activation of interstitial procollagenase and prostromelysin in the presence of plasminogen. This activation occurs through a uPA-plasmin-dependent pathway in which plasmin catalyzes the first step in activation of both collagenase and stromelysin by amino

  5. Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

    Directory of Open Access Journals (Sweden)

    Abdelnasser S.S. Ibrahim

    2015-05-01

    Conclusions: A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.

  6. Trichosporon inkin biofilms produce extracellular proteases and exhibit resistance to antifungals.

    Science.gov (United States)

    de Aguiar Cordeiro, Rossana; Serpa, Rosana; Flávia Uchoa Alexandre, Camila; de Farias Marques, Francisca Jakelyne; Vladia Silva de Melo, Charlline; da Silva Franco, Jônatas; José de Jesus Evangelista, Antonio; Pires de Camargo, Zoilo; Samia Nogueira Brilhante, Raimunda; Fabio Gadelha Rocha, Marcos; Luciano Bezerra Moreira, José; de Jesus Pinheiro Gomes Bandeira, Tereza; Júlio Costa Sidrim, José

    2015-11-01

    The aim of this study was to determine experimental conditions for in vitro biofilm formation of clinical isolates of Trichosporon inkin, an important opportunistic pathogen in immunocompromised patients. Biofilms were formed in microtitre plates in three different media (RPMI, Sabouraud and CLED), with inocula of 104, 105 or 106 cells ml- 1, at pH 5.5 and 7.0, and at 35 and 28 °C, under static and shaking conditions for 72 h. Growth kinetics of biofilms were evaluated at 6, 24, 48 and 72 h. Biofilm milieu analysis were assessed by counting viable cells and quantification of nucleic acids released into biofilm supernatants. Biofilms were also analysed for proteolytic activity and antifungal resistance against amphotericin B, caspofungin, fluconazole, itraconazole and voriconazole. Finally, ultrastructural characterization of biofilms formed in microtitre plates and catheter disks was performed by scanning electron microscopy. Greater biofilm formation was observed with a starter inoculum of 106 cells ml- 1, at pH 7.0 at 35 °C and 80 r.p.m., in both RPMI and Sabouraud media. Growth kinetics showed an increase in both viable cells and biomass with increasing incubation time, with maximum production at 48 h. Biofilms were able to disperse viable cells and nucleic acids into the supernatant throughout the developmental cycle. T. inkin biofilms produced more protease than planktonic cells and showed high tolerance to amphotericin B, caspofungin and azole derivatives. Mature biofilms were formed by different morphotypes, such as blastoconidia, arthroconidia and hyphae, in a strain-specific manner. The present article details the multicellular lifestyle of T. inkin and provides perspectives for further research.

  7. Impact of individual extracellular proteases on Staphylococcus aureus biofilm formation in diverse clinical isolates and their isogenic sarA mutants.

    Science.gov (United States)

    Loughran, Allister J; Atwood, Danielle N; Anthony, Allison C; Harik, Nada S; Spencer, Horace J; Beenken, Karen E; Smeltzer, Mark S

    2014-12-01

    We demonstrate that the purified Staphylococcus aureus extracellular proteases aureolysin, ScpA, SspA, and SspB limit biofilm formation, with aureolysin having the greatest impact. Using protease-deficient derivatives of LAC, we confirmed that this is due to the individual proteases themselves. Purified aureolysin, and to a lesser extent ScpA and SspB, also promoted dispersal of an established biofilm. Mutation of the genes encoding these proteases also only partially restored biofilm formation in an FPR3757 sarA mutant and had little impact on restoring virulence in a murine bacteremia model. In contrast, eliminating the production of all of these proteases fully restored both biofilm formation and virulence in a sarA mutant generated in the closely related USA300 strain LAC. These results confirm an important role for multiple extracellular proteases in S. aureus pathogenesis and the importance of sarA in repressing their production. Moreover, purified aureolysin limited biofilm formation in 14 of 15 methicillin-resistant isolates and 11 of 15 methicillin-susceptible isolates, while dispersin B had little impact in UAMS-1, LAC, or 29 of 30 contemporary isolates of S. aureus. This suggests that the role of sarA and its impact on protease production is important in diverse strains of S. aureus irrespective of their methicillin resistance status.

  8. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing....... These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host....

  9. Factor VII activating protease. Single nucleotide polymorphisms light the way.

    Science.gov (United States)

    Kanse, S M; Etscheid, M

    2011-08-01

    Factor VII activating protease (FSAP) is a circulating serine protease with high homology to fibrinolytic enzymes. A role in the regulation of coagulation and fibrinolysis is suspected based on in vitro studies demonstrating activation of FVII or pro-urokinase plasminogen activator (uPA). However, considering the paucity of any studies in animal models or any correlative studies in humans the role of FSAP in haemostasis remains unclear. In relation to vascular remodeling processes or inflammation it has been convincingly shown that FSAP interacts with growth factors as well as protease activated receptors (PAR). Against this sparse background there are a plethora of studies which have investigated the linkage of single nucleotide polymorphisms (SNP) in the FSAP gene (HABP2) to various diseases. The G534E SNP of FSAP is associated with a low proteolytic activity due to an amino acid exchange in the protease domain. This and other SNPs have been linked to carotid stenosis, stroke as well as thrombosis in the elderly and plaque calcification. These SNP analyses indicate an important role for FSAP in the regulation of the haemostasis system as well as fibroproliferative inflammatory processes.

  10. Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

    DEFF Research Database (Denmark)

    Christiansen, Torben; Nielsen, Jens

    2002-01-01

    for product formation. The dynamics of the production of Savinuse(R) were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate...... was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific......The production of the extracellular alkaline protease Savinase(R) (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum...

  11. Purification, crystallization and preliminary X-ray diffraction analysis of the Staphylococcus epidermidis extracellular serine protease Esp.

    Science.gov (United States)

    Vengadesan, Krishnan; Macon, Kevin; Sugumoto, Shinya; Mizunoe, Yoshimitsu; Iwase, Tadayuki; Narayana, Sthanam V L

    2013-01-01

    Esp, an extracellular serine protease from Staphylococcus epidermidis, has been shown to inhibit S. aureus biofilm formation and nasal colonization. The full-length 27 kDa pro-Esp was purified and digested with thermolysin to obtain mature Esp. The mature Esp containing 216 residues crystallized in space group P2(1), with unit-cell parameters a = 39.5, b = 61.2, c = 42.5 Å, β = 98.2° and one molecule in the asymmetric unit, with an estimated solvent content of 42%. A diffraction data set has been collected to 1.8 Å resolution on a rotating-anode home-source facility.

  12. Operating Conditions Effects Onenzyme Activity: Case Enzyme Protease

    Directory of Open Access Journals (Sweden)

    Adel Oueslati

    2014-09-01

    Full Text Available The Proteases an enzyme added to detergents to degrade the protein spots origin.Their action is manifested through its activity the middle of washing clothes. This activity depends on the operating conditions. In this article, the effects of temperature and pH of the reaction and the substrate concentration and time of washing medium on the enzyme activity were studied. There action mechanism has been shown. The activity measurements were made by absorption spectrometry

  13. Activity profiling of papain-like cysteine proteases in plants

    NARCIS (Netherlands)

    Hoorn, van der R.A.L.; Leeuwenburgh, M.A.; Bogyo, M.; Joosten, M.H.A.J.; Peck, S.C.

    2004-01-01

    Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttransl

  14. Intervention with Serine Protease Activity with Small Peptides

    DEFF Research Database (Denmark)

    Xu, Peng

    2015-01-01

    , plasma kallikrein, which contributes to the pathogenesis in hereditary angioedema. According to the X-ray crystal structure analysis, we proposed a principle for designing inhibitors of other serine proteases from mupain-1. In order to be able to evaluate the inhibitory activities of our peptides in vivo...

  15. LPS counter regulates RNA expression of extracellular proteases and their inhibitors in murine macrophages

    DEFF Research Database (Denmark)

    Hald, Andreas; Rønø, Birgitte; Lund, Leif R

    2012-01-01

    in extracellular matrix metabolism in the mouse derived-macrophage cell line RAW 264.7 following stimulation with LPS. Our results revealed that LPS induces the expression of matrix metalloproteinases while at the same time decreased the expression of matrix metalloproteinase inhibitors. The opposite scenario...

  16. Extracellular polymers of ozonized waste activated sludge.

    Science.gov (United States)

    Liu, J C; Lee, C H; Lai, J Y; Wang, K C; Hsu, Y C; Chang, B V

    2001-01-01

    Effect of ozonation on characteristics of waste activated sludge was investigated in the current study. Concentrations of cell-bound extracellular polymers (washed ECPs) did not change much upon ozonation, whereas the sum of cell-bound and soluble extracellular polymers (unwashed ECPs) increased with increasing ozone dose. Washed ECPs in original sludge as divided by molecular weight distribution was 39% 10,000 Da (high MW). It was observed that the low-MW fraction decreased, and the high-MW fraction increased in ozonized sludge. The unwashed ECPs were characterized as 44% in low MW, 30% in medium MW, and 26% in high MW. Both low-MW and medium-MW fractions of unwashed ECPs decreased while high-MW fraction increased in ozonized sludge. The dewaterability of ozonized sludge, assessed by capillary suction time (CST) and specific resistance to filtration (SRF), deteriorated with ozone dose. The optimal dose of cationic polyelectrolyte increased with increasing ozone dose. The production rate and the accumulated amount of methane gas of ozonized sludge were also higher.

  17. 蛋白酶激活受体%Research progress of protease-activated receptors

    Institute of Scientific and Technical Information of China (English)

    余果宇; 张勇; 江萍; 张云

    2011-01-01

    蛋门酶激活受体(protease-activated receptor,PAR)属于G蛋白偶联受体家族,包括4个成员,除PAR2为胰蛋白酶受体外,其他三个都是凝血酶受体,PAR通过形成或暴露新的N末端被激活.PAR广泛表达于全身各组织,尤其在消化系统表现出多种功能;通过促进细胞增殖、迁移、浸润、血管生成以及组织重构(通过促进细胞增殖、迁移,浸润和血管生成),同时抑制细胞的分化和凋亡等因素参与肿瘤的发生和发展,这为临床诊治和顶后评判提供了有力的手段.%Protease-activated receptors are G-protein-coupled receptors that are responded to extracellular proteases and activated by forming or exposing a tethered ligand. There are four protease-activated receptors (PARs) encoded in the mammalian genome. PAR1, PAR3 and PAR4 are respond to thrombin, whereas PAR2 is activated by trypsin. PARs are expressed extensively in many organs, and involved in various functions in digestive system. They play important roles in cancer development by promoting cell proliferation, migration, invasion, angiogenesis, and inhibiting cell differentiation, apoptosis, and might have some clinical implications in cancer's diagnosis, therapy and prognosis.

  18. The Expression of Soluble and Active Recombinant Haemophilus influenzae IgA1 Protease in E. coli

    Directory of Open Access Journals (Sweden)

    Shinong Long

    2010-01-01

    Full Text Available Immunoglobulin A1 (IgA1 proteases from Haemophilus influenzae are extracellular proteases that specifically cleave the hinge region of human IgA1, the predominant class of immunoglobulin present on mucosal membranes. The IgA1 proteases may have the potential to cleave IgA1 complexes in the kidney and be a therapeutic agent for IgA1 nephropathy (IgAN, a disease characterized by deposition of the IgA1 antibody in the glomerulus. We have screened for the expression of recombinant H. influenzae IgA1 protease by combining various expression plasmids, IgA1 protease constructs, and E. coli strains under multiple conditions. Using the method we have developed, approximately 20–40 mg/L of soluble and active H. influenzae IgA1 protease can be produced from E. coli strain C41(DE3, a significant increase in yield compared to the yield upon expression in H. influenzae or other related bacteria.

  19. Development of an integrated system for activity-based profiling of matrix metallo-proteases

    NARCIS (Netherlands)

    Freije, Jan Robert

    2006-01-01

    Matrix metallo-proteases constitute a family of extracellular zinc-dependent endopeptidases that are involved in degradation of extracellular matrix (ECM) components and other bioactive non-ECM molecules. A plethora of studies have implicated important roles for MMPs in many diseases (including

  20. Development of an integrated system for activity-based profiling of matrix metallo-proteases

    NARCIS (Netherlands)

    Freije, Jan Robert

    2006-01-01

    Matrix metallo-proteases constitute a family of extracellular zinc-dependent endopeptidases that are involved in degradation of extracellular matrix (ECM) components and other bioactive non-ECM molecules. A plethora of studies have implicated important roles for MMPs in many diseases (including canc

  1. Diverse impact of acute and long-term extracellular proteolytic activity on plasticity of neuronal excitability.

    Science.gov (United States)

    Wójtowicz, Tomasz; Brzdąk, Patrycja; Mozrzymas, Jerzy W

    2015-01-01

    Learning and memory require alteration in number and strength of existing synaptic connections. Extracellular proteolysis within the synapses has been shown to play a pivotal role in synaptic plasticity by determining synapse structure, function, and number. Although synaptic plasticity of excitatory synapses is generally acknowledged to play a crucial role in formation of memory traces, some components of neural plasticity are reflected by nonsynaptic changes. Since information in neural networks is ultimately conveyed with action potentials, scaling of neuronal excitability could significantly enhance or dampen the outcome of dendritic integration, boost neuronal information storage capacity and ultimately learning. However, the underlying mechanism is poorly understood. With this regard, several lines of evidence and our most recent study support a view that activity of extracellular proteases might affect information processing in neuronal networks by affecting targets beyond synapses. Here, we review the most recent studies addressing the impact of extracellular proteolysis on plasticity of neuronal excitability and discuss how enzymatic activity may alter input-output/transfer function of neurons, supporting cognitive processes. Interestingly, extracellular proteolysis may alter intrinsic neuronal excitability and excitation/inhibition balance both rapidly (time of minutes to hours) and in long-term window. Moreover, it appears that by cleavage of extracellular matrix (ECM) constituents, proteases may modulate function of ion channels or alter inhibitory drive and hence facilitate active participation of dendrites and axon initial segments (AISs) in adjusting neuronal input/output function. Altogether, a picture emerges whereby both rapid and long-term extracellular proteolysis may influence some aspects of information processing in neurons, such as initiation of action potential, spike frequency adaptation, properties of action potential and dendritic

  2. Diverse impact of acute and long-term extracellular proteolytic activity on plasticity of neuronal excitability

    Directory of Open Access Journals (Sweden)

    Tomasz eWójtowicz

    2015-08-01

    Full Text Available Learning and memory require alteration in number and strength of existing synaptic connections. Extracellular proteolysis within the synapses has been shown to play a pivotal role in synaptic plasticity by determining synapse structure, function, and number. Although synaptic plasticity of excitatory synapses is generally acknowledged to play a crucial role in formation of memory traces, some components of neural plasticity are reflected by nonsynaptic changes. Since information in neural networks is ultimately conveyed with action potentials, scaling of neuronal excitability could significantly enhance or dampen the outcome of dendritic integration, boost neuronal information storage capacity and ultimately learning. However, the underlying mechanism is poorly understood. With this regard, several lines of evidence and our most recent study support a view that activity of extracellular proteases might affect information processing in neuronal networks by affecting targets beyond synapses. Here we review the most recent studies addressing the impact of extracellular proteolysis on plasticity of neuronal excitability and discuss how enzymatic activity may alter input-output/transfer function of neurons, supporting cognitive processes. Interestingly, extracellular proteolysis may alter intrinsic neuronal excitability and excitation/inhibition balance both rapidly (time of minutes to hours and in long-term window. Moreover, it appears that by cleavage of extracellular matrix constituents, proteases may modulate function of ion channels or alter inhibitory drive and hence facilitate active participation of dendrites and axon initial segments in adjusting neuronal input/output function. Altogether, a picture emerges whereby both rapid and long-term extracellular proteolysis may influence some aspects of information processing in neurons, such as initiation of action potential, spike frequency adaptation, properties of action potential and dendritic

  3. The Role of Protease-Activated Receptors for the Development of Myocarditis: Possible Therapeutic Implications.

    Science.gov (United States)

    Weithauser, Alice; Witkowski, Marco; Rauch, Ursula

    2016-01-01

    Protease-activated receptors (PARs) are a unique group of four G-protein coupled receptors. They are widely expressed within the cardiovascular system and the heart. PARs are activated via cleavage by serine proteases. In vitro and in vivo studies showed that the activation of PAR1 and PAR2 plays a crucial role in virus induced inflammatory diseases. The receptors enable cells to recognize pathogen-derived changes in the extracellular environment. An infection with Coxsackie-virus B3 (CVB3) can cause myocarditis. Recent studies have been shown that PAR1 signaling enhanced the antiviral innate immune response via interferon β (IFNβ) and thus limited the virus replication and cardiac damage. In contrast, PAR2 signaling decreased the antiviral innate immune response via IFNβ und thus increased the virus replication, which caused severe myocarditis. Along with CVB3 other viruses such as influenza A virus (IAV) and herpes simplex virus (HSV) can induce myocarditis. The role of PAR signaling in IAV infections is contrarily discussed. During HSV infections PARs facilitate the virus infection of the host cell. These studies show that PARs might be interesting drug targets for the treatment of virus infections and inflammatory heart diseases. First studies with PAR agonists, antagonists, and serine protease inhibitors have been conducted in mice. The inhibition of thrombin the main PAR1 activating protease decreased the IFNβ response and increased the virus replication in CVB3-induced myocarditis. This indicates that further studies with direct PAR agonists and antagonists are needed to determine whether PARs are useful drug targets for the therapy of virus-induced heart diseases.

  4. Protease activity in cockroach and basidiomycete allergen extracts.

    Science.gov (United States)

    Wongtim, S; Lehrer, S B; Salvaggio, J E; Horner, W E

    1993-01-01

    Inherent proteolytic activity was estimated in cockroach and basidiomycete extracts by quantifying acid soluble peptides that were released by incubating extracts with 1% bovine serum albumin as measured by Lowry (Sigma). Reference proteases released 740 (Proteinase K, 0.1 U), 248 (Trypsin, 1.0 U), and 533 micrograms/ml (Pronase, 0.5 U) of soluble peptides. American whole body cockroach extract (0.1 mg dry weight) released 330 micrograms/ml of soluble peptides, representing 13 trypsin equivalent units (TEU)/mg. Extracts from spores of the mushroom Pleurotus ostreatus released 230 micrograms/ml (0.9 TEU/mg) and Pleurotus cap extract released 112 micrograms/ml (0.5 TEU/mg). Mycelium of Pleurotus and the mushroom Psilocybe cubensis and spores of Psilocybe and the puffball Calvatia cyathiformis showed negligible amounts of proteolytic activity. The protease inhibitor phenylmethylsulfonyl flouride reduced the proteolytic activity of American whole body cockroach extract by 80% (@1 mM) and the inhibitor ethylene diaminetetraacetic acid inhibited the proteolytic activity of Pleurotus spores by 95% (@1 mM). Loss of allergen activity as determined by RAST inhibition and immunoprinting correlated with protease activity. Thus, in the preparation and handling of allergen extracts, one should employ conditions that minimize proteolysis.

  5. Organ Length Control by an ADAMTS Extracellular Protease in Caenorhabditis elegans

    Science.gov (United States)

    Shibata, Yukimasa; Kawakado, Yuri; Hori, Noriyoshi; Tanaka, Kota; Inoue, Ryo; Takano, Tomomi; Kubota, Yukihiko; Nishiwaki, Kiyoji

    2016-01-01

    MIG-17, a secreted protease of the ADAMTS family, acts in the directed migration of gonadal distal tip cells (DTCs) through regulation of the gonadal basement membrane in Caenorhabditis elegans. Here, we show that MIG-17 is also required for the control of pharynx elongation during animal growth. We found that the pharynx was elongated in mig-17 mutants compared with wild type. MIG-17 localized to the pharyngeal basement membrane as well as to the gonadal basement membrane. The number of nuclei in the pharynx, and the pumping rate of the pharynx, were not affected in mig-17 mutants, suggesting that cells constituting the pharynx are elongated, although the pharynx functions normally in these mutants. In contrast to the control of DTC migration, MIG-18, a secreted cofactor of MIG-17, was not essential for pharynx length regulation. In addition, the downstream pathways of MIG-17 involving LET-2/type IV collagen, FBL-1/fibulin-1, and NID-1/nidogen, partly diverged from those in gonad development. These results indicate that basement membrane remodeling is important for organ length regulation, and suggest that MIG-17/ADAMTS functions in similar but distinct molecular machineries in pharyngeal and gonadal basement membranes. PMID:26994289

  6. Peptide-modified optical filters for detecting protease activity.

    Science.gov (United States)

    Kilian, Kristopher A; Böcking, Till; Gaus, Katharina; Gal, Michael; Gooding, J Justin

    2007-11-01

    The organic derivatization of silicon-based nanoporous photonic crystals is presented as a method to immobilize peptides for the detection of protease enzymes in solution. A narrow-line-width rugate filter, a one-dimensional photonic crystal, is fabricated that exhibits a high-reflectivity optical resonance that is sensitive to small changes in the refractive index at the pore walls. To immobilize peptide in the pore of the photonic crystal, the hydrogen-terminated silicon surface was first modified with the alkene 10-succinimidyl undecenoate via hydrosilylation. The monolayer with the succinimide ester moiety at the distal end served the dual function of protecting the underlying silicon from oxidation as well as providing a surface suitable for subsequent derivatization with amines. The surface was further modified with 1-aminohexa(ethylene glycol) (EG(6)) to resist nonspecific adsorption of proteins common in complex biological samples. The distal hydroxyl of the EG(6) is activated using the solid-phase coupling reagent disuccinimidyl carbonate for selective immobilization of peptides as protease recognition elements. X-ray photoelectron spectroscopy analysis reveals high activation and coupling efficiency at each stage of the functionalization. Exposure of the peptide-modified crystals to the protease subtilisin in solution causes a change in the refractive index, resulting in a shift of the resonance to shorter wavelengths, indicating cleavage of organic material within the pores. The lowest detected concentration of enzyme was 37 nM (7.4 pmol in 200 microL).

  7. Moringa oleifera Lam.: Protease activity against blood coagulation cascade

    Directory of Open Access Journals (Sweden)

    A Satish

    2012-01-01

    Full Text Available Background : The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae leaf (MOL and root (MOR. Materials and Methods : Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Results : Caseinolytic activity of MOL was significantly higher (P ≤ 0.05 than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05 plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05 in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen and fibrinolytic activity (human plasma clot was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aa and Bb subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. Conclusions : These findings support the traditional usage of M. oleifera extracts for wound healing.

  8. Antiretroviral activity of protease inhibitors against Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Lianet Monzote

    2013-02-01

    Full Text Available The introduction of highly active antiretroviral therapy (HAART has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE. These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

  9. Protease-activated receptor-1 modulates hippocampal memory formation and synaptic plasticity.

    Science.gov (United States)

    Almonte, Antoine G; Qadri, Laura H; Sultan, Faraz A; Watson, Jennifer A; Mount, Daniel J; Rumbaugh, Gavin; Sweatt, J David

    2013-01-01

    Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity. © 2012 International Society for Neurochemistry.

  10. Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

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    Lai Meng-Jiun

    2010-08-01

    Full Text Available Abstract Enterovirus type 71 (EV71 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

  11. Cysteine protease activation and apoptosis in Murine norovirus infection

    Directory of Open Access Journals (Sweden)

    Ettayebi Khalil

    2009-09-01

    Full Text Available Abstract Background Noroviruses are the leading cause of viral gastroenteritis. Because a suitable in vitro culture system for the human virus has yet to be developed, many basic details of the infection process are unknown. Murine norovirus (MNV serves as a model system for the study of norovirus infection. Recently it was shown that infection of RAW 264.7 cells involved a novel apoptotic pathway involving survivin. Results Using a different set of approaches, the up-regulation of caspases, DNA condensation/fragmentation, and membrane blebbing, all of which are markers of apoptosis, were confirmed. Live cell imaging and activity-based protein profiling showed that activation of caspase-like proteases occurred within two hours of infection, followed by morphological changes to the cells. MNV infection in the presence of caspase inhibitors proceeded via a distinct pathway of rapid cellular necrosis and reduced viral production. Affinity purification of activity-based protein profiling targets and identification by peptide mass fingerprinting showed that the cysteine protease cathepsin B was activated early in infection, establishing this protein as an upstream activator of the intrinsic apoptotic pathway. Conclusion This work adds cathepsin B to the noncanonical programmed cell death induced by MNV, and provides data suggesting that the virus may induce apoptosis to expand the window of time for viral replication. This work also highlights the significant power of activity-based protein profiling in the study of viral pathogenesis.

  12. Serine protease inhibitor kazal-type 6 inhibits tumorigenesis of human hepatocellular carcinoma cells via its extracellular action

    Science.gov (United States)

    Wang, Wei; Gu, Meigang; Dai, Xinchuan; Xu, Yuqiang; Wu, Hongyu; Li, Guodong; Lu, Hairong; Zhong, Jiang; Huang, Qingshan

    2017-01-01

    Hepatocellular carcinoma (HCC) causes significant medical burdens worldwide. Diagnosis, especially in the early stages, is still challenging. Therapeutic options are limited and often ineffective. Although several risk factors have been known important for development of HCC, the molecular basis of the process is rather complex and has not been fully understood. We have found that a subpopulation of HCC cells which are resistant to oncolytic parvovirus H1 superinfection highly express serine protease inhibitor Kazal-type 6 (SPINK6). This protein is specifically reduced in all HCC cell lines and tissues we analyzed. When upregulated, SPINK6 could suppress the malignant phenotypes of the HCC cells in several in vitro models. The putative tumor suppression role of SPINK6 is, however, independent of its protease inhibitory activity. To suppress the malignancy of HCC cells, SPINK6 has to be secreted to trigger signals which regulate an intracellular signaling molecule, ERK1/2, as well as a series of downstream factors involved in cell cycle progression, apoptosis and migration. Our study supports that SPINK6 is an important tumor suppressor in liver, and further investigations may help develop more effective diagnostic and therapeutic approaches. PMID:27999203

  13. Protease inhibitors activity in lepromatous leprosy and lepra reaction.

    Science.gov (United States)

    Yemul, V L; Sengupta, S R; Dhole, T N

    1983-01-01

    Serum alpha one antitrypsin levels were measured in 50 healthy age and sex matched controls with 45 lepromatous leprosy cases and 5 cases of lepra reaction. It was noted that the mean level in healthy controls was 281.00 mg%, while the mean levels in LL patients was 421.00 mg% and in LR 570.00 mg%. The elevation of Alpha one antitrypsin was statistically significant in LL patients. It is possible that the rise is a reaction to release of proteases and or higher complement activity, which are the results of a high bacillary loading to formation of immune complexes.

  14. Cloning, Expression and Activity Analysis of a Novel Fibrinolytic Serine Protease fromArenicola cristata

    Institute of Scientific and Technical Information of China (English)

    ZHAO Chunling; JU Jiyu

    2015-01-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplifi-cation of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encod-ing a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (<40%) to other serine proteases. The gene encoding the active form ofA. cristata serine protease was cloned and expressed inE. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result sug-gested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clotin vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene inA. cristata.

  15. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    Science.gov (United States)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  16. Protease-mediated drug delivery

    Science.gov (United States)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  17. Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

    Science.gov (United States)

    Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

    2014-07-01

    Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure.

  18. MOFzyme: Intrinsic protease-like activity of Cu-MOF.

    Science.gov (United States)

    Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

    2014-10-24

    The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu₂(C₉H₃O₆)₄/₃ MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

  19. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    Science.gov (United States)

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. The protease activity of the paracaspase MALT1 is controlled by monoubiquitination.

    Science.gov (United States)

    Pelzer, Christiane; Cabalzar, Katrin; Wolf, Annette; Gonzalez, Montserrat; Lenz, Georg; Thome, Margot

    2013-04-01

    The protease activity of the paracaspase MALT1 is central to lymphocyte activation and lymphomagenesis, but how this activity is controlled remains unknown. Here we identify a monoubiquitination of MALT1 on Lys644 that activated the protease function of MALT1. Monoubiquitinated MALT1 had enhanced protease activity, whereas a ubiquitination-deficient MALT1 mutant with replacement of that lysine with arginine (MALT1(K644R)) had less protease activity, which correlated with impaired induction of interleukin 2 (IL-2) via the T cell antigen receptor in activated T cells. Expression of MALT1(K644R) diminished the survival of cells derived from diffuse large B cell lymphoma of the activated B cell-like subtype (ABC DLBCL), which require constitutive protease activity of MALT1 for survival. Thus, monoubiquitination of MALT1 is essential for its catalytic activation and is therefore a potential target for the treatment of ABC-DLBCL and for immunomodulation.

  1. TRY-5 is a sperm-activating protease in Caenorhabditis elegans seminal fluid.

    Directory of Open Access Journals (Sweden)

    Joseph R Smith

    2011-11-01

    Full Text Available Seminal fluid proteins have been shown to play important roles in male reproductive success, but the mechanisms for this regulation remain largely unknown. In Caenorhabditis elegans, sperm differentiate from immature spermatids into mature, motile spermatozoa during a process termed sperm activation. For C. elegans males, sperm activation occurs during insemination of the hermaphrodite and is thought to be mediated by seminal fluid, but the molecular nature of this activity has not been previously identified. Here we show that TRY-5 is a seminal fluid protease that is required in C. elegans for male-mediated sperm activation. We observed that TRY-5::GFP is expressed in the male somatic gonad and is transferred along with sperm to hermaphrodites during mating. In the absence of TRY-5, male seminal fluid loses its potency to transactivate hermaphrodite sperm. However, TRY-5 is not required for either hermaphrodite or male fertility, suggesting that hermaphrodite sperm are normally activated by a distinct hermaphrodite-specific activator to which male sperm are also competent to respond. Within males, TRY-5::GFP localization within the seminal vesicle is antagonized by the protease inhibitor SWM-1. Together, these data suggest that TRY-5 functions as an extracellular activator of C. elegans sperm. The presence of TRY-5 within the seminal fluid couples the timing of sperm activation to that of transfer of sperm into the hermaphrodite uterus, where motility must be rapidly acquired. Our results provide insight into how C. elegans has adopted sex-specific regulation of sperm motility to accommodate its male-hermaphrodite mode of reproduction.

  2. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses

    Science.gov (United States)

    Yu, Jong W.; Hoffman, Sandy; Beal, Allison M.; Dykon, Angela; Ringenberg, Michael A.; Hughes, Anna C.; Dare, Lauren; Anderson, Amber D.; Finger, Joshua; Kasparcova, Viera; Rickard, David; Berger, Scott B.; Ramanjulu, Joshi; Emery, John G.; Gough, Peter J.; Bertin, John; Foley, Kevin P.

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo. PMID:25965667

  3. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

    Directory of Open Access Journals (Sweden)

    Jong W Yu

    Full Text Available CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

  4. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

    Science.gov (United States)

    Yu, Jong W; Hoffman, Sandy; Beal, Allison M; Dykon, Angela; Ringenberg, Michael A; Hughes, Anna C; Dare, Lauren; Anderson, Amber D; Finger, Joshua; Kasparcova, Viera; Rickard, David; Berger, Scott B; Ramanjulu, Joshi; Emery, John G; Gough, Peter J; Bertin, John; Foley, Kevin P

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

  5. Managing Brain Extracellular K(+) during Neuronal Activity

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

    2016-01-01

    reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia...... parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na(+)/K(+)-ATPase in the regulation of extracellular K(+) in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better...

  6. Antiretroviral activity of protease inhibitors against Toxoplasma gondii Terapia antiretroviral de inibidores da protease contra Toxoplasma gondii

    OpenAIRE

    Lianet Monzote; Marta Rodríguez; Yenisey Alfonso; Raymundo Cox

    2013-01-01

    The introduction of highly active antiretroviral therapy (HAART) has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE). These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API) on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. F...

  7. Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

    Science.gov (United States)

    Sun, Qian; Zhang, Bin; Yan, Qiao-Juan; Jiang, Zheng-Qiang

    2016-12-15

    In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.

  8. Development of activity-based probes for trypsin-family serine proteases.

    Science.gov (United States)

    Pan, Zhengying; Jeffery, Douglas A; Chehade, Kareem; Beltman, Jerlyn; Clark, James M; Grothaus, Paul; Bogyo, Matthew; Baruch, Amos

    2006-06-01

    A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.

  9. Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases.

    Directory of Open Access Journals (Sweden)

    Andrea Seper

    Full Text Available The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

  10. Some Investigations on Protease Enzyme Production Kinetics Using Bacillus licheniformis BBRC 100053 and Effects of Inhibitors on Protease Activity

    Directory of Open Access Journals (Sweden)

    Zahra Ghobadi Nejad

    2014-01-01

    Full Text Available Due to great commercial application of protease, it is necessary to study kinetic characterization of this enzyme in order to improve design of enzymatic reactors. In this study, mathematical modeling of protease enzyme production kinetics which is derived from Bacillus licheniformis BBRC 100053 was studied (at 37°C, pH 10 after 73 h in stationary phase, and 150 rpm. The aim of the present paper was to determine the best kinetic model and kinetic parameters for production of protease and calculating Ki (inhibition constant of different inhibitors to find the most effective one. The kinetic parameters Km (Michaelis-Menten constant and Vm (maximum rate were calculated 0.626 mM and 0.0523 mM/min. According to the experimental results, using DFP (diisopropyl fluorophosphate and PMSF (phenylmethanesulfonyl fluoride as inhibitors almost 50% of the enzyme activity could be inhibited when their concentrations were 0.525 and 0.541 mM, respectively. Ki for DFP and PMSF were 0.46 and 0.56 mM, respectively. Kinetic analysis showed that the Lineweaver-Burk model was the best fitting model for protease production kinetics DFP was more effective than PMSF and both of them should be covered in the group of noncompetitive inhibitors.

  11. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused...... to the protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N...... 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...

  12. Proteomics study of extracellular fibrinolytic proteases from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from Indonesian fermented food

    Science.gov (United States)

    Nur Afifah, Diana; Rustanti, Ninik; Anjani, Gemala; Syah, Dahrul; Yanti; Suhartono, Maggy T.

    2017-02-01

    This paper presents the proteomics study which includes separation, identification and characterization of proteins. The experiment on Indonesian fermented food such as extracellular fibrinolytic protease from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from red oncom and tempeh gembus was conducted. The experimental works comprise the following steps: (1) a combination of one- and two-dimensional electrophoresis analysis, (2) mass spectrometry analysis using MALDI-TOF-MS and (3) investigation using protein database. The result suggested that there were new two protein fractions of B. licheniformis RO3 and three protein fractions of B. pumilus 2.g. These result has not been previously reported.

  13. A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

    Energy Technology Data Exchange (ETDEWEB)

    Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F., E-mail: saeed@southernresearch.org

    2016-09-15

    The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

  14. The threonine protease activity of testes-specific protease 50 (TSP50 is essential for its function in cell proliferation.

    Directory of Open Access Journals (Sweden)

    Yu-Yin Li

    Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50, a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation. CONCLUSION: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.

  15. An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I.

    Science.gov (United States)

    Le, Tien; Lee, Hak Jin; Jin, Hyung Jong

    2015-08-15

    A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium.

    Science.gov (United States)

    Böttcher-Friebertshäuser, Eva; Klenk, Hans-Dieter; Garten, Wolfgang

    2013-11-01

    Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA-activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS2 in the human airway epithelium. In addition, we outline investigations of HA-activating proteases as potential drug targets for influenza treatment. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  17. Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

    Science.gov (United States)

    Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

    2011-08-15

    Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.

  18. Protease Activities of Semi-purified Pseudomonas fluorescensin Protein Degradation of Pasteurized Milk at Storage

    Directory of Open Access Journals (Sweden)

    Tatik Khusniati

    2012-10-01

    Full Text Available Protein on stored milks spoiled due to protease activities of Pseudomonas fluorescens. To know protease effect on milks, protease activities of semi-purified P. fluorescens on protein degradation in stored pasteurized milks were detected. Protease was semi-purified by ethanol 70%. Protease activities were detected by modified Lowry method, protein degradation by formol titration, and protein content by Kjeldahl method. Milk storage' times were conducted on 0 day (4 days before expired date up to 12 days (8 days after expired date. The results show that the longer the storage times the higher protease activities and protein degradation of milks. At storage 12 days, protease activities on control were 0.2556 Unit/mL (skim and 0.2116 Unit/mL (whole, and on inoculated milk (crude was 2.2044 Unit/mL (skim and 1.5378 Unit/mL (whole; while on inoculated milk (semi-purified was 3.5355 Unit/mL (skim and 1.6778 Unit/mL (whole, respectively. The decrease of inoculated milk' homogeneous were faster than that of control. Protein degradation on control, inoculated skim milks (crude and semi-purified on 12 days were 4.48%, 7.28% and 7.62%, while that on whole milks were 3.81%, 7.28% and 6.04%, respectively. Based on protease, protein degradation and homogeneous, it can be concluded that skim milk spoiled faster than whole milk.

  19. Matricryptins and matrikines: biologically active fragments of the extracellular matrix.

    Science.gov (United States)

    Ricard-Blum, Sylvie; Salza, Romain

    2014-07-01

    Numerous extracellular proteins and glycosaminoglycans (GAGs) undergo limited enzymatic cleavage resulting in the release of fragments exerting biological activities, which are usually different from those of the full-length molecules. In this review, we define matrikines and matricryptins, which are bioactive fragments released from the extracellular matrix proteins, proteoglycans and GAGs and report their major biological activities. These fragments regulate a number of physiopathological processes including angiogenesis, cancer, fibrosis, inflammation, neurodegenerative diseases and wound healing. The challenges to translate these fragments from molecules biologically active in vitro and in experimental models to potential drugs are discussed in the last part of the review.

  20. Serine Protease Autotransporters of Enterobacteriaceae (SPATEs: Biogenesis and Function

    Directory of Open Access Journals (Sweden)

    Nathalie Dautin

    2010-05-01

    Full Text Available Serine Protease Autotransporters of Enterobacteriaceae (SPATEs constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.

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

    NARCIS (Netherlands)

    Konig, T.; Troder, S.E.; Bakka, K.; Korwitz, A.; Richter-Dennerlein, R.; Lampe, P.A.; Patron, M.; Muhlmeister, M.; Guerrero-Castillo, S.; Brandt, U.; Decker, T.; Lauria, I.; Paggio, A.; Rizzuto, R.; Rugarli, E.I.; Stefani, D. De; Langer, T.

    2016-01-01

    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

  2. Hormone therapy affects plasma measures of factor VII-activating protease in younger postmenopausal women

    DEFF Research Database (Denmark)

    Mathiasen, Jørn Sidelmann; Skouby, S.O.; Vitzthum, F.;

    2010-01-01

    Objectives Current reviews indicate that hormone therapy (HT) has a protective role in coronary heart disease (CHD) in younger postmenopausal women, whereas HT contributes to CHD in older women Factor VII-activating protease (FSAP) is a serine protease that accumulates in unstable atherosclerotic...

  3. A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes.

    Science.gov (United States)

    Iketani, Aya; Nakamura, Mayumi; Suzuki, Yuya; Awai, Koichiro; Shioi, Yuzo

    2013-03-01

    A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.

  4. Functional dissection of the alphavirus capsid protease: sequence requirements for activity

    Directory of Open Access Journals (Sweden)

    Pützer Brigitte M

    2010-11-01

    Full Text Available Abstract Background The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Results Amongst alphaviruses, the C-terminal amino acid tryptophan (W261 is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. Conclusions We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

  5. Protease activated receptor-2 contributes to heart failure.

    Directory of Open Access Journals (Sweden)

    Silvio Antoniak

    Full Text Available Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs, in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.

  6. Extracellular enzymatic activities of Bipolaris sorokiniana isolates.

    Science.gov (United States)

    Geimba, Mercedes P; Brandelli, Adriano

    2002-01-01

    Several enzymatic activities were investigated in six isolates of the fungus Bipolaris sorokiniana, originating from different areas of Brazil. Among the glycosidases studied, beta-glucosidase, beta-N-acetylglucosaminidase, beta-xylosidase, cellobiohydrolase, and chitobiohydrolase were the major activities. In some isolates, beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activities were also present. Polysaccharide-hydrolyzing enzymes, such as pectin lyase and carboxymethyl cellulase were detected in significant amounts, and their activities were variable among the different isolates. Other enzymes, namely phosphatases, proteinases and phenol oxidase, were also examined, showing variable amounts depending on the isolate. The pH dependence of all enzymes tested was investigated. Endoproteinase, carboxymethyl cellulase, and phenoloxidase had maximum activity in the pH range of 6-8, whilst all other enzymes showed maximum activity at pH 4-6.

  7. Intestinal protease-activated receptor-2 and fecal serine protease activity are increased in canine inflammatory bowel disease and may contribute to intestinal cytokine expression.

    Science.gov (United States)

    Maeda, Shingo; Ohno, Koichi; Uchida, Kazuyuki; Igarashi, Hirotaka; Goto-Koshino, Yuko; Fujino, Yasuhito; Tsujimoto, Hajime

    2014-08-01

    Serine proteases elicit cellular responses via protease-activated receptor-2 (PAR-2) which is known to regulate inflammation and the immune response. Although the gastrointestinal tract is exposed to large amounts of proteolytic enzymes, the role of PAR-2 in canine inflammatory bowel disease (IBD) remains unclear. The objective of this study was to investigate the effects of PAR-2 activation on inflammatory cytokine/chemokine gene expression in canine intestine and the expression of intestinal PAR-2 and fecal serine protease activity in dogs with IBD. Duodenal biopsies from healthy dogs were cultured and treated ex vivo with trypsin or PAR-2 agonist peptide, and inflammatory cytokine/chemokine gene expression in the tissues was then quantified by real-time PCR. PAR-2 mRNA and protein expression levels in the duodenal mucosa were examined by real-time PCR and immunohistochemistry, respectively. Fecal serine protease activity was determined by azocasein assay. In ex vivo-cultured duodenum, trypsin and PAR-2 agonist peptide induced significant up-regulation of mRNA expression levels of interleukin-1 β (IL-1β), IL-8, mucosae-associated epithelial chemokine (MEC) and fractalkine, and this up-regulation was inhibited by a serine protease inhibitor. Duodenal PAR-2 mRNA and protein expression levels were higher in dogs with IBD than in healthy control dogs. Fecal serine protease activity was significantly elevated in dogs with IBD, and the level of activity correlated positively with the clinical severity score. These results suggest that PAR-2 may contribute to the pathogenesis of canine IBD by inducing expression of inflammatory mediators in response to luminal serine proteases.

  8. Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom.

    Science.gov (United States)

    Ma, Hakim; Xiao-Peng, Tang; Yang, Shi-Long; Lu, Qiu-Min; Lai, Ren

    2016-08-01

    It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  9. Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

    Energy Technology Data Exchange (ETDEWEB)

    He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Li, Xiao-Dong [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Hong, Mo-Na [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Chen, Qi-Zhi [Shanghai Institute of Hypertension, Shanghai (China); Han, Wei-Qing, E-mail: whan020@gmail.com [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Gao, Ping-Jin, E-mail: gaopingjin@sibs.ac.cn [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China)

    2016-04-29

    Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

  10. Detection of extracellular enzymatic activity in microorganisms ...

    African Journals Online (AJOL)

    sunny t

    2015-09-18

    Sep 18, 2015 ... bacterial and 14 fungi strains that presented positive lipolytic activity were obtained by detection through Rhodamine B ... The samples were placed in plastic bags, then ..... The ecology of chitin degradation. In: Marshall KC.

  11. Inhibition of activity of the protease from bovine leukemia virus.

    Science.gov (United States)

    Ménard, A; Leonard, R; Llido, S; Geoffre, S; Picard, P; Berteau, F; Precigoux, G; Hospital, M; Guillemain, B

    1994-06-13

    In view of the close similarity between bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I) we investigated the possibility of developing specific inhibitors of the proteases of these retroviruses using the purified enzyme from BLV. We tested the ability of this protease to specifically cleave various short oligopeptide substrates containing cleavage sites of BLV and HTLV-I proteases, as well as a recombinant BLV Gag precursor. The best substrate, a synthetic decapeptide bearing the natural cleavage site between the matrix and the capsid proteins of BLV Gag precursor polyprotein, was used to develop an inhibition assay. We determined the relative inhibitory effect of synthetic Gag precursor-like peptides in which the cleavable site was replaced by a non-hydrolyzable moiety. The encouraging inhibitory effect of these compounds indicates that potent non-peptidic inhibitors for retroviral proteases are not unattainable.

  12. Synthesis and extended activity of triazole-containing macrocyclic protease inhibitors

    DEFF Research Database (Denmark)

    Pehere, A.D.; Pietsch, M.; Gütschow, M.;

    2013-01-01

    Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole- containing macrocyclic protease inhibitors pre-organized into a b-strand conformation and an evaluation...... of their activity against a panel of proteases. Acyclic azidoalkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpain II, cathepsin L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent...

  13. Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production

    NARCIS (Netherlands)

    Kauffman, HF; Tomee, JFC; van de Riet, MA; Timmerman, AJB; Borger, P

    2000-01-01

    Background: Proteases in extracts of Aspergillus fumigatus cause epithelial cell desquamation and release of proinflammatory cytokines. Objective: We sought to assess protease activity in Alternaria alternata, Cladosporium herbarum, and Aspergillus fumigatus extracts and study the ability of these e

  14. Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

    Science.gov (United States)

    Andrews, R E; Bibilos, M M; Bulla, L A

    1985-01-01

    Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable. Images PMID:3909962

  15. In vitro inhibition of protease-activated receptors 1, 2 and 4 demonstrates that these receptors are not involved in an Acanthamoeba castellanii keratitis isolate-mediated disruption of the human brain microvascular endothelial cells.

    Science.gov (United States)

    Iqbal, Junaid; Naeem, Komal; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2014-11-01

    Granulomatous amoebic encephalitis is a rare but serious human disease leading almost always to death. The pathophysiology of amoebic encephalitis is better understood, while events leading to the constitution of brain infection are largely unknown. Traversal of the blood-brain barrier is a key step in amoebae invasion of the central nervous system and facilitated by amoebic extracellular proteases. By using specific inhibitors of protease-activated receptors 1, 2 and 4, here we studied the role of these host receptors in Acanthamoeba castellanii-mediated damage to human brain microvasculature endothelial cells (HBMEC), which constitute the blood-brain barrier. The primary HBMEC were incubated with A. castellanii-conditioned medium in the presence or absence of FR-171113 (selective inhibitor of protease-activated receptor 1), FSLLRY-NH2 (inhibitor of protease-activated receptor 2), and tcY-NH2 (inhibitor of protease-activated receptor 4). The HBMEC monolayer disruptions were assessed by microscopy using Eosin staining, while host cell cytotoxicity was determined by measuring the release of cytoplasmic lactate dehydrogenase. Zymographic assays were performed to determine the effects of inhibitors of protease-activated receptors on the extracellular proteolytic activities of A. castellanii. A. castellanii-conditioned medium produced severe HBMEC monolayer disruptions within 60 min. The selective inhibitors of protease-activated receptors tested did not affect HBMEC monolayer disruptions. On the contrary, pre-treatment of A. castellanii-conditioned medium with phenylmethylsulfonyl fluoride, a serine protease inhibitor, or heating for 10 min at 95°C abolished HBMEC monolayer disruptions. Additionally, inhibitors of protease-activated receptors tested, failed to block A. castellanii-mediated HBMEC cytotoxicity and did not affect extracellular proteolytic activities of A. castellanii. Protease-activated receptors 1, 2 and 4 do not appear to play a role in A. castellanii

  16. Protease-Activated Receptor-2 Activation Contributes to House Dust Mite-Induced IgE Responses in Mice

    NARCIS (Netherlands)

    Post, Sijranke; Heijink, Irene; Petersen, A H; de Bruin, Harold G.; van Oosterhout, Antoon J. M.; Nawijn, Martijn C.

    2014-01-01

    Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium,

  17. Protease-activated receptor-2 activation contributes to house dust mite-induced IgE responses in mice

    NARCIS (Netherlands)

    Post, Sijranke; Heijink, Irene H; Petersen, Arjen H; de Bruin, Harold G; van Oosterhout, Antoon J M; Nawijn, Martijn C

    2014-01-01

    Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium,

  18. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    OpenAIRE

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally ...

  19. Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

    Science.gov (United States)

    Jing, Tengyang; Lai, Zhangxing; Wu, Lidan; Han, Jongyoon; Lim, Chwee Teck; Chen, Chia-Hung

    2016-12-06

    Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

  20. Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

    Directory of Open Access Journals (Sweden)

    Dienger Krista

    2011-09-01

    Full Text Available Abstract Background A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2; however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear. Methods Mice (wild type and PAR-2-deficient were sensitized using German cockroach (GC feces (frass, the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production, serum IgE levels and airway hyperresponsiveness (AHR were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry. Results Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice

  1. THE EFFECT OF FEEDING Lactobacillus ON GROWTH, SURVIVAL RATE AND PROTEASE ACTIVITY OF Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    Nunak Nafiqoh

    2011-12-01

    Full Text Available This study examined the effect of two Lactobacillus bacteria on protease activity and growth rate of Litopenaeus vannamei. An experiment was conducted to examine protease activity and growth rate. The experiment consisted of two treatment tanks, the first tank was provided with artemia immersed in 2.6 x 1016 cfu/mL of bacteria solution, the second tank served as the control tank. After 20 days, the L. vannamei in the tank that received Lactobacillus have significantly different in growth, survival rate and protease activity (P<0.05 compared to the control, but no significant difference between Lactobacillus casei and Lactobacillus plantarum treatments. Within the digestive organ, protease activity of hepatopancreas and stomach demonstrated significant higher activity (P<0.05 compared to the intestine.

  2. Skip Regulates TGF-β1-Induced Extracellular Matrix Degrading Proteases Expression in Human PC-3 Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Victor Villar

    2013-01-01

    Full Text Available Purpose. To determine whether Ski-interacting protein (SKIP regulates TGF-β1-stimulated expression of urokinase-type plasminogen activator (uPA, matrix metalloproteinase-9 (MMP-9, and uPA Inhibitor (PAI-1 in the androgen-independent human prostate cancer cell model. Materials and Methods. PC-3 prostate cancer cell line was used. The role of SKIP was evaluated using synthetic small interference RNA (siRNA compounds. The expression of uPA, MMP-9, and PAI-1 was evaluated by zymography assays, RT-PCR, and promoter transactivation analysis. Results. In PC-3 cells TGF-β1 treatment stimulated uPA, PAI-1, and MMP-9 expressions. The knockdown of SKIP in PC-3 cells enhanced the basal level of uPA, and TGF-β1 treatment inhibited uPA production. Both PAI-1 and MMP-9 production levels were increased in response to TGF-β1. The ectopic expression of SKIP inhibited both TGF-β1-induced uPA and MMP-9 promoter transactivation, while PAI-1 promoter response to the factor was unaffected. Conclusions. SKIP regulates the expression of uPA, PAI-1, and MMP-9 stimulated by TGF-β1 in PC-3 cells. Thus, SKIP is implicated in the regulation of extracellular matrix degradation and can therefore be suggested as a novel therapeutic target in prostate cancer treatment.

  3. Sulfur activation-related extracellular proteins of Acidithiobacillus ferrooxidans

    Institute of Scientific and Technical Information of China (English)

    ZHANG Cheng-gui; ZHANG Rui-yong; XIA Jin-lan; ZHANG Qian; NIE Zhen-yuan

    2008-01-01

    The fractions of the extracellular proteins of Acidithiobacillus ferrooxidans grown on two different energy substrates,elemental sulfur and ferrous sulfate,were selectively prepared with hot water treatment and distinctly shown by two-dimensional gel electrophoresis.Some protein spots with apparently higher abundance in sulfur energy substrate than in ferrous sulfate energy substrate were identified by using MALDI-TOF/TOF.Based on peptide mass fingerprints and bioinformatical analysis,the extracellular proteins were classified according to their functions as conjugal transfer protein,pilin,vacJ lipoprotein,polysaccharide deacetylase family protein,Ser/Thr protein phosphatase family protein and hypothetical proteins.Several extracellular proteins were found abundant in thiol groups and with CXXC functional motif,these proteins may be directly involved in the sulfur activation by use of their thiol group (Pr-SH) to bond the elemental sulfur.

  4. Hyaluronidase and protease activities from Indian snake venoms: neutralization by Mimosa pudica root extract.

    Science.gov (United States)

    Girish, K S; Mohanakumari, H P; Nagaraju, S; Vishwanath, B S; Kemparaju, K

    2004-06-01

    The aqueous root extract of Mimosa pudica dose dependently inhibited the hyaluronidase and protease activities of Indian snakes (Naja naja, Vipera russelii and Echis carinatus) venom. Copyright 2004 Elsevier B.V.

  5. Proteolytic activity of extracellular products from Arthrobotrys musiformis and their effect in vitro against Haemonchus contortus infective larvae

    Science.gov (United States)

    Acevedo-Ramírez, Perla María del Carmen; Figueroa-Castillo, Juan Antonio; Ulloa-Arvizú, Raúl; Martínez-García, Luz Gisela; Guevara-Flores, Alberto; Rendón, Juan Luis; Valero-Coss, Rosa Ofelia; Mendoza-de Gives, Pedro; Quiroz-Romero, Héctor

    2015-01-01

    Arthrobotrys musiformis is a nematophagous fungus with potential for the biological control of Haemonchus contortus larvae. This study aimed to identify and demonstrate the proteolytic activity of extracellular products from A musiformis cultured in a liquid medium against H contortus infective larvae. A musiformis was cultured on a solid medium and further grown in a liquid medium, which was then processed through ion exchange and hydrophobic interaction chromatography. The proteolytic activity of the purified fraction was assayed with either gelatin or bovine serum albumin as substrate. Optimum proteolytic activity was observed at pH 8 and a temperature of 37°C. Results obtained with specific inhibitors suggest the enzyme belongs to the serine-dependent protease family. The purified fraction concentrate from A musiformis was tested against H contortus infective larvae. A time-dependent effect was observed with 77 per cent immobility after 48 hours incubation, with alteration of the sheath. It is concluded that A musiformis is a potential candidate for biological control because of its resistant structures and also because of its excretion of extracellular products such as proteases. The present study contributes to the identification of one of the in vitro mechanisms of action of Amusiformis, namely the extracellular production of proteases against H contortus infective larvae. More investigations should be undertaken into how these products could be used to decrease the nematode population in sheep flocks under field conditions, thereby improving animal health while simultaneously diminishing the human and environmental impact of chemical-based drugs. PMID:26392902

  6. 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.

  7. Comparative characterization of protease activity in cultured spotted rose snapper juveniles (Lutjanus guttatus

    Directory of Open Access Journals (Sweden)

    Emyr Peña

    2015-09-01

    Full Text Available Partial characterizations of digestive proteases were studied in three life stages of spotted rose snapper: early (EJ, middle (MJ and late juvenile (LJ with corresponding average weights of 21.3 ± 2.6 g (3 months after hatching, MAH, 190 ± 4.4 g (7 MAH, and 400 ± 11.5 g (12 MAH. At sampling points, the digestive tract was dissected into the stomach (St, pyloric caeca (PC, and the intestine in three sections (proximal (PI, middle (MI and distal intestine (DI. The effect of pH and temperature and specific inhibitors were evaluated for acid and alkaline proteases. Total acid and alkaline protease activity showed a tendency to increase with juvenile life stage of fish while trypsin activity decreased. Differences were found in acid and alkaline protease activities at different pH and temperatures during juvenile stages. Pepstatin A inhibited total activity in the stomach extract in all juvenile stages. Activity in total alkaline protease inhibition was significantly higher in EJ using TLCK, PMSF, SBTI, Phen and Ovo than in MJ and LJ, while no significant differences were found with TPCK inhibition. Therefore increases in protease activities with fish growth through juvenile stages in which a substitution or diversification in the type of alkaline enzymes exist. These results lead a better comprehension of changes in digestive potential of Lutjanidae fish.

  8. Protease inhibitor

    DEFF Research Database (Denmark)

    2009-01-01

    The present invention relates to a polypeptide exhibiting a protease inhibitory activity and uses of said polypeptide in methods for inhibiting, directly or indirectly, one or more proteases of the blood clotting cascade. The invention also relates to use of said polypeptide as a pharmaceutical e...

  9. Design, synthesis, and activity of nanocellulosic protease sensors

    Science.gov (United States)

    Here we contrast the molecular assembly, and biochemical utility of nanocellulosic materials prepared from cotton and wood as protease sensors. The cotton-based nanocellulosic substrates were prepared in a variety of ways to produce nanocrystals, films and aerogels, which were derivatized with eithe...

  10. Milk Clotting Activity of Protease, Extracted from Rhizome of Taffin ...

    African Journals Online (AJOL)

    MBI

    2017-03-07

    Mar 7, 2017 ... substituting calf rennet in the food industries, particularly in cheese making processes. Keywords: Ginger ..... practice (Macedo et al., 1993). Ginger ... evaluation procedure, the protease MCA/PA ratio has been a ... Quantities of Protein Utilizing the Principle .... proteolysis and sensory quality of Peshawari.

  11. Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease.

    Science.gov (United States)

    Ogino, Hiroyasu; Tsuchiyama, Shotaro; Yasuda, Masahiro; Doukyu, Noriyuki

    2010-03-01

    The PST-01 protease is highly stable and catalyzes the synthesis of the aspartame precursor with high reaction yields in the presence of organic solvents. However, the synthesis rate using the PST-01 protease was slower than that observed when thermolysin was used. Structural comparison of both enzymes showed particular amino acid differences near the active center. These few residue differences in the PST-01 protease were mutated to match those amino acid types found in thermolysin. The mutated PST-01 proteases at the 114th residue from tyrosine to phenylalanine showed enhancement of synthetic activity. This activity was found to be similar to thermolysin. In addition, mutating the residue in the PST-01 protease with arginine and serine showed more improvement of the activity. The mutant PST-01 protease should be more useful than thermolysin for the synthesis of the aspartame precursor, because this enzyme has higher stability and activity in the presence of organic solvents. The results show the potential of organic solvent-stable enzymes as industrial catalysts.

  12. Purification and characterization of a prothrombin-activating protease from Nephila clavata.

    Science.gov (United States)

    Joo, Han-Seung; Park, Gun-Chun; Cho, Woo Ri; Tak, Eunsik; Paik, Seung R; Chang, Chung-Soon

    2002-03-01

    We report upon the purification and characterization of a novel prothrombin-activating enzyme from the body fluid (total homogenates of isolated digestive tract without eggs, spinnerets and silk glands) of the spider, Nephila clavata by a combination of acetone fractionation, ion exchange, and Soybean trypsin inhibitor-Sepharose chromatography. Analysis of the purified enzyme with SDS-PAGE and gel filtration revealed a single polypeptide chain with an apparent molecular weight of 24kDa. The proteolytic activity of the enzyme was stable up to 50 degrees C, however, it became unstable over 55 degrees C. The enzyme had an optimum pH of 8, and Ca(2+) was not required for the enzyme activity. According to inhibition profiles obtained with several serine protease inhibitors such as PMSF and benzamidine, the purified protease is a member of the serine proteases. Bz-Ile-Glu(gamma-OR)- Gly-Arg-pNA and Z-Arg-Gly-Arg-pNA which are known as substrates for factor Xa, were hydrolyzed favorably by the enzyme. And the Nephila protease could produce thrombin from prothrombin at nM range, and form the turbid ring using fibrinogen-agarose plate. The results obtained confirmed that the purified protease is a potent prothrombin-activating activity belonging to the family of serine protease.

  13. Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

    Science.gov (United States)

    Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M

    2016-07-08

    Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific.

  14. Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates

    OpenAIRE

    Saisawang, Chonticha; Sillapee, Pornpan; Sinsirimongkol, Kwanhathai; Ubol, Sukathida; Smith, Duncan R.; Ketterman, Albert J.

    2015-01-01

    Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. ...

  15. Extracellular electrical activity from the photoreceptors of midge

    Indian Academy of Sciences (India)

    A A Babrekar; G R Kulkarni; B B Nath; P B Vidyasagar

    2004-09-01

    The ontogeny of photosensitivity has been studied in a holometabolous insect, the midge Chironomus ramosus. The life cycle of midges shifts from an aquatic environment to a non-aquatic environment. Extracellular electrical activity of photoreceptor organs was recorded at larval and adult stages. We found an increase in photosensitivity as the larva metamorphosed to the adult stage. This is the first report of changes in photosensitivity during the development of any insect described in an ecological context.

  16. Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

    Science.gov (United States)

    Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

    2011-01-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

  17. Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

    Science.gov (United States)

    Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

    2011-01-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

  18. Multiplex Detection of Protease Activity with Quantum Dot Nanosensors Prepared by Intein-Mediated Specific Bioconjugation

    Science.gov (United States)

    Xia, Zuyong; Xing, Yun; So, Min-Kyung; Koh, Ai Leen; Sinclair, Robert; Rao, Jianghong

    2009-01-01

    We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few ng/ml, and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown. PMID:18922019

  19. The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Lata; Leduc, Yvonne; Hayakawa, Koto; Delbaere, Louis T.J. (Saskatchewan)

    2008-06-27

    V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

  20. Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology

    DEFF Research Database (Denmark)

    Bæk, Kristoffer Torbjørn; Vegge, Christina Skovgaard; Skórko-Glonek, Joanna;

    2011-01-01

    The microaerophilic bacterium Campylobacter jejuni is the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope such as HtrA and SurA. HtrA displays both chaperone and protease activity......, but little is known about how each of these activities contributes to stress tolerance in bacteria. In vitro experiments showed temperature dependent protease and chaperone activities of C. jejuni HtrA. A C. jejuni mutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone...... activity is sufficient for growth at high temperature or oxidative stress, whereas the HtrA protease activity is only essential at conditions close to the growth limit for C. jejuni. However, the protease activity was required to prevent induction of the cytoplasmic heat-shock response even at optimal...

  1. Lectin Activation in Giardia lamblia by Host Protease: A Novel Host-Parasite Interaction

    Science.gov (United States)

    Lev, Boaz; Ward, Honorine; Keusch, Gerald T.; Pereira, Miercio E. A.

    1986-04-01

    A lectin in Giardia lamblia was activated by secretions from the human duodenum, the environment where the parasite lives. Incubation of the secretions with trypsin inhibitors prevented the appearance of lectin activity, implicating proteases as the activating agent. Accordingly, lectin activation was also produced by crystalline trypsin and Pronase; other proteases tested were ineffective. When activated, the lectin agglutinated intestinal cells to which the parasite adheres in vivo. The lectin was most specific to mannose-6-phosphate and apparently was bound to the plasma membrane. Activation of a parasite lectin by a host protease represents a novel mechanism of hostparasite interaction and may contribute to the affinity of Giardia lamblia to the infection site.

  2. Protease activity in gut of Daphnia magna: evidence for trypsin and chymotrypsin enzymes.

    Science.gov (United States)

    von Elert, Eric; Agrawal, Manish Kumar; Gebauer, Christine; Jaensch, Heike; Bauer, Ulrike; Zitt, Anja

    2004-03-01

    Two major protease activities were present in gut homogenates of the cladoceran crustacean Daphnia magna: (i) a trypsin activity that hydrolysed the synthetic substrate N-benzoyl-dl-arginine p-nitroanilide and was strongly inhibited by N-p-tosyl-lysine chloroketone (TLCK) and 4-(amidinophenyl)methanesulfonyl fluoride (APMSF) and not inhibited by chymostatin; and (ii) a chymotrypsin activity that hydrolysed synthetic chymotrypsin substrates containing more than one amino acid, did not hydrolyse N-benzoyl-l-tyrosine p-nitroanilide, and was strongly inhibited by chymostatin and not by TLCK and APMSF. Both activities had alkaline pH optima (pH 7-10), but were shown to be due to distinct types of proteases. These two enzyme activities accounted for 75-83% of the proteolytic activity of gut contents. Substrate SDS-polyacrylamide gel electrophoresis revealed nine different proteases ranging from 15 to 73 kDa.

  3. Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

    Institute of Scientific and Technical Information of China (English)

    Beverly Z Packard; Akira Komoriya

    2008-01-01

    Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clinical efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.

  4. Secretion of extracellular hsp90α via exosomes increases cancer cell motility: a role for plasminogen activation

    Directory of Open Access Journals (Sweden)

    Chan Doug

    2010-06-01

    Full Text Available Abstract Background Metastasis is a multi-step process that is responsible for the majority of deaths in cancer patients. Current treatments are not effective in targeting metastasis. The molecular chaperone hsp90α is secreted from invasive cancer cells and activates MMP-2 to enhance invasiveness, required for the first step in metastasis. Methods We analyzed the morphology and motility of invasive cancer cells that were treated with exogenous exosomes in the presence or absence of hsp90α. We performed mass spectrometry and immunoprecipitation to identify plasminogen as a potential client protein of extracellular hsp90α. Plasmin activation assays and migration assays were performed to test if plasminogen is activated by extracellular hsp90α and has a role in migration. Results We found that hsp90α is secreted in exosomes in invasive cancer cells and it contributes to their invasive nature. We identified a novel interaction between hsp90α and tissue plasminogen activator that together with annexin II, also found in exosomes, activates plasmin. Extracellular hsp90α promotes plasmin activation as well as increases plasmin dependent cell motility. Conclusions Our data indicate that hsp90α is released by invasive cancer cells via exosomes and implicates hsp90α in activating plasmin, a second protease that acts in cancer cell invasion.

  5. Classification of the extracellular fields produced by activated neural structures

    Directory of Open Access Journals (Sweden)

    Perry Danielle

    2005-09-01

    Full Text Available Abstract Background Classifying the types of extracellular potentials recorded when neural structures are activated is an important component in understanding nerve pathophysiology. Varying definitions and approaches to understanding the factors that influence the potentials recorded during neural activity have made this issue complex. Methods In this article, many of the factors which influence the distribution of electric potential produced by a traveling action potential are discussed from a theoretical standpoint with illustrative simulations. Results For an axon of arbitrary shape, it is shown that a quadrupolar potential is generated by action potentials traveling along a straight axon. However, a dipole moment is generated at any point where an axon bends or its diameter changes. Next, it is shown how asymmetric disturbances in the conductivity of the medium surrounding an axon produce dipolar potentials, even during propagation along a straight axon. Next, by studying the electric fields generated by a dipole source in an insulating cylinder, it is shown that in finite volume conductors, the extracellular potentials can be very different from those in infinite volume conductors. Finally, the effects of impulses propagating along axons with inhomogeneous cable properties are analyzed. Conclusion Because of the well-defined factors affecting extracellular potentials, the vague terms far-field and near-field potentials should be abandoned in favor of more accurate descriptions of the potentials.

  6. Differential cleavage of the norovirus polyprotein precursor by two active forms of the viral protease.

    Science.gov (United States)

    Scheffler, Ulrike; Rudolph, Wolfram; Gebhardt, Julia; Rohayem, Jacques

    2007-07-01

    Protein translation in noroviruses requires translational processing of a polyprotein precursor by the viral protease. So far, the molecular mechanisms of catalytic cleavage by the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the viral protease were examined in vitro by using synthetic peptides (11-15 residues) corresponding to the cleavage sites of the norovirus polyprotein. Both predicted forms of the viral protease, the 3C-like protease (3C(pro)) and the 3CD-like protease polymerase protein (3CD(propol)), displayed a specific trans cleavage activity of peptides bearing Gln-Gly at the scissile bond. In contrast, peptides bearing Glu-Gly at the scissile bond (p20/VPg and 3C(pro)/3D(pol) junctions) were resistant to trans-cleavage by 3C(pro) and 3CD(propol). Interestingly, the VPg/3C(pro) scissile bond (Glu-Ala) was cleaved only by 3CD(propol), and examination of relative cleavage efficiencies revealed significant differences in processing of peptides, indicating differential cleavage patterns for 3C(pro) and 3CD(propol).

  7. Triclabendazole Effect on Protease Enzyme Activity in the Excretory- Secretory Products of Fasciola hepatica in Vitro.

    Directory of Open Access Journals (Sweden)

    Yosef Shrifi

    2014-03-01

    Full Text Available Fasciola hepatica is one of the most important helminthes parasites and triclabendazole (TCBZ is routinely used for treatment of infected people and animals. Secreted protease enzymes by the F. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. The aim of study was to determine the protease activity of excretory- secretory products (ESP of F. hepatica in the presence of TCBZ anthelmintic.F. hepatica helminthes were collected and cultured within RPMI 1640 [TCBZ treated (test and untreated (control] for 6 h at 37 °C. ESP of treated and control were collected, centrifuged and supernatants were stored at -20°C. Protein concentrations were measured according to Bradford method. Protease enzymes activities of ESP samples were estimated by using sigma's non-specific protease activity assay. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE.Mean protein concentrations in control and treated of ESP samples were determined 196.1 ±14.52 and 376.4 ±28.20 μg/ml, respectively. Mean protease enzymes activities in control and treated were 0.37 ±0.1 and 0.089 ±0.03 U/ml, respectively. Significant difference between proteins concentrations and protease enzymes activities of two groups was observed (P<0.05. SDS-PAGE showed different patterns of protein bands between treated and control samples.The TCBZ reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues.

  8. MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation.

    Science.gov (United States)

    Schmitt, Anja; Grondona, Paula; Maier, Tabea; Brändle, Marc; Schönfeld, Caroline; Jäger, Günter; Kosnopfel, Corinna; Eberle, Franziska C; Schittek, Birgit; Schulze-Osthoff, Klaus; Yazdi, Amir S; Hailfinger, Stephan

    2016-04-01

    The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as well as silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases.

  9. Matriptase-2 mutations in iron-refractory iron deficiency anemia patients provide new insights into protease activation mechanisms.

    Science.gov (United States)

    Ramsay, Andrew J; Quesada, Victor; Sanchez, Mayka; Garabaya, Cecilia; Sardà, María P; Baiget, Montserrat; Remacha, Angel; Velasco, Gloria; López-Otín, Carlos

    2009-10-01

    Mutations leading to abrogation of matriptase-2 proteolytic activity in humans are associated with an iron-refractory iron deficiency anemia (IRIDA) due to elevated hepcidin levels. Here we describe two novel heterozygous mutations within the matriptase-2 (TMPRSS6) gene of monozygotic twin girls exhibiting an IRIDA phenotype. The first is the frameshift mutation (P686fs) caused by the insertion of the four nucleotides CCCC in exon 16 (2172_2173insCCCC) that is predicted to terminate translation before the catalytic serine. The second mutation is the di-nucleotide substitution c.467C>A and c.468C>T in exon 3 that causes the missense mutation A118D in the SEA domain of the extracellular stem region of matriptase-2. Functional analysis of both variant matriptase-2 proteases has revealed that they lead to ineffective suppression of hepcidin transcription. We also demonstrate that the A118D SEA domain mutation causes an intra-molecular structural imbalance that impairs matriptase-2 activation. Collectively, these results extend the pattern of TMPRSS6 mutations associated with IRIDA and functionally demonstrate that mutations affecting protease regions other than the catalytic domain may have a profound impact in the regulatory role of matriptase-2 during iron deficiency.

  10. Supermarket Proteases.

    Science.gov (United States)

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  11. Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors

    Institute of Scientific and Technical Information of China (English)

    HEShao-Heng; CHENPu; CHENHan-Qiu

    2003-01-01

    AIM: To investigate the actions of protease inhibitors on the enzymatic activities of tryptase and chymase in similarexperimental systems. METHODS: Human lung tryptase and human skin chymase were purified by a similarprocedure involving high salt extraction of tryptase, heparin agarose affinity chromatography, and S-200 Sephacrylgel filtration chromatography. Actions of protease inhibitors on tryptase and chymase activities were examined byenzyme assays. RESULTS: The specific activities of tryptase and chymase were 2.1 kU/g protein and 4.9 kU/g protein, respectively. Both preparations showed a single diffuse band on SDS-PAGE. Among non-native proteaseinhibitors, N-(1-hydroxy-2-naphthoyl)-L- arginyl-L-prolinamide hydrochloride (HNAP), leupeptin, antipain,benzamidine, and protamine inhibited more than 90 % enzymatic activity of tryptase, whereas soy bean trypsininhibitor (SBTI), Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPM) and chymostatin inhibited more than 95 % enzymaticactivity of chymase. Native protease inhibitors α-antitrypsin and secretory leukocyte protease inhibitor (SLPI)inhibited more than 90 % enzymatic activity of chymase, but lactoferrin appeared to enhance chymase enzymaticactivity. All the 3 inhibitors had weak inhibitory actions on tryptase. CONCLUSION: The protease inhibitorstested had relatively good selectivity to either tryptase or chymase.

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

    Science.gov (United States)

    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.

  13. Protease activated receptors (PARS) mediation in gyroxin biological activity; Mediacao dos receptores ativados por proteases (PARs) em atividades biologicas da giroxina

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Jose Alberto Alves da

    2009-07-01

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH{sub 2}, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  14. Factor VII-activating protease in patients with acute deep venous thrombosis

    DEFF Research Database (Denmark)

    Sidelmann, Johannes J; Vitzthum, Frank; Funding, Eva;

    2008-01-01

    Factor VII-activating protease (FSAP) is involved in haemostasis and inflammation. FSAP cleaves single chain urokinase-type plasminogen activator (scu-PA). The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene leads to the expression of a FSAP variant with reduced ability to activate scu...

  15. Extracellular enzyme activity in a willow sewage treatment system.

    Science.gov (United States)

    Brzezinska, Maria Swiontek; Lalke-Porczyk, Elżbieta; Kalwasińska, Agnieszka

    2012-12-01

    This paper presents the results of studies on the activity of extra-cellular enzymes in soil-willow vegetation filter soil which is used in the post-treatment of household sewage in an onsite wastewater treatment system located in central Poland. Wastewater is discharged from the detached house by gravity into the onsite wastewater treatment system. It flows through a connecting pipe into a single-chamber septic tank and is directed by the connecting pipe to a control well to be further channelled in the soil-willow filter by means of a subsurface leaching system. Soil samples for the studies were collected from two depths of 5 cm and 1 m from three plots: close to the wastewater inflow, at mid-length of the plot and close to its terminal part. Soil samples were collected from May to October 2009. The activity of the extra-cellular enzymes was assayed by the fluorometric method using 4-methylumbelliferyl and 7-amido-4-methylcoumarin substrate. The ranking of potential activity of the assayed enzymes was the same at 5 cm and 1 m soil depths, i.e. esterase > phosphmomoesterase > leucine-aminopeptidase > β-glucosidase > α-glucosidase. The highest values of enzymatic activity were recorded in the surface layer of the soil at the wastewater inflow and decreased with increasing distance from that point.

  16. Four different types of protease-activated receptors are widely expressed in the brain and are up-regulated in hippocampus by severe ischemia.

    Science.gov (United States)

    Striggow, F; Riek-Burchardt, M; Kiesel, A; Schmidt, W; Henrich-Noack, P; Breder, J; Krug, M; Reymann, K G; Reiser, G

    2001-08-01

    A variety of extracellular serine proteases are expressed in the central nervous system or might permeate the blood-brain barrier under pathological conditions. However, their intracerebral targets and physiological functions are largely unknown. Here, we show that four distinct subtypes of protease-activated receptors (PARs) are abundantly expressed in the adult rat brain and in organotypic hippocampal slice cultures. PAR-1 expression was significant in the hippocampus, cortex and amygdala. Highest densities of PAR-2 and PAR-3 were observed in hippocampus, cortex, amygdala, thalamus, hypothalamus and striatum. Apart from the striatum, a similar localization was found for PAR-4. Within the hippocampal formation, each PAR subtype was predominantly localized in the pyramidal cell layers. Additionally, we identified PAR-2 in mossy fibers between dentate gyrus and CA3, PAR-3 in the subiculum and PAR-4 in CA3 and in mossy fibres as well as in the stratum lacunosum moleculare. After exposing hippocampal slice cultures to a severe experimental ischemia (oxygen-glucose deprivation), the expression of PARs 1-3 was up-regulated with subtype-specific kinetics. The localization of PARs in brain regions particularly vulnerable to ischemic insults as well as distinct alterations in the expression pattern after experimental ischemia support the notion of an important role of extracellular serine proteases and PARs in cerebral ischemia.

  17. Protein Kinase D and Gβγ Subunits Mediate Agonist-evoked Translocation of Protease-activated Receptor-2 from the Golgi Apparatus to the Plasma Membrane.

    Science.gov (United States)

    Jensen, Dane D; Zhao, Peishen; Jimenez-Vargas, Nestor N; Lieu, TinaMarie; Gerges, Marina; Yeatman, Holly R; Canals, Meritxell; Vanner, Stephen J; Poole, Daniel P; Bunnett, Nigel W

    2016-05-20

    Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca(2+) signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases.

  18. Hippocampal expression of apoptotic protease activating factor-1 following diffuse axonal injury under mild hypothermia

    Institute of Scientific and Technical Information of China (English)

    Peng Yang; Limin Zhang; Yunhe Zhang; Xifeng Zou; Qunxi Li; Yun Li; Jun Zhu; Jianmin Li; Aijun Fu; Qingjun Liu; Tong Chen; Zelin Sun; Zhiyong Zhang

    2011-01-01

    The influence of mild hypothermia on neural cell apoptosis remains poorly understood. Therefore, the present study established rat models of diffuse axonal injury (DAI) at 33 °C. Morris water maze results demonstrated significantly better learning and memory functions in DAI rats with hypothermia compared with DAI rats with normothermia. Expression of apoptotic protease activating factor-1 in the hippocampal CA1 region was significantly lower in the DAI hypothermia group compared with the DAI normothermia group. Expression of apoptotic protease activating factor-1 positively correlated with latency, but negatively correlated with platform location times and time of swimming in the quadrant area. Results suggested that post-traumatic mild hypothermia in a rat model of DAI could provide cerebral protection by attenuating expression of apoptotic protease activating factor-1.

  19. Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates.

    Science.gov (United States)

    Saisawang, Chonticha; Sillapee, Pornpan; Sinsirimongkol, Kwanhathai; Ubol, Sukathida; Smith, Duncan R; Ketterman, Albert J

    2015-04-22

    Alphavirus nsP2 proteins are multifunctional and essential for viral replication. The protease role of nsP2 is critical for virus replication as only the virus protease activity is used for processing of the viral non-structural polypeptide. Chikungunya virus is an emerging disease problem that is becoming a world-wide health issue. We have generated purified recombinant chikungunya virus nsP2 proteins, both full length and a truncated protease domain from the C-terminus of the nsP2 protein. Enzyme characterization shows that the protease domain alone has different properties compared with the full length nsP2 protease. We also show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity. Moreover, the chikungunya nsP2 also appears to differ from other alphavirus nsP2 in its distinctive ability to recognize small peptide substrates.

  20. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

    Science.gov (United States)

    Watson, Steve P

    2009-01-01

    The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

  1. Activity of protease-activated receptors in primary cultured human myenteric neurons

    Directory of Open Access Journals (Sweden)

    Eva Maria Kugler

    2012-09-01

    Full Text Available Activity of the four known protease-activated receptors (PARs has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (AP. Application of the PAR1-AP (TFLLR or PAR4-AP (GYPGQV evoked spike discharge in 79% or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs of PAR1/PAR2 in 51%, PAR1/PAR4 in 43% and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system.

  2. Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals.

    Science.gov (United States)

    Wagstaff, Carol; Leverentz, Michael K; Griffiths, Gareth; Thomas, Brian; Chanasut, Usawadee; Stead, Anthony D; Rogers, Hilary J

    2002-02-01

    The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.

  3. Activation of human pro-urokinase by unrelated proteases secreted by Pseudomonas aeruginosa.

    NARCIS (Netherlands)

    Beaufort, N.; Seweryn, P.; Bentzmann, S. de; Tang, A.; Kellermann, J.; Grebenchtchikov, N.J.; Schmitt, M.; Sommerhoff, C.P.; Pidard, D.; Magdolen, V.

    2010-01-01

    Pathogenic bacteria, including Pseudomonas aeruginosa, interact with and engage the host plasminogen (Plg) activation system, which encompasses the urokinase (uPA)-type Plg activator, and is involved in extracellular proteolysis, including matrilysis and fibrinolysis. We hypothesized that secreted b

  4. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors.

    Science.gov (United States)

    Burtoloso, Antonio C B; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C; Gonçalez, Cristiana; Kenny, Peter W; Leitão, Andrei; Montanari, Carlos A; Quilles, José Carlos; Ribeiro, Jean F R; Rocha, Josmar R

    2017-02-01

    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases.

  5. Extracellular enzyme activity and biogeochemical cycling in restored prairies

    Science.gov (United States)

    Lynch, L.; Hernandez, D.; Schade, J. D.

    2011-12-01

    Winter microbial activity in mid-latitude prairie ecosystems is thermally sensitive and significantly influenced by snow depth. Snow insulates the soil column facilitating microbial processing of complex organic substrates. Previous studies in forests and tundra ecosystems suggest patterns of substrate utilization and limitation are seasonal; above freezing, soil microbes access fresh litter inputs and sugar exudates from plant roots, while under frozen condition they recycle nutrients incorporated in microbial biomass. In order to liberate nutrients required for carbon degradation, soil microbes invest energy in the production of extracellular enzymes that cleave monomers from polymer bonds. The inverse relationship between relative enzyme abundance and substrate availability makes enzyme assays a useful proxy to assess changes in resources over time. Our objective in this study was to assess patterns in microbial biomass, nutrient availability, and extracellular enzyme activity in four snow exclosure sites over a seven-month period. Over the past three years, we have maintained a snow removal experiment on two restored prairies in central Minnesota. In each prairie, snow was continuously removed annually from two 4 x 4 m plots by shoveling after each snow event. Extractable C, N and P, and microbial C, N and P in soil samples were measured in samples collected from these snow removal plots, as well as in adjacent unmanipulated prairie control plots. Pools of C, N, and P were estimated using standard extraction protocols, and microbial pools were estimated using chloroform fumigation direct extraction (CFDE). We conducted fluorometric extracellular enzyme assays (EEA) to assess how the degradation potential of cellulose (cellobiohydrolase, CBH), protein (leucine aminopeptidase, LAP), and phosphate esters (phosphatase, PHOS) changed seasonally. Microbial C and N declined between October and June, while microbial P declined during the fall and winter, but increased

  6. THE CONSISTOGRAPHIC DETERMINATION OF ENZYME ACTIVITY OF PROTEASE ON THE WAFFLE

    Directory of Open Access Journals (Sweden)

    Ioan DAVID

    2014-12-01

    Full Text Available The purpose of this research is to analise the action of protease, an exogenous enzyme used in different dosages on the waffle dough. Proteases hydrolyze the peptide bond which are found in the protein molecules, preferable at the level of positive aminoacids, which explaines their reduced action on the wheat flour proteins where the bazic aminoacids are in low proportion. In dough, this leads to weakening of the gluten chains. To avoid weakening the gluten structure, which result in an uneven texture of the dough structure, protease has to be added to the dough in the mixing phase. In these conditions, it will not reduce mixing time because the enzyme has not enough time to hydrolyze more gluten.The waffle doughs contain small amounts of water and fat, which makes them dense and therefore difficult to laminate layers. The addition of proteolytic enzymes allows adjustment of the rheological characteristics of dough according to the needs of the technological process. The consistograph method used in this study shows the rheological characteristics of the waffle dough presenting the effects caused by protease in: a low dosage, a correct dose and an overdose. The dough sample that has a low dose of protease does not have a significant improvement on the quality of the dough. On the other hand to much protease influences the dough handling during the technological process due to a wet and sticky content of the dough which leads to a low porosity, high humidity and a higher water activity. Using the correct dose of protease has positive effects on freshness, on the porosity and color of the crust.

  7. Antiretroviral activity of protease inhibitors against Toxoplasma gondii Terapia antiretroviral de inibidores da protease contra Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Lianet Monzote

    2013-02-01

    Full Text Available The introduction of highly active antiretroviral therapy (HAART has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE. These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.La introducción de la terapia antirretroviral de alta efectividad ha causada una marcada reducción en la ocurrencia y curso clínico de las infecciones parasitarias, incluyendo la toxoplasmosis encefálica (TE. Estos cambios han sido atribuidos a la restauración celular. Este estudio fue desarrollado para examinar la actividad de seis inhibidores de proteasas antirretrovirales (IPA sobre taquizoitos de Toxoplasma gondii. Los seis IPA mostraron actividad anti-Toxoplasma, con valores de CI50 entre 1.4 y 6.6 µg/mL. Futuros estudios a nivel molecular deben ser realizados, los cuales podrán delucidar si el uso de IPA pudiera beneficiar o no a los pacientes que sufren de TE.

  8. Genome-Wide Survey of Pseudomonas aeruginosa PA14 Reveals a Role for the Glyoxylate Pathway and Extracellular Proteases in the Utilization of Mucin.

    Science.gov (United States)

    Flynn, Jeffrey M; Phan, Chi; Hunter, Ryan C

    2017-08-01

    Chronic airway infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. Although this bacterium has been extensively studied for its virulence determinants, biofilm growth, and immune evasion mechanisms, comparatively little is known about the nutrient sources that sustain its growth in vivo Respiratory mucins represent a potentially abundant bioavailable nutrient source, although we have recently shown that canonical pathogens inefficiently use these host glycoproteins as a growth substrate. However, given that P. aeruginosa, particularly in its biofilm mode of growth, is thought to grow slowly in vivo, the inefficient use of mucin glycoproteins may be relevant to its persistence within the CF airways. To this end, we used whole-genome fitness analysis, combining transposon mutagenesis with high-throughput sequencing, to identify genetic determinants required for P. aeruginosa growth using intact purified mucins as a sole carbon source. Our analysis reveals a biphasic growth phenotype, during which the glyoxylate pathway and amino acid biosynthetic machinery are required for mucin utilization. Secondary analyses confirmed the simultaneous liberation and consumption of acetate during mucin degradation and revealed a central role for the extracellular proteases LasB and AprA. Together, these studies describe a molecular basis for mucin-based nutrient acquisition by P. aeruginosa and reveal a host-pathogen dynamic that may contribute to its persistence within the CF airways. Copyright © 2017 American Society for Microbiology.

  9. Characterizing proteases in an Antarctic Janthinobacterium sp. isolate:Evidence of a protease horizontal gene transfer event

    Institute of Scientific and Technical Information of China (English)

    Cecilia Martinez-Rosales; Juan Jos Marizcurrena; Andrs Iriarte; Natalia Fullana; Hctor Musto; Susana Castro-Sowinski

    2015-01-01

    We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AU11 (growth between 4°C and 30°C) produces a single cold-active extracellular protease (ExPAU11), differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as an alkaline metallo-protease (70% coverage with an extracellular protease of Janthinobacterium sp. PI12), and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene (named JSP8A) showing 60% identity (98%query coverage) to subtilisin peptidases belonging to the S8 family (S8A subfamily) of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from S8A bacterial sequences of other phyla (including its own). An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer (HGT) from a cyanobacterium. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

  10. Learning and memory deficits in mice lacking protease activated receptor-1.

    Science.gov (United States)

    Almonte, Antoine G; Hamill, Cecily E; Chhatwal, Jasmeer P; Wingo, Thomas S; Barber, Jeremy A; Lyuboslavsky, Polina N; David Sweatt, J; Ressler, Kerry J; White, David A; Traynelis, Stephen F

    2007-10-01

    The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1-/- animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally motivated learning.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Broad spectrum activity of a lectin-like bacterial serine protease family on human leukocytes.

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    Jorge Luis Ayala-Lujan

    Full Text Available The serine protease autotransporter from Enterobacteriaceae (SPATE family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system.

  13. A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex.

    Science.gov (United States)

    Siritapetawee, Jaruwan; Thumanu, Kanjana; Sojikul, Punchapat; Thammasirirak, Sompong

    2012-07-01

    A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis.

  14. Detection and localisation of the abalone probiotic Vibrio midae SY9 and its extracellular protease, VmproA, within the digestive tract of the South African abalone, Haliotis midae.

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    Robert J Huddy

    Full Text Available Probiotics have been widely reported to increase the growth rate of commercially important fish and shellfish by enhancing the digestion of ingested feed through the production of extracellular enzymes such as proteases and alginases. In order to investigate this further, the objective of this study was to localise the bacterial probiont Vibrio midae SY9 and one of the extracellular proteases it produces in the digestive tract of the South African abalone Haliotis midae. This was accomplished by inserting a promotorless gfp gene into the chromosome of the bacterium which was incorporated in an artificial, fishmeal-based abalone feed. In situ histological comparison of abalone fed either a basal diet or the basal diet supplemented with V. midae SY9::Tn10.52 using a cocktail of DNA probes to the gfp gene localised the probiont to the crop/stomach and intestinal regions of the H. midae digestive tract. Generally, the ingested probiotic bacterium occurred in association with feed and particulate matter within the crop/stomach and intestinal regions, as well as adhered to the wall of the crop/stomach. Histological immunohistochemical examination using polyclonal anti-VmproA antibodies localised an extracellular protease produced by V. midae SY9 to the H. midae crop/stomach and intestine where it appeared to be associated with feed and/or other particulate matter in the abalone gut. Thus the data suggests that V. midae SY9 colonises and/or adheres to the mucous lining of the abalone gut. Furthermore, the close association observed between the bacterium, its extracellular protease and ingested feed particles supports the theory that V. midae SY9 elevates in situ digestive enzyme levels and thus enhances feed digestion in farmed abalone.

  15. Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation

    DEFF Research Database (Denmark)

    Sondergaard, B C; Henriksen, K; Wulf, H

    2006-01-01

    explants were stimulated with oncostatin M (OSM) 10 ng/ml and tumor necrosis factor-alpha (TNF-alpha) 20 ng/ml in the presence or absence of the broad-spectrum MMP inhibitor GM6001 and the cysteine protease inhibitor, E64. Cartilage degradation was evaluated in the conditioned medium by glycosaminoglycans...... in vivo in CK null mice. CONCLUSION: Inhibition of MMP activity reduced both proteoglycan loss and type II collagen degradation. In contrast, inhibition of cysteine proteases resulted in an increase rather than a decrease in MMP derived fragments of collagen type II degradation, CTX-II, suggesting altered...

  16. AtDeg2 – a chloroplast protein with dual protease/chaperone activity

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    Przemysław Jagodzik

    2014-07-01

    Full Text Available Chloroplast protease AtDeg2 (an ATP-independent serine endopeptidase is cytosolically synthesized as a precursor, which is imported into the chloroplast stroma and deprived of its transit peptide. Then the mature protein undergoes routing to its functional location at the stromal side of thylakoid membrane. In its linear structure AtDeg2 molecule contains the protease domain with catalytic triad (HDS and two PDZ domains (PDZ1 and PDZ2. In vivo AtDeg2 most probably exists as a supposedly inactive haxamer, which may change its oligomeric stage to form active 12-mer, or 24-mer. AtDeg2 has recently been demonstrated to exhibit dual protease/chaperone function. This review is focused on the current awareness with regard to AtDeg2 structure and functional significance.

  17. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

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    Jun-Ha Hwang

    Full Text Available Mesenchymal stem cell (MSC differentiation is regulated by the extracellular matrix (ECM through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  18. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Science.gov (United States)

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  19. House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

    Directory of Open Access Journals (Sweden)

    Timmerman J André B

    2006-03-01

    Full Text Available Abstract House dust mite allergens (HDM cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1 and unknown enzymatic activity (Der p 2, Der p 5 induce biological responses in a human airway-derived epithelial cell line (A549, and if so, to elucidate the underlying mechanism(s of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

  20. Protease activated receptor-1 regulates macrophage-mediated cellular senescence : a risk for idiopathic pulmonary fibrosis

    NARCIS (Netherlands)

    Lin, Cong; Rezaee, Farhad; Waasdorp, Maaike; Shi, Kun; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a destructive disease in part resulting from premature or mature cellular aging. Protease-activated receptor-1 (PAR-1) recently emerged as a critical component in the context of fibrotic lung diseases. Therefore, we aimed to study the role of macrophages in PAR

  1. Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous nepenthes plants.

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    Franziska Buch

    Full Text Available Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep. Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

  2. Novel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine Proteases

    NARCIS (Netherlands)

    Verhelst, Steven H.L.; Witte, Martin D.; Arastu-Kapur, Shirin; Fonovic, Marko; Bogyo, Matthew

    2006-01-01

    Recent characterization of multiple classes of functionalized azapeptides as effective covalent inhibitors of cysteine proteases prompted us to investigate O-acyl hydroxamates and their azapeptide analogues for use as activity-based probes (ABPs). We report here a new class of azaglycine-containing

  3. Pharmacological targeting of protease-activated receptor 2 affords protection from bleomycin-induced pulmonary fibrosis

    NARCIS (Netherlands)

    C. Lin (Cong); J. von der Thusen (Jan); J. Daalhuisen (Joost); M. Ten Brink (Marieke); B. Crestani (Bruno); T. van der Poll (Tom); K. Borensztajn (Keren); C. Arnold Spek (C.)

    2015-01-01

    textabstractIdiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whet

  4. Blood coagulation-(in)dependent protease activated receptor signaling in pancreatic cancer

    NARCIS (Netherlands)

    K. Shi

    2015-01-01

    There is no doubt that pancreatic cancer is one of the most lethal diseases in general and current treatment options are limited. The studies described in this thesis pinpoint Protease activated receptors (PARs) as key players in pancreatic cancer progression. Inhibition of PAR-1 may be clinical rel

  5. Factor B structure provides insights into activation of the central protease of the complement system

    NARCIS (Netherlands)

    Milder, F.J.; Gomes, L.; Schouten, A.; Janssen, B.J.C.; Huizinga, E.G.; Romijn, R.A.; Hemrika, W.; Roos, A; Daha, M.R.; Gros, P.

    2007-01-01

    Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-A° resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domai

  6. Extracellular lipolytic enzyme activity of a newly isolated Debaryomyces hansenii.

    Science.gov (United States)

    Takaç, Serpil; Sengel, Banu S

    2010-01-01

    A strain isolated from waste of a milk products plant and exhibited extracellular lipolytic activity was identified as Debaryomyces hansenii by 5.8S rRNA and 28S rRNA gene sequence analyses. Lipolytic activity was assayed spectrophotometrically by using p-nitrophenylpalmitate. Higher specific lipolytic activities were obtained in the presence of tristearin (0.68 U/mg prot), oleic acid (0.56 U/mg prot), and soybean oil (0.36 U/mg prot) than other triglycerides, fatty acids, and vegetable oils considered as carbon sources. Cheese whey appeared to be a good alternative to lipidic substances for lipolytic activity. Among various organic and inorganic nitrogen sources, soy flour was found to attain the lipolytic activity similar to that provided by universal yeast medium components. This work is the first report on the discussion of lipolytic activity enhancement by D. hansenii through modulating the cultivation medium. It also proposes low cost medium nutrients that could be of industrial value and could serve as basal nutrients for further optimization studies on the lipase production by D. hansenii.

  7. Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities.

    Science.gov (United States)

    Sainsbury, Frank; Rhéaume, Ann-Julie; Goulet, Marie-Claire; Vorster, Juan; Michaud, Dominique

    2012-12-01

    Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.

  8. The effects of hypochlorous acid and neutrophil proteases on the structure and function of extracellular superoxide dismutase

    DEFF Research Database (Denmark)

    Morales, Karla; Olesen, Mads Nikolaj; Poulsen, Ebbe Toftgaard;

    2015-01-01

    environment in which enzymatic activity in general is challenged. In this study, we show that EC-SOD exposed to physiologically relevant concentrations of HOCl remains enzymatically active and retains the heparin-binding capacity, although HOCl exposure established oxidative modification of the N......-SOD irrespective of HOCl oxidation. Although the cleavage by elastase did not affect the quaternary structure, the cleavage by cathepsin G dissociated the molecule to produce EC-SOD monomers. The present data suggest that EC-SOD is stable and active at the site of inflammation and that neutrophils have...

  9. Protease activity in Giardia duodenalis trophozoites of axenic strains isolated from symptomatic and asymptomatic patients

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    Guimarães Semíramis

    2003-01-01

    Full Text Available We have examined by gelatin-SDS-PAGE the protease activity in cell lysates of Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic patient (BTU-11 and an asymptomatic carrier (BTU-10, and the reference strain Portland 1 (P1. The proteolysis band patterns showed differences among strains isolated from asymptomatic and symptomatic individuals. The lysate of the strain BTU-10, showed only five hydrolysis bands, while a greater number of bands (10-11 bands was seen in strains BTU-11 and P1. The protease activity in all lysates was inhibited by cysteine (E-64 and iodoacetamide and serine proteases (TPCK and TLCK inhibitors, but not by PMSF and EDTA. In general, the results revealed protease activities in G. duodenalis trophozoites of Brazilian axenic strains and the predominance of cysteine proteinases. It should be stressed the inter-strain difference in hydrolysis band patterns observed between strains isolated from symptomatic patients and the strain obtained from an asymptomatic carrier.

  10. Activity, specificity, and probe design for the smallpox virus protease K7L.

    Science.gov (United States)

    Aleshin, Alexander E; Drag, Marcin; Gombosuren, Naran; Wei, Ge; Mikolajczyk, Jowita; Satterthwait, Arnold C; Strongin, Alex Y; Liddington, Robert C; Salvesen, Guy S

    2012-11-16

    The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

  11. The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection

    OpenAIRE

    Kawabata, Atsufumi; Kinoshita, Mitsuhiro; Nishikawa, Hiroyuki; Kuroda, Ryotaro; Nishida, Minoru; Araki, Hiromasa; Arizono, Naoki; Oda, Yasuo; Kakehi, Kazuaki

    2001-01-01

    Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2–activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsai...

  12. Active endocannabinoids are secreted on extracellular membrane vesicles.

    Science.gov (United States)

    Gabrielli, Martina; Battista, Natalia; Riganti, Loredana; Prada, Ilaria; Antonucci, Flavia; Cantone, Laura; Matteoli, Michela; Maccarrone, Mauro; Verderio, Claudia

    2015-02-01

    Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.

  13. Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas

    DEFF Research Database (Denmark)

    Pedersen, Tanja Xenia; Pennington, Caroline J; Almholt, Kasper;

    2005-01-01

    of urokinase plasminogen activator, plasminogen activator inhibitor 1 and the matrix metalloproteases MMP-2, -3, -11, -13 and -14, and found that all these seven genes are predominantly expressed by stromal cells. Our results were qualitatively supported by in situ hybridization analysis of the expression of m......RNAs for MMP-2, -3 and -13 in the PyMT tumors. Statistical analyses indicated that the quantitative expression patterns observed in cancer and stromal cells isolated from individual tumors from different PyMT mice are quite reproducible. The methodology described in this study provides excellent tools to study...

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

    Science.gov (United States)

    Friis, Stine; Sales, Katiuchia Uzzun; Schafer, Jeffrey Martin; Vogel, Lotte K; Kataoka, Hiroaki; Bugge, Thomas H

    2014-08-01

    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 the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial 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 was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.

  15. Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Dennis J Grab

    Full Text Available BACKGROUND: Using human brain microvascular endothelial cells (HBMECs as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain. In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs known as protease activated receptors (PARs that might be implicated in calcium signaling by African trypanosomes. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA interference (RNAi we found that in vitro PAR-2 gene (F2RL1 expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%-49% and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Galpha(q with Pasteurella multocida toxin (PMT. PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified. CONCLUSIONS/SIGNIFICANCE: Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.

  16. Methods for producing dairy products, in particular cheese using lactic acid bacteria provided with additional neutral protease activity

    NARCIS (Netherlands)

    Mierau, Igor; van den Burg, Lambertus; Kok, Jan; Venema, Gerhardus

    1997-01-01

    The present invention relates to a method for carrying out a process of biotransformation of a substrate whereby at least one lactic acid bacterium comprising a gene encoding a neutral protease having an altered resulting activity, e.g. a neutral protease having an altered stability and/or specifici

  17. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Raheem Ullah

    Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  18. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Science.gov (United States)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  19. Protection of protease-activated receptor 2 mediated vasodilatation against angiotensin II-induced vascular dysfunction in mice

    OpenAIRE

    Chia, Elizabeth; Kagota, Satomi; Wijekoon, Enoka P; McGuire, John J

    2011-01-01

    Background Under conditions of cardiovascular dysfunction, protease-activated receptor 2 (PAR2) agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase-2, nitric oxide synthase and calcium-activated potassium channel (SK3.1) activities. Protease-activated receptor 2 agonist mediated vasodilatation is unknown under conditions of dysfunction caused by angiotensin II. The main purpose of our study was to determine whether PAR2-induced vasodilatation of re...

  20. Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants.

    Science.gov (United States)

    Kovács, Judit; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2016-06-01

    The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

  1. Comparison of alpha-amylase and protease activities of a zoophytophagous and two phytozoophagous Heteroptera.

    Science.gov (United States)

    Zeng, F; Cohen, A C

    2000-05-01

    To better understand the nature of facultative phytophagy in the zoophytophagous Geocoris punctipes (Say), and facultative zoophagy in phytozoophagous Lygus hesperus (Knight) and Lygus lineolaris (Palisot de Beauvois), we compared the activities of both the starch digesting enzyme alpha-amylase and of general proteases in these species. The alpha-amylases and proteases were demonstrated in L. hesperus, L. lineolaris and G. punctipes. The presence of alpha-amylase in the salivary gland complexes of G. punctipes indicates a disposition of this species toward utilization of nutrients that can be derived only from plants, either directly from ingestion of plant macromolecules or from second-hand ingestion of plant material from the digestive system of their prey. The alpha-amylase activity in G. punctipes was much less than those of phytozoophagous L. hesperus and L. lineolaris. The relative importance of amylolytic activity and proteolytic activity is also discussed.

  2. Pharmacological inhibition of MALT1 protease activity protects mice in a mouse model of multiple sclerosis

    OpenAIRE

    Mc Guire, Conor; Elton, Lynn; Wieghofer, Peter; Staal, Jens; Voet, Sofie; Demeyer, Annelies; Nagel, Daniel; Krappmann, Daniel; Prinz, Marco; Beyaert, Rudi; van Loo, Geert

    2014-01-01

    Background: The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is crucial for lymphocyte activation through signaling to the transcription factor NF-kappa B. Besides functioning as a scaffold signaling protein, MALT1 also acts as a cysteine protease that specifically cleaves a number of substrates and contributes to specific T cell receptor-induced gene expression. Recently, small molecule inhibitors of MALT1 proteolytic activity were identified and sho...

  3. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses

    OpenAIRE

    Jong W Yu; Sandy Hoffman; Allison M Beal; Angela Dykon; Michael A Ringenberg; Anna C Hughes; Lauren Dare; Amber D Anderson; Joshua Finger; Viera Kasparcova; David Rickard; Scott B Berger; Joshi Ramanjulu; John G Emery; Peter J Gough

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 pr...

  4. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  5. Delivery of a Protease-Activated Cytolytic Peptide Prodrug by Perfluorocarbon Nanoparticles.

    Science.gov (United States)

    Jallouk, Andrew P; Palekar, Rohun U; Marsh, Jon N; Pan, Hua; Pham, Christine T N; Schlesinger, Paul H; Wickline, Samuel A

    2015-08-19

    Melittin is a cytolytic peptide derived from bee venom that inserts into lipid membranes and oligomerizes to form membrane pores. Although this peptide is an attractive candidate for treatment of cancers and infectious processes, its nonspecific cytotoxicity and hemolytic activity have limited its therapeutic applications. Several groups have reported the development of cytolytic peptide prodrugs that only exhibit cytotoxicity following activation by site-specific proteases. However, systemic administration of these constructs has proven difficult because of their poor pharmacokinetic properties. Here, we present a platform for the design of protease-activated melittin derivatives that may be used in conjunction with a perfluorocarbon nanoparticle delivery system. Although native melittin was substantially hemolytic (HD50: 1.9 μM) and cytotoxic (IC50: 2.4 μM), the prodrug exhibited 2 orders of magnitude less hemolytic activity (HD50: > 100 μM) and cytotoxicity (IC50: > 100 μM). Incubation with matrix metalloproteinase-9 (MMP-9) led to cleavage of the prodrug at the expected site and restoration of hemolytic activity (HD50: 3.4 μM) and cytotoxicity (IC50: 8.1 μM). Incubation of the prodrug with perfluorocarbon nanoparticles led to stable loading of 10,250 peptides per nanoparticle. Nanoparticle-bound prodrug was also cleaved and activated by MMP-9, albeit at a fourfold slower rate. Intravenous administration of prodrug-loaded nanoparticles in a mouse model of melanoma significantly decreased tumor growth rate (p = 0.01). Because MMPs and other proteases play a key role in cancer invasion and metastasis, this platform holds promise for the development of personalized cancer therapies directed toward a patient's individual protease expression profile.

  6. Thalidomide combined with irradiation alters the activity of two proteases.

    Science.gov (United States)

    Şimşek, Ece; Aydemir, Esra; Korcum, Aylin Fidan; Fişkın, Kayahan

    2015-02-01

    The aim of the present study was to investigate the effects of thalidomide, a drug known for its anti‑angiogenic and antitumor properties, at its cytotoxic dose previously determined as 40 µg/ml (according to four cytotoxic test results). The effect of the drug alone and in combination with radiotherapy using Cobalt 60 (60Co) at 45 Gy on the enzymatic activity of substance‑P degrading A disintegrin and metalloproteinase (ADAM)10 and neprilysin (NEP) was investigated in the mouse breast cancer cell lines 4T1 and 4T1 heart metastases post‑capsaicin (4THMpc). Thalidomide (40 µg/ml) exerted differing effects on the activities of ADAM10 and NEP enzymes. In 4T1 cells, 40 µg/ml thalidomide alone did not alter ADAM10 enzyme activity. 60Co irradiation at 45 Gy alone caused a 42% inhibition in ADAM10 activity, however, the inhibition increased to 89% when combined therapy was used. By contrast, in the 4THMpc cell line, 40 µg/ml thalidomide alone induced a 66.6% increase in ADAM10 enzyme activity. Radiotherapy alone and thalidomide with 60Co combined therapy caused a 33.3 and 40% inhibition of ADAM10 activity, respectively. In 4T1 cells, thalidomide alone caused a 40.9% increase in NEP activity. Radiation therapy alone or in combination with the drug caused a 40.7% increase in NEP activity. In more aggressive 4THMpc cells, thalidomide alone caused a 26.6% increase in NEP activity. Radiotherapy alone and combined therapy caused a 33.3 and 37% increase in enzyme activity, respectively. To the best of our knowledge, the present study is the first to demonstrate that thalidomide alone or in combination with radiotherapy exhibits significant cytotoxic effects on 4T1 and 4THMpc mouse breast cancer cell lines indicating that this drug affects the enzymatic activity of ADAM10 and NEP in vitro.

  7. Factor Xa stimulates proinflammatory and profibrotic responses in fibroblasts via protease-activated receptor-2 activation

    NARCIS (Netherlands)

    Borensztajn, Keren; Stiekema, Jurrieen; Nijmeijer, Sebastiaan; Reitsmalf, Pieter H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

    Coagulation proteases have been suggested to play a role in the pathogenesis of tissue remodeling and fibrosis. We therefore assessed the proinflammatory and fibroproliferative effects of coagulation protease factor (F)Xa. We show that FXa elicits a signaling response in C2C12 and NIH3T3

  8. The total protein content, protein fractions and proteases activities of drone prepupae of Apis mellifera due to varrosis.

    Science.gov (United States)

    Zółtowska, Krystyna; Lipiński, Zbigniew; Dmitryjuk, Małgorzata

    2005-01-01

    The proteins level and activities of acid and alkaline proteases in whole body extracts of drone prepupae of Apis mellifera naturally infested with Varroa destructor were studied. The infested and a non-infested group did not differ significantly in their total protein content. However, some differences in protein profiles were found. A lack of three protein fractions of moderate and lower molecular weight in infested prepupae was noted. Moreover, some differences in the quantity of protein in most of the fractions were observed. The activity of acid proteases from infested prepupae was lower (p drone had higher activity of alkaline proteases than non-infested but this difference was not statisticaly significant.

  9. Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

    Science.gov (United States)

    Perez, Michel; Lamothe, Marie; Maraval, Catherine; Mirabel, Etienne; Loubat, Chantal; Planty, Bruno; Horn, Clemens; Michaux, Julien; Marrot, Sebastien; Letienne, Robert; Pignier, Christophe; Bocquet, Arnaud; Nadal-Wollbold, Florence; Cussac, Didier; de Vries, Luc; Le Grand, Bruno

    2009-10-08

    Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

  10. Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives.

    Science.gov (United States)

    Sun, Changsheng; Liang, Jiongqiu; Shi, Rui; Gao, Xuna; Zhang, Ruijuan; Hong, Fulin; Yuan, Qihang; Wang, Shengbin

    2012-03-01

    Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.

  11. Hemoglobinase activity of the lysine gingipain protease (Kgp) of Porphyromonas gingivalis W83.

    Science.gov (United States)

    Lewis, J P; Dawson, J A; Hannis, J C; Muddiman, D; Macrina, F L

    1999-08-01

    Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the alpha chain and 10 Lys residues in the beta chain. In contrast, there are only three Arg residues in each of the alpha and beta chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalis is a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.

  12. Protease activity of MALT1: a mystery unravelled.

    Science.gov (United States)

    Kirchhofer, Daniel; Vucic, Domagoj

    2012-06-01

    Constitutive NF-κB (nuclear factor κB) activation in B-cell lymphomas relies greatly on the CARMA1 [CARD (caspase recruitment domain)-containing MAGUK (membrane-associated guanylate kinase) 1]-Bcl10-MALT1 (mucosa-associated lymphoid tissue translocation gene 1) signalling complex. Within this protein complex, MALT1 possesses a rather unique enzymatic activity, which allows it to cleave Bcl10, RelB and CYLD, among other substrates. The catalytic activity of MALT1 promotes activation of canonical and non-canonical NF-κB as well as other signalling pathways. However, even after a decade of intense research on MALT1, many mechanistic aspects of its enzymatic activity remain elusive. A recent article by Hachmann, Snipas, van Raam, Cancino, Houlihan, Poreba, Kasperkiewicz, Drag and Salvesen [(2012) Biochem. J. 443, 287-295] provides novel insight into the activation mechanism and the substrate specificity of MALT1. These intriguing findings convincingly demonstrate the importance of MALT1 dimerization for its catalytic activity and pave the way for novel therapeutic approaches that target this crucial regulator of lymphoma survival and proliferation.

  13. Epicutaneous Administration of Papain Induces IgE and IgG Responses in a Cysteine Protease Activity-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Hideo Iida

    2014-01-01

    Conclusions: We demonstrated that the epicutaneous administration of protease not only disrupted skin barrier function, but also induced IgE and IgG responses in a manner dependent on its protease activity. These results suggest that protease activity contained in environmental sources contributes to sensitization through an epicutaneous route.

  14. Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP).

    Science.gov (United States)

    Zhao, Hong-Yan; Wu, Li-Ying; Liu, Gang; Feng, Hong

    2016-12-01

    To engineer dehairing alkaline protease (DHAP) variants to improve cold activity and increase thermostability so these variants are suitable for the leather processing industry. Based on previous studies with bacterial alkaline proteases, double-site mutations (W106K/V149I and W106K/M124L) were introduced into the DHAP from Bacillus pumilus. Compared with the wild-type DHAP hydrolytic activity, the double-site variant W106K/V149I showed an increase in specific hydrolytic activity at 15 °C by 2.3-fold toward casein in terms of hydrolytic rate and 2.7-fold toward the synthetic peptide AAPF-pN by means of kcat/Km value. The thermostability of the variant (W106K/V149I) was improved with the half-life at 60 and 70 °C increased by 2.7- and 5.0-fold, respectively, when compared with the thermostability of the wild-type DHAP. Conclusively, an increase in the cold activity and thermostability of a bacterial alkaline protease was achieved by protein engineering.

  15. Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soils provide numerous essential ecosystem services such as carbon cycling...... the functional diversity and activity of the microorganisms involved in decomposition processes. Their activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysacharides as cellulose, hemicellulose...

  16. The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2

    OpenAIRE

    2011-01-01

    Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inh...

  17. Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

    Energy Technology Data Exchange (ETDEWEB)

    Lupardus, P.J.; Shen, A.; Bogyo, M.; Garcia, K.C.

    2009-05-19

    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  18. Structure-activity relationships of heteroaromatic esters as human rhinovirus 3C protease inhibitors.

    Science.gov (United States)

    Im, Isak; Lee, Eui Seung; Choi, Soo Jeong; Lee, Ju-Yeon; Kim, Yong-Chul

    2009-07-01

    Human rhinovirus 3C protease (HRV 3C(pro)) is known to be a promising target for development of therapeutic agents against the common cold because of the importance of the protease in viral replication as well as its expression in a large number of serotypes. To explore non-peptidic inhibitors of HRV 3C(pro), a series of novel heteroaromatic esters was synthesized and evaluated for inhibitory activity against HRV 3C(pro), to determine the structure-activity relationships. The most potent inhibitor, 7, with a 5-bromopyridinyl group, had an IC(50) value of 80nM. In addition, the binding mode of a novel analog, 19, with the 4-hydroxyquinolinone moiety, was explored by molecular docking, suggesting a new interaction in the S1 pocket.

  19. Hemolysin, Protease, and EPS Producing Pathogenic Aeromonas hydrophila Strain An4 Shows Antibacterial Activity against Marine Bacterial Fish Pathogens

    Directory of Open Access Journals (Sweden)

    Anju Pandey

    2010-01-01

    Full Text Available A pathogenic Aeromonas hydrophila strain An4 was isolated from marine catfish and characterized with reference to its proteolytic and hemolytic activity along with SDS-PAGE profile (sodium dodecyl sulphate-Polyacrylamide gel electrophoresis of ECPs (extracellular proteins showing hemolysin (approximately 50 kDa. Agar well diffusion assay using crude cell extract of the bacterial isolate clearly demonstrated antibacterial activity against indicator pathogenic bacteria, Staphylococcus arlettae strain An1, Acinetobacter sp. strain An2, Vibrio parahaemolyticus strain An3, and Alteromonas aurentia SE3 showing inhibitory zone >10 mm well comparable to common antibiotics. Further GC-MS analysis of crude cell extract revealed several metabolites, namely, phenolics, pyrrolo-pyrazines, pyrrolo-pyridine, and butylated hydroxytoluene (well-known antimicrobials. Characterization of EPS using FTIR indicated presence of several protein-related amine and amide groups along with peaks corresponding to carboxylic and phenyl rings which may be attributed to its virulent and antibacterial properties, respectively. Besides hemolysin, EPS, and protease, Aeromonas hydrophila strain An4 also produced several antibacterial metabolites.

  20. Increasing the alkaline protease activity of Bacillus cereus and ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... activities of B. polymyxa and B. cereus were measured at the death phase of the growth curve and at the start of the ... in glucose synthetic medium at 30°C with 100 rpm aeration speed for 144 h. .... surface, gray to light brown.

  1. Cockroach protease allergen induces allergic airway inflammation via epithelial cell activation

    Science.gov (United States)

    Kale, Sagar L.; Agrawal, Komal; Gaur, Shailendra Nath; Arora, Naveen

    2017-01-01

    Protease allergens are known to enhance allergic inflammation but their exact role in initiation of allergic reactions at mucosal surfaces still remains elusive. This study was aimed at deciphering the role of serine protease activity of Per a 10, a major cockroach allergen in initiation of allergic inflammation at mucosal surfaces. We demonstrate that Per a 10 increases epithelial permeability by disruption of tight junction proteins, ZO-1 and occludin, and enhances the migration of Monocyte derived dendritic cell precursors towards epithelial layer as exhibited by trans-well studies. Per a 10 exposure also leads to secretion of IL-33, TSLP and intracellular Ca2+ dependent increase in ATP levels. Further, in vivo experiments revealed that Per a 10 administration in mice elevated allergic inflammatory parameters along with high levels of IL-33, TSLP, IL-1α and uric acid in the mice lungs. We next demonstrated that Per a 10 cleaves CD23 (low affinity IgE receptor) from the surface of PBMCs and purified B cells and CD25 (IL-2 receptor) from the surface of PBMCs and purified T cells in an activity dependent manner, which might favour Th2 responses. In conclusion, protease activity of Per a 10 plays a significant role in initiation of allergic airway inflammation at the mucosal surfaces. PMID:28198394

  2. Differentiating Intracellular from Extracellular Alkaline Phosphatase Activity in Soil by Sonication

    NARCIS (Netherlands)

    Qin, S.P.; Hu, C.S.; Oenema, O.

    2013-01-01

    Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio

  3. Structure of the Mature Streptococcal Cysteine Protease Exotoxin mSpeB in Its Active Dimeric Form

    DEFF Research Database (Denmark)

    Olsen, Johan G; Dagil, Robert; Niclasen, Louise Meinert

    2009-01-01

    Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 A, disclosing....... Based on the present structure, the active site of clan CA cysteine proteases is expanded and a detailed mechanism of the deacylation mechanism is proposed. The results may have applications for the development of protease inhibitors specific to bacterial cysteine proteases....... a homodimer. A serine from one subunit inserts into the active site of the other to donate to the oxyanion hole and coordinates the ligand proximal to the active-site cysteine. Dimerization is unique to the mature form and is clearly a prerequisite for catalysis. The present structure supports a tripartite...

  4. Inactivation of genes encoding extracellular proteases in bacillus halodurans BhFC01 and the impact on its modified flagellin type III secretion pathway towards improving peptide expression

    CSIR Research Space (South Africa)

    Berger, E

    2009-01-01

    Full Text Available C-NC6 containing fractions were collected and protein precipitation was 21 performed with 10% TCA as described above. 22 23 24 25 11 One-dimensional SDS-PAGE and protease zymography. 1 Protein electrophoresis was performed in a vertical gel...). Following electrophoresis, the gels were stained using 5 colloidal Coomassie Blue G-250 gel stain (8). 6 7 For protease zymograms the different protease deficient strains were grown into stationary 8 phase (24 hours) in LB pH 8.5 at 37oC. Cells were...

  5. Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort.

    Science.gov (United States)

    Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke; Lendeckel, Uwe

    2017-03-01

    Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the "Greifswald Approach to Individualized Medicine" (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin-angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is

  6. Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity reflects the functional diversity and activity of the microorganisms involved in decomposition processes which are essential processes for soil functioning...... and soil ecosystem services. The soil enzyme activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysaccharides as cellulose, hemicellulose and chitin, while degradation of proteins has been...... followed by amino-methyl-coumaric substrates (AMC). The already developed Extracellular Enzyme Activity (EEA) assay was further optimized as a microwell based assay for the activity of enzymes involved in degradation of polysaccharides and proteins. Using specific MUF and AMC substrates on European soils...

  7. Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    of soil microbial functions is still needed. In soil, enzymes originate from a variety of organisms, notably fungi and bacteria and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity will reflect...... the functional diversity and activity of the microorganisms involved in decomposition processes. Their activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysacharides as cellulose, hemicellulose...... and chitin, while degradation of proteins has been followed by amino-methyl-coumaric substrates (AMC). Based on these fluorogenic substrates the Extracellular Enzyme Activity assay was optimized as a microwell based standardized assay for the activity of enzymes involved in degradation of polysaccharides...

  8. β-Amyloid induces nuclear protease-mediated lamin fragmentation independent of caspase activation.

    Science.gov (United States)

    Ramasamy, Vijay Sankar; Islam, Md Imamul; Haque, Md Aminul; Shin, Song Yub; Park, Il-Seon

    2016-06-01

    β-Amyloid (Aβ), a hallmark peptide of Alzheimer's disease, induces both caspase-dependent apoptosis and non-apoptotic cell death. In this study, we examined caspase-independent non-apoptotic cell death preceding caspase activation in Aβ42-treated cells. We first determined the optimal treatment conditions for inducing cell death without caspase activation and selected a double-treatment method involving the incubation of cells with Aβ42 for 4 and 6 h (4+6 h sample). We observed that levels of lamin A (LA) and lamin B (LB) were reduced in the 4+6 h samples. This reduction was decreased by treatment with suc-AAPF-CMK, an inhibitor of nuclear scaffold (NS) protease, but not by treatment with z-VAD-FMK, a pan-caspase inhibitor. In addition, suc-AAPF-CMK decreased the changes in nuclear morphology observed in cells in the 4+6 h samples, which were different from nuclear fragmentation observed in STS-treated cells. Furthermore, suc-AAPF-CMK inhibited cell death in the 4+6 h samples. LA and LB fragmentation occurred in the isolated nuclei and was also inhibited by suc-AAPF-CMK. Together, these data indicated that the fragmentation of LA and LB in the Aβ42-treated cells was induced by an NS protease, whose identity is not clearly determined yet. A correlation between Aβ42 toxicity and the lamin fragmentation by NS protease suggests that inhibition of the protease could be an effective method for controlling the pathological process of AD.

  9. INFLUENCE OF VARIOUS ENVIRONMENTAL PARAMETERS ON PROTEASE SECRETION FROM BACILLUS SUBTILIS DKMNR

    Directory of Open Access Journals (Sweden)

    D. Kezia

    2011-03-01

    Full Text Available A protease producing microorganism was isolated from soil collected from garden soil samples around the department of Chemical Engineering, Andhra University, Vizag, A.P, India and identified as bacillus species. The isolate DKMNR produced alkaline protease the optimum conditions for protease activity was 330C at pH 9 with 2% inoculum in the medium after 24 h of incubation with an agitation speed of 200 rpm and EDTA was found to be inhibitor of alkaline protease. The extracellular production of the enzyme, its alkaline nature and compatibility with most commercial detergents are features which suggest its application in the detergent industry.

  10. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    DEFF Research Database (Denmark)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban

    2016-01-01

    and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three......Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its...

  11. Staphylococcal SplB serine protease utilizes a novel molecular mechanism of activation.

    Science.gov (United States)

    Pustelny, Katarzyna; Zdzalik, Michal; Stach, Natalia; Stec-Niemczyk, Justyna; Cichon, Przemyslaw; Czarna, Anna; Popowicz, Grzegorz; Mak, Pawel; Drag, Marcin; Salvesen, Guy S; Wladyka, Benedykt; Potempa, Jan; Dubin, Adam; Dubin, Grzegorz

    2014-05-30

    Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process.

  12. Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular Enzyme Activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soil enzymes originate from a variety of organisms, notably fungi and bacteria...... and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity reflects the functional diversity and activity of the microorganisms involved in decomposition processes which are essential processes for soil functioning......, experimental conditions of extraction of enzymes from soils, buffer and pH, substrate concentration, temperature and the necessary controls were optimized and standardized. This has resulted in an optimized standard operating procedure of EEA, which are being tested as an indicator of soil functional diversity...

  13. Partial characterization of cold active amylases and proteases of Streptomyces sp. from Antarctica

    Directory of Open Access Journals (Sweden)

    Mihaela Cotârleţ

    2011-09-01

    Full Text Available The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100% identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20ºC, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20ºC. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures.

  14. Structural Insights into the Activation and Inhibition of Histo-Aspartic Protease from Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi; Gustchina, Alla; Kiso, Yoshiaki; Yada, Rickey Y.; Wlodawer, Alexander (Guelph); (Kyoto); (NCI)

    2012-09-17

    Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 {angstrom} resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.

  15. Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus.

    Science.gov (United States)

    Salles, Hévila Oliveira; Braga, Ana Carolina Linhares; Nascimento, Maria Thayana dos Santos Canuto do; Sousa, Ana Márjory Paiva; Lima, Adriano Rodrigues; Vieira, Luiz da Silva; Cavalcante, Antônio Cézar Rocha; Egito, Antonio Silvio do; Andrade, Lúcia Betânia da Silva

    2014-01-01

    Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (Libidibia ferrea, Moringa oleifera and Ricinus communis (P0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

  16. Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus

    Directory of Open Access Journals (Sweden)

    Hévila Oliveira Salles

    Full Text Available Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (0.05, Bonferroni test. Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

  17. On Blastocystis secreted cysteine proteases: a legumain-activated cathepsin B increases paracellular permeability of intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Nourrisson, C; Wawrzyniak, I; Cian, A; Livrelli, V; Viscogliosi, E; Delbac, F; Poirier, P

    2016-11-01

    Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.

  18. Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

    Directory of Open Access Journals (Sweden)

    Hideki Hayashi

    Full Text Available Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026;We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy.Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

  19. The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Ashis K Mukherjee

    Full Text Available Proteases from Russell's viper venom (RVV induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼ 42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

  20. Variation in activity of root extracellular phytase between genotypes of barley

    DEFF Research Database (Denmark)

    Asmar, Mohammad Farouq

    1997-01-01

    Barley genotypes grown in nutrient solution under P nutrient stress and sterile conditions were compared in activity of root-associated and root-released extracellular phytase. The activity of root-associated phytase of all genotypes was about 10 times higher than that of root-released phytase...... and the genotypes performed differently with regard to the activity of the enzymes. The winter barley genotype, Marinka had the highest activity of root-associated extracellular phytase which differed significantly from Alexis and Senate, but not from Regatta. Alexis showed the lowest activity of root......-released extracellular phytase which differed significantly from those of Marinka and Regatta, but not from Senate. Generally, there was a significant correlation between the activity of root-associated and released extracellular phytase....

  1. Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

    LENUS (Irish Health Repository)

    Gleeson, Eimear M

    2013-07-19

    Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

  2. Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01.

    Science.gov (United States)

    Divakar, K; Suryia Prabha, M; Nandhinidevi, G; Gautam, P

    2017-04-21

    The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking-Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820 × 10(3) U/L and extracellular protease activity of 172 × 10(3) U/L were obtained at the 16th hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.

  3. Extracellular engagement of ADAM12 induces clusters of invadopodia with localized ectodomain shedding activity

    DEFF Research Database (Denmark)

    Albrechtsen, Reidar; Hansen, Dorte Stautz; Sanjay, Archana;

    2011-01-01

    -Src interaction site in the ADAM12 cytoplasmic domain, but was independent of the catalytic activity of ADAM12. Caveolin-1 and transmembrane protease MMP14/MT1-MMP were both present in the ADAM12-induced clusters of invadopodia, and cholesterol depletion prevented their formation, suggesting that lipid-raft...

  4. Searching for halo-alkalophilic proteases maintaining stability and activity in hydrophilic aprotic solvents as biocatalysts in carbohydrate chemistry

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Mørkholt, Camilla Kær; Nielsen, Carsten Bue

    2012-01-01

    and other carbohydrates (1, 2). Therefore, we are searching for new bacterial halo-alkaline proteases from extreme environments in Denmark. So far we have identified a number of interesting isolates showing proteolytic activity at pH 7 and 10. Whole genome Illumina Hiseq 2000 amplicon sequencing, de novo...... assembly of the genome and automatic annotation of genes in the genome was carried out by RAST to find the genes responsible for protease production. The amino-acid sequence of the expressed proteolytic proteins was analyzed using tandem MS. The isolates identified so far belong to Bacillus sp. One...... of the interesting genomes with a total length of 4.1 Mb was assembled in 42 contigs with a total of 3070 known genes of which 39 are encoding for proteases (one serine endopeptidase). Another genome was assembled in 219 contigs with 4004 coding sequences and 2821 known genes of which 36 are encoding for proteases...

  5. MERS-CoV papain-like protease has deISGylating and deubiquitinating activities.

    Science.gov (United States)

    Mielech, Anna M; Kilianski, Andy; Baez-Santos, Yahira M; Mesecar, Andrew D; Baker, Susan C

    2014-02-01

    Coronaviruses encode papain-like proteases (PLpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (DUB)/deISGylating activity, which is hypothesized to modify the innate immune response to infection. Here, we investigate the predicted DUB activity of the PLpro domain of the recently described Middle East Respiratory Syndrome Coronavirus (MERS-CoV). We found that expression of MERS-CoV PLpro reduces the levels of ubiquitinated and ISGylated host cell proteins; consistent with multifunctional PLpro activity. Further, we compared the ability of MERS-CoV PLpro and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) PLpro to block innate immune signaling of proinflammatory cytokines. We show that expression of SARS-CoV and MERS-CoV PLpros blocks upregulation of cytokines CCL5, IFN-β and CXCL10 in stimulated cells. Overall these results indicate that the PLpro domains of MERS-CoV and SARS-CoV have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis.

  6. Urinary serine proteases and activation of ENaC in kidney

    DEFF Research Database (Denmark)

    Svenningsen, Per; Andersen, Henrik; Nielsen, Lise Hald

    2015-01-01

    pressure, while gain-of-function mutations lead to impaired Na(+) excretion, hypertension, and hypokalemia. We review an emerging pathophysiological concept that aberrant glomerular filtration of plasma proteases, e.g., plasmin, prostasin, and kallikrein, contributes to proteolytic activation of ENaC, both...... with albuminuria compatible with impaired renal Na(+) excretion: hypertension and volume retention is secondary to proteinuria in, e.g., preeclampsia and nephrotic syndrome; plasma concentrations of renin, angiotensin II, and aldosterone are frequently suppressed in proteinuric conditions, e.g., preeclampsia...

  7. Design and structure-activity relationships of novel inhibitors of human rhinovirus 3C protease.

    Science.gov (United States)

    Kawatkar, S P; Gagnon, M; Hoesch, V; Tiong-Yip, C; Johnson, K; Ek, M; Nilsson, E; Lister, T; Olsson, L; Patel, J; Yu, Q

    2016-07-15

    Human rhinovirus (HRV) is a primary cause of common cold and is linked to exacerbation of underlying respiratory diseases such as asthma and COPD. HRV 3C protease, which is responsible for cleavage of viral polyprotein in to proteins essential for viral life-cycle, represents an important target. We have designed proline- and azetidine-based analogues of Rupintrivir that target the P2 pocket of the binding site. Potency optimization, aided with X-ray crystallography and quantum mechanical calculations, led to compounds with activity against a broad spectrum of HRV serotypes. Altogether, these compounds represent alternative starting points to identify promising leads in our continual efforts to treat HRV infections.

  8. Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae

    Science.gov (United States)

    Mondal, Ayan; Tapader, Rima; Chatterjee, Nabendu Sekhar; Ghosh, Amit; Sinha, Ritam; Koley, Hemanta; Saha, Dhira Rani; Chakrabarti, Manoj K.; Wai, Sun Nyunt

    2016-01-01

    Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses. PMID:26930702

  9. Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae.

    Science.gov (United States)

    Mondal, Ayan; Tapader, Rima; Chatterjee, Nabendu Sekhar; Ghosh, Amit; Sinha, Ritam; Koley, Hemanta; Saha, Dhira Rani; Chakrabarti, Manoj K; Wai, Sun Nyunt; Pal, Amit

    2016-05-01

    Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. Isolation of microorganisms with chinitase, protease and keratinase activities from petroleum contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Cervantes-Gonzalez, E.; Rojas-Avelizapa, L.; Cruz-Camarillo, R. [1 Escuela Nacional de Ciencias Biologicas Departamento de Microbiologia, Laboratorio de Enzimas Microbianas, Mexico City (Mexico); Rojas-Avelizapa, N.G. [Programa de Biotecnologia del Petroleo, Instituto Mexicano del Petroleo, Mexico City (Mexico)

    2005-07-01

    The most important part in one process of bio-remediation are the microorganisms with the capacities to degrade target compounds, this research is based to find microorganisms hydrocarbon-clastic with enzyme activities to degrade chicken feather (keratinolytic activity) which is also a contaminant and has been used such as sorbent of petroleum and can be composted after the oil spill cleanup is complete, the isolation was also to degrade shrimp waste (chitinolitic and proteolitic activity) which is waste material that can be used in compost or such as sorbent of petroleum too. We isolated mesofilic aerobic microorganisms from mexican soils located in Tabasco, Mexico. We achieved to isolate 105 bacteria from 10 soils, 90% was Bacillus Gram (-) which are common in soils and all were hydrocarbon-clastic, only 7 different bacteria had protease and chitinase activity and 12 bacteria had keratinase activity. So we found three fungi and one actinomycete with capacity to degrade hydrocarbons and presence of chitinase activity. The results of growth and enzyme activities in liquid culture showed that the protease activity was produced between 18 and 48 h in almost all bacteria, the chitinase activity started at 12 h but was slight , only 0.5 U/ml, and the keratinase activity was produced after 6 h of incubation and there were correlation between logarithmic phase of growth and enzymes production. With this study we showed the existence of some enzyme activities from microorganisms that live in hostile habitats. This, can be useful in bio-treatment soils by the possible use of this type of residues that can be bio-degraded at the same time that the hydrocarbons increasing the speed or the quality of cleanup in soils. (authors)

  11. 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.

  12. Protease-activated receptor-2 activation contributes to house dust mite-induced IgE responses in mice.

    Directory of Open Access Journals (Sweden)

    Sijranke Post

    Full Text Available Aeroallergens such as house dust mite (HDM, cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR-2, on the airway epithelium, thereby potentially inducing allergic sensitization at the expense of inhalation tolerance. We hypothesized that the proteolytic activity of allergens may play an important factor in the allergenicity to house dust mite and is essential to overcome airway tolerance. Here, we aimed to investigate the role of PAR-2 activation in allergic sensitization and HDM-induced allergic airway inflammation. In our study, Par-2 deficient mice were treated with two different HDM extracts containing high and low serine protease activities twice a week for a period of 5 weeks. We determined airway inflammation through quantification of percentages of mononuclear cells, eosinophils and neutrophils in the bronchial alveolar lavage fluid and measured total IgE and HDM-specific IgE and IgG1 levels in serum. Furthermore, Th2 and pro-inflammatory cytokines including IL-5, IL-13, Eotaxin-1, IL-17, KC, Chemokine (C-C motif ligand 17 (CCL17 and thymic stromal lymphopoietin (TSLP, were measured in lung tissue homogenates. We observed that independent of the serine protease content, HDM was able to induce elevated levels of eosinophils and neutrophils in the airways of both wild-type (WT and Par-2 deficient mice. Furthermore, we show that induction of pro-inflammatory cytokines by HDM exposure is independent of Par-2 activation. In contrast, serine protease activity of HDM does contribute to enhanced levels of total IgE, but not HDM-specific IgE. We conclude that, while Par-2 activation contributes to the development of IgE responses, it is largely dispensable for the HDM-induced induction of pro-inflammatory cytokines and airway inflammation in an experimental mouse model of HDM

  13. Inhibition of sulfur mustard-increased protease activity by niacinamide, N-acetyl-L-cysteine or dexamethasone

    Energy Technology Data Exchange (ETDEWEB)

    Cowan, F.M.; Broomfield, C.A.; Smith, W.J.

    1991-03-11

    The pathologic mechanism of sulfur mustard-induced skin vesication is as yet undefined. Papirmeister et al. have postulated a biochemical mechanism for sulfur mustard-induced cutaneous injury involving sequelae of DNA alkylation, metabolic disruption resulting in NAD+ depletion and activation of protease. The authors have utilized a chromogenic peptide substrate assay to establish that human peripheral blood lymphocytes exposed 24 hr previously to sulfur mustard exhibited an increase in proteolytic activity. Doses of compounds known to alter the biochemical events associated with sulfur mustard exposure or reduce protease activity were tested in this system for their ability to block the sulfur mustard-induced protease activity. Treatment with niacinamide 1 hr after or with N-acetyl-L-cysteine or dexamethasone 24 hr prior to sulfur mustard exposure resulted in a decrease of 39%, 33% and 42% respectively of sulfur mustard-increased protease activity. These data suggest that therapeutic intervention into the biochemical pathways that culminate in protease activation might serve as an approach to treatment of sulfur mustard-induced pathology.

  14. Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications.

    Science.gov (United States)

    Kumar Vr, Santhosh; Darisipudi, Murthy N; Steiger, Stefanie; Devarapu, Satish Kumar; Tato, Maia; Kukarni, Onkar P; Mulay, Shrikant R; Thomasova, Dana; Popper, Bastian; Demleitner, Jana; Zuchtriegel, Gabriele; Reichel, Christoph; Cohen, Clemens D; Lindenmeyer, Maja T; Liapis, Helen; Moll, Solange; Reid, Emma; Stitt, Alan W; Schott, Brigitte; Gruner, Sabine; Haap, Wolfgang; Ebeling, Martin; Hartmann, Guido; Anders, Hans-Joachim

    2016-06-01

    Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

  15. In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti

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    Isoe Jun

    2011-08-01

    Full Text Available Abstract Background The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes. Results We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin. Conclusions These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.

  16. Protease with collagenolytic activity produced by Bacillus sp. DPUA 1728 from Amazonian soil

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    Lorena A. Lima

    2015-12-01

    Full Text Available Qualitative analyses were carried out on solid medium with insoluble collagen 0.25% (w/v to detect proteases with collagenolytic activity produced by Bacillus sp. In cultures incubated for 24 h, a 23 full factorial design with four repetitions at the center point was developed to analyze the effects and interactions between initial pH, temperature and the concentration of gelatin. Based on the results of the first 23 full factorial design, a successive 23 full factorial design was performed. The most favorable production conditions were found to be 1.5% (w/v gelatin, pH 9.0 and 37 °C with enzymatic activity of 86.27 U/mL. The enzyme showed optimal activity at 50 °C and pH 9.0, and it was stable over wide pH (7.2-10.0 and temperature (45 °C-60 °C ranges. These results indicate that Bacillus sp DPUA 1728 is a potential source for producing collagenolytic protease with possible biotechnological applications, such as in the food, cosmetics and leather industries.

  17. Systematic functional analysis and application of a cold-active serine protease from a novel Chryseobacterium sp.

    Science.gov (United States)

    Mageswari, Anbazhagan; Subramanian, Parthiban; Chandrasekaran, Suganthi; Karthikeyan, Sivashanmugam; Gothandam, Kodiveri Muthukaliannan

    2017-02-15

    Psychrotolerant bacteria isolated from natural and artificially cold environments were screened for synthesis of cold-active protease. The strain IMDY showing the highest protease production at 5°C was selected and phylogenetic analysis revealed that IMDY as novel bacterium with Chryseobacterium soli(T) as its nearest neighbor. Classical optimization enhanced the protease production from 18U/mg to 26U/mg and the enzyme was found to be active at low temperature, activity enhanced by CaCl2, inhibited by PMSF, stable against NaCl, and its activity retained in the presence of surfactants, organic solvents and detergents. On testing, the meat tenderization, myofibril fragmentation, pH, and TBA values were favorable in IMDY-protease treated meat compared to control. SDS profiling and SEM analysis also showed tenderization in meat samples. Hence, this study proposes to consider the cold-active protease from Chryseobacterium sp. IMDY as a pertinent candidate to develop potential applications in food processing industry.

  18. Essential role of mitogen-activated protein kinase pathways in protease activated receptor 2-mediated nitric-oxide production from rat primary astrocytes.

    Science.gov (United States)

    Park, Gyu Hwan; Jeon, Se Jin; Ryu, Jae Ryun; Choi, Min Sik; Han, Seol-Heui; Yang, Sung-Il; Ryu, Jong Hoon; Cheong, Jae Hoon; Shin, Chan Young; Ko, Kwang Ho

    2009-09-01

    Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.

  19. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

    Science.gov (United States)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

    2016-02-10

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  20. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    Science.gov (United States)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  1. The coagulation factor Xa/protease activated receptor-2 axis in the progression of liver fibrosis : a multifaceted paradigm

    NARCIS (Netherlands)

    Borensztajn, Keren; von der Thusen, Jan H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

    2010-01-01

    Introduction Activation of the coagulation cascade during liver fibrosis: a puzzling paradox Protease-activated receptors: the link between coagulation cascade activation and liver fibrosis Expression and distribution of human PAR-2 in normal and pathological liver tissue FXa signalling on PAR-2 exp

  2. In vitro and comparative study on the extracellular enzyme activity of molds isolated from keratomycosis and soil

    Institute of Scientific and Technical Information of China (English)

    Arumugam; Mythili; Yendranbam; Randhir; Babu; Singh; Ravindran; Priya; Anamangadan; Shafeeq; Hassan; Palanisamy; Manikandan; Kanesan; Panneerselvam; Venkatapathy; Narendran; Coimbatore; Subramanian; Shobana

    2014-01-01

    AIM:To isolate and identify the molds involved in mycotic keratitis; to isolate corresponding species from soil samples; to compare the extracellular enzyme activity indices of the molds isolated from keratitis cases and the corresponding soil isolates.METHODS:The specimens were collected from the target patients attending the microbiology laboratory of tertiary eye hospital in Coimbatore, Tamilnadu state,India. The isolates were subjected for identification based on the growth on solid media, direct microscopy and lacto phenol cotton blue wet mount preparation.Extracellular enzymes such as lipase, deoxyribonuclease(DNase), α-amylase, protease, cellulase and pectinase produced by the fungal isolates were screened on solid media supplemented with the corresponding substrates.Based on growth and zone diameter, the enzyme activity indices were calculated and were compared with that of the soil fungal isolates.RESULTS:A total of 108 clinical samples were collected from a tertiary eye care hospital and out of which 60 fungal isolates were obtained. Among these,Fusarium spp.(n =30), non sporulating molds(n =9),Aspergillus flavus(n =6), Bipolaris spp.(n =6),Exserohilum spp.(n =4), Curvularia spp.(n =3),Alternaria spp.(n =1) and Exophiala spp.(n =1) were identified and designated as FS1-30, NSM1-9, AF1-6,BS1-6, ES1-4, CS1-3, AS1 and EX1, respectively. For comparative analysis, soil samples were also collected from which, one isolate of each Fusarium spp.,Aspergillus flavus, Bipolaris spp., Exserohilum spp., and Curvularia spp., respectively were selected. Highest lipase activity was seen in corneal isolate NSM2(EAI= 2.14).The DNase activity was higher in NSM9(EAI =1.88). In case of protease, Fusarium spp.(FS9) had prominent enzyme activity index of 1.38; α-amylase activity was also superior in corneal isolate FS13 with EAI of 1.63 when compared to other isolates. The enzyme activity index for cellulase was also noted to be higher in corneal isolates i.e. NSM7 with EAI of

  3. Stimulation of Naive Monocytes and PBMCs with Coagulation Proteases Results in Thrombin-Mediated and PAR-1-Dependent Cytokine Release and Cell Proliferation in PBMCs Only

    NARCIS (Netherlands)

    Nieuwenhuizen, L.; Falkenburg, W. J. J.; Schutgens, R. E. G.; Roosendaal, G.; van Veghel, K.; Biesma, D. H.; Lafeber, F. P. J. G.

    2013-01-01

    Protease-activated receptors (PARs) are stimulated by proteolytic cleavage of their extracellular domain. Coagulation proteases, such as FVIIa, the binary TF-FVIIa complex, free FXa, the ternary TF-FVIIa-FXa complex and thrombin, are able to stimulate PARs. Whereas the role of PARs on platelets is w

  4. Antimicrobial Activity of a Halocidin-Derived Peptide Resistant to Attacks by Proteases

    Science.gov (United States)

    Shin, Yong Pyo; Park, Ho Jin; Shin, Seo Hwa; Lee, Young Shin; Park, Seungmi; Jo, Sungho; Lee, Yong Ho; Lee, In Hee

    2010-01-01

    Cationic antimicrobial peptides (AMPs) have attracted a great deal of interest as a promising candidate for a novel class of antibiotics that might effectively treat recalcitrant infections caused by a variety of microbes that are resistant to currently available drugs. However, the AMPs are inherently limited in that they are inevitably susceptible to attacks by proteases generated by human and pathogenic microbes; this vulnerability severely hinders their pharmaceutical use in human therapeutic protocols. In this study, we report that a halocidin-derived AMP, designated HG1, was found to be resistant to proteolytic degradation. As a result of its unique structural features, HG1 proved capable of preserving its antimicrobial activity after incubation with trypsin, chymotrypsin, and human matrix metalloprotease 7 (MMP-7). Additionally, HG1 was observed to exhibit profound antimicrobial activity in the presence of fluid from human skin wounds or proteins extracted from the culture supernatants of Staphylococcus aureus and Pseudomonas aeruginosa. Greater understanding of the structural motifs of HG1 required for its protease resistance might provide feasible ways to solve the problems intrinsic to the development of an AMP-based antibiotic. PMID:20385874

  5. Interspecies differences in membrane-associated protease activities of thyrocytes and their relevance for thyroid cancer studies

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    Fröhlich Eleonore

    2012-05-01

    Full Text Available Abstract Background To understand the role of proteases involved in human thyroid cancer progression and tissue invasion, thyrocytes from other species could potentially be used provided their characteristics are similar. It is not known whether dipeptidyl peptidase IV and aminopeptidase N activities, which are overexpressed in human thyroid cancer, are, as in human, also absent in normal thyrocytes of other species, making them suitable models for studies on the regulation of these proteases. Methods To assess the role of these proteases, activity was measured in thyroid tissue of human, mouse, rat, porcine, bovine and ovine origin. The lysosomal protease, dipeptidyl peptidase II, was used for comparison. Results Murine, rat, ovine, bovine and human thyrocytes all lacked dipeptidyl peptidase IV and aminopeptidase N activity, but porcine thyrocytes were found to possess both. In contrast, lysosomal dipeptidyl peptidase II was strongly expressed in all species. These activity patterns were maintained in cultured cells. Cultured porcine thyrocytes formed follicles with typical morphology upon stimulation with TSH but differed from human thyrocytes in their response to thiamazole. Conclusions These species differences in the expression of dipeptidyl peptidase IV and aminopeptidase N, indicate that porcine thyrocytes cannot be considered appropriate for the study of proteases in human cancer development.

  6. Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

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    M Holzhausen

    2005-03-01

    Full Text Available Proteinase-activated receptor-2 (PAR2 belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

  7. Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

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    Seiichi Mawatari

    Full Text Available BACKGROUND: It has been hypothesized that persistent hepatitis C virus (HCV infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4, composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation. METHODS: Human C4 was incubated with HCV nonstructural (NS 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined. RESULTS: HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4. CONCLUSIONS: C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.

  8. Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

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    Abdelnasser Salah Shebl Ibrahim

    2016-01-01

    Full Text Available The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH2 nanospheres showed highest immobilization yield (75.6% and loading capacity (88.1 μg protein/mg carrier and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher Vmax, kcat and kcat/Km, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

  9. Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres.

    Science.gov (United States)

    Ibrahim, Abdelnasser Salah Shebl; Al-Salamah, Ali A; El-Toni, Ahmed M; Almaary, Khalid S; El-Tayeb, Mohamed A; Elbadawi, Yahya B; Antranikian, Garabed

    2016-01-29

    The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS-NH₂ nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V(max), k(cat) and k(cat)/K(m), than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

  10. Antibacterial Activity of Fistulin: A Protease Inhibitor Purified from the Leaves of Cassia fistula.

    Science.gov (United States)

    Arulpandi, I; Sangeetha, R

    2012-01-01

    Plant protease inhibitors (PPIs) are one of the important components of a plant's defense machinery. PPIs are active against the insects and microbes which invade the plant. Cassia species possess anti-insecticidal and antimicrobial properties and this study was aimed at investigating the antibacterial efficacy of a PPI present in the leaves of Cassia fistula. A PPI, fistulin, was isolated from the leaves of C. fistula and purified by gel filtration chromatography. The antibacterial activity of the purified fistulin was studied against five bacterial strains, namely, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli. The PPI was found to be very active against S. aureus, E. coli, B. subtilis, and K. pneumonia, and its efficacy was comparable to the standard drug, streptomycin sulphate.

  11. Distal hinge of plasminogen activator inhibitor-1 involves its latency transition and specificities toward serine proteases

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    Shaltiel Shmuel

    2003-07-01

    Full Text Available Abstract Background The plasminogen activator inhibitor-1 (PAI-1 spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347, which interacts with serine residue of tissue-type plasminogen activator (tPA with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases. Results To understand more about the role of distal hinge for PAI-1 specificities towards serine proteases and for its conformational transition, wild type PAI-1 and its mutants were expressed in baculovirus system. WtPAI-1 was found to be about 12 fold more active than the fibrosarcoma PAI-1. Single site mutants within the Asp355-Arg356-Pro357 segment of PAI-1 yield guanidine activatable inhibitors (a that can still form SDS stable complexes with tPA and urokinase plasminogen activator (uPA, and (b that have inhibition rate constants towards plasminogen activators which resemble those of the fibrosarcoma inhibitor. More importantly, latency conversion rate of these mutants was found to be ~3–4 fold faster than that of wtPAI-1. We also tested if Glu351 is important for serine protease specificity. The functional stability of wtPAI-1, Glu351Ala, Glu351Arg was about 18 ± 5, 90 ± 8 and 14 ± 3 minutes, respectively, which correlated well with both their corresponding specific activities (84 ± 15 U/ug, 112 ± 18 U/ug and 68 ± 9 U/ug, respectively and amount of SDS-stable complex formed with tPA after denatured by Guanidine-HCl and dialyzed against 50 mM sodium acetate at 4°C. The second-order rate constants of inhibition for uPA, plasmin and thrombin by Glu351Ala and Glu351Arg were increased about 2–10 folds compared to wtPAI-1, but there was no change for tPA. Conclusion The Asp355-Pro357 segment and Glu351 in distal hinge are involved in maintaining the inhibitory conformation of PAI-1. Glu351 is a specificity

  12. Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

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    Assaf Shapira

    Full Text Available The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins". In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA and Ricin A chain (RTA, respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

  13. Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

    Science.gov (United States)

    Shapira, Assaf; Gal-Tanamy, Meital; Nahary, Limor; Litvak-Greenfeld, Dana; Zemel, Romy; Tur-Kaspa, Ran; Benhar, Itai

    2011-01-14

    The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins"). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

  14. Searching for halo-alkalophilic proteases maintaining stability and activity in hydrophilic aprotic solvents as biocatalysts in carbohydrate chemistry

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Mørkholt, Camilla Kær; Nielsen, Carsten Bue

    2012-01-01

    BIOCAT2012, Hamburg University of Technology Book of Abstracts, p70 ISBN 987-3-941492 L12) Searching for halo-alkalophilic proteases maintaining stability and activity in hydrophilic aprotic solvents as biocatalysts in carbohydrate chemistry. Lars Haastrup Pedersen, Camilla Kær Mørkholt, Carsten...... Bue Nielsen Department of Biotechnology, Chemistry and Environmental Engineering Aalborg University Denmark Halo-alkalophilic proteases have proven relatively stable maintaining activity in hydrophilic aprotic solvents sufficiently to be useful as biocatalysts for regiospecific acylation of sucrose...... and other carbohydrates (1, 2). Therefore, we are searching for new bacterial halo-alkaline proteases from extreme environments in Denmark. So far we have identified a number of interesting isolates showing proteolytic activity at pH 7 and 10. Whole genome Illumina Hiseq 2000 amplicon sequencing, de novo...

  15. Skeletal muscle protease activities in the early growth and development of wild Atlantic salmon (Salmo salar L.).

    Science.gov (United States)

    Lysenko, Liudmila A; Kantserova, Nadezda P; Kaivarainen, Elena I; Krupnova, Marina Yu; Nemova, Nina N

    2017-09-01

    Growth-related dynamics of intracellular protease activities in four year classes of the Atlantic salmon (Salmo salar L. 1758) parr and smolts inhabiting salmon rivers of northwestern Russia (the White Sea basin) were studied. Cathepsin B, cathepsin D, proteasome, and calpain activities in the skeletal muscles of salmon were assessed to investigate their relative contribution to the total protein degradation as well as to young fish growth process. It was confirmed that calpain activity dominates in salmon muscles while proteasome plays a minor role, in contrast to terrestrial vertebrates. Calpain and proteasome activities were maximal at the early post-larval stage (in parrs 0+) and declined with age (parrs 1+ through 2+) dropping to the lowest level in salmon smolts. Annual growth increments and proteolytic activities of calpains and proteasome in the muscles of salmon juveniles changed with age in an orchestrated manner, while lysosomal cathepsin activities increased with age. Comparing protease activities and growth increments in salmon parr and smolts we suggested that the partial suppression of the protein degradation could be a mechanism stimulating efficient growth in smoltifying salmon. Growth and smoltification-related dynamics of protease activities was quite similar in salmon populations from studied spawning rivers, such as Varzuga and Indera; however, some habitat-related differences were observed. Growth increments and protease activities varied in salmon parr 0+ (but not on later ages) inhabiting either main rivers or small tributaries apparently due to habitat difference on the resources for fish growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Differentiating intracellular from extracellular alkaline phosphatase activity in soil by sonication.

    Directory of Open Access Journals (Sweden)

    Shuping Qin

    Full Text Available Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v and power density  =  15 watt ml(-1], the activity of alkaline phosphomonoesterase (phosphatase in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80% of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.

  17. Differentiating intracellular from extracellular alkaline phosphatase activity in soil by sonication.

    Science.gov (United States)

    Qin, Shuping; Hu, Chunsheng; Oenema, Oene

    2013-01-01

    Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v) and power density  =  15 watt ml(-1)], the activity of alkaline phosphomonoesterase (phosphatase) in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80%) of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.

  18. Basis Tetrapeptides as Potent Intracellular Inhibitors of type A Botulinum Neurotoxin Protease Activity

    Energy Technology Data Exchange (ETDEWEB)

    Hale, M.; Swaminathan, S.; Oyler, G.; Ahmed, S. A.

    2011-01-21

    Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development.

  19. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    Directory of Open Access Journals (Sweden)

    Nagata Tatsuya

    2010-06-01

    Full Text Available Abstract Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL, and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat, were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.

  20. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    Energy Technology Data Exchange (ETDEWEB)

    Gottipati, Keerthi; Acholi, Sudheer [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States); Ruggli, Nicolas [Institute of Virology and Immunology, CH-3147 Mittelhäusern (Switzerland); Choi, Kyung H., E-mail: kychoi@utmb.edu [Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0647 (United States)

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  1. Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity.

    Science.gov (United States)

    Lane, Michael D; Seelig, Burckhard

    2016-05-01

    Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures.

  2. The role of Serine Proteases and Serine Protease Inhibitors in the migration of Gonadotropin-Releasing Hormone neurons

    Directory of Open Access Journals (Sweden)

    Silverman Ann-Judith

    2002-02-01

    Full Text Available Abstract Background Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes. Results Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1, and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction. Conclusions These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.

  3. Post-prandial protease activity in the digestive tract of African catfish, Clarias gariepinus, larvae fed decapsulated cysts of Artemia

    NARCIS (Netherlands)

    García Ortega, A.; Verreth, J.; Segner, H.

    2000-01-01

    The alkaline proteolytic activity in the gut of African catfish larvae was studied during short time ranges from 30 min to 4 h after ingestion of decapsulated Artemia cysts. The variation in total protease and trypsin activities during the day was monitored during starvation, after one single meal i

  4. Protease and lipase activities of fungal and bacterial strains derived from an artisanal raw ewe's milk cheese

    NARCIS (Netherlands)

    Ozturkoglu Budak, Sebnem; Wiebenga, Ad; Bron, Peter A; de Vries, Ronald P

    2016-01-01

    We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic

  5. Serum cysteine proteases and their inhibitors in rheumatoid arthritis: relation to disease activity and radiographic progression

    DEFF Research Database (Denmark)

    Kos, Janko; Krašovec, Marta; Troelsen, Lone

    2011-01-01

    This study aims to investigate the serum levels of cysteine proteases cathepsins B and H and their inhibitors stefin A, stefin B, and cystatin C, as well as traditional inflammatory markers such as C-reactive protein in patients with rheumatoid arthritis and to correlate these markers with scores...... of disease activity and radiographic progression. Seventy-two patients with rheumatoid arthritis were included from two previously described cohorts of patients with chronic polyarthritis. At inclusion, disease activity was assessed by a 28-joint count, patient global assessment, and serum C-reactive protein....... Erosive status of hands and wrists was expressed by the Larsen score and recorded at inclusion and after 1 year. Serum levels of cathepsin B, cathepsin H, stefin A, stefin B, and cystatin C were determined by enzyme-linked immunosorbent assay. Neither cathepsin B nor cathepsin H serum levels were...

  6. Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.

    Science.gov (United States)

    Shrestha, Madan K; Peri, Irena; Smirnoff, Patricia; Birk, Yehudith; Golan-Goldhirsh, Avi

    2002-09-25

    The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.

  7. Degradation of plasma proteins by the trypsin-like enzyme of Porphyromonas gingivalis and inhibition of protease activity by a serine protease inhibitor of human plasma.

    Science.gov (United States)

    Fishburn, C S; Slaney, J M; Carman, R J; Curtis, M A

    1991-08-01

    The interaction between Porphyromonas gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and IgG light chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of P. gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not alpha 1-antitrypsin nor alpha 2-macroglobulin inhibited the trypsin-like activity in the fluorometric assay.

  8. Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte*

    Science.gov (United States)

    Ruecker, Andrea; Shea, Michael; Hackett, Fiona; Suarez, Catherine; Hirst, Elizabeth M. A.; Milutinovic, Katarina; Withers-Martinez, Chrislaine; Blackman, Michael J.

    2012-01-01

    The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte. PMID:22984267

  9. OMP Peptides Activate the DegS Stress-Sensor Protease by a Relief of Inhibition Mechanism

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-03-19

    In the E. coli periplasm, C-terminal peptides of misfolded outer-membrane porins (OMPs) bind to the PDZ domains of the trimeric DegS protease, triggering cleavage of a transmembrane regulator and transcriptional activation of stress genes. We show that an active-site DegS mutation partially bypasses the requirement for peptide activation and acts synergistically with mutations that disrupt contacts between the protease and PDZ domains. Biochemical results support an allosteric model, in which these mutations, active-site modification, and peptide/substrate binding act in concert to stabilize proteolytically active DegS. Cocrystal structures of DegS in complex with different OMP peptides reveal activation of the protease domain with varied conformations of the PDZ domain and without specific contacts from the bound OMP peptide. Taken together, these results indicate that the binding of OMP peptides activates proteolysis principally by relieving inhibitory contacts between the PDZ domain and the protease domain of DegS.

  10. Balancing speed and accuracy of polyclonal T cell activation: a role for extracellular feedback

    Directory of Open Access Journals (Sweden)

    Savir Yonatan

    2012-08-01

    Full Text Available Abstract Background Extracellular feedback is an abundant module of intercellular communication networks, yet a detailed understanding of its role is still lacking. Here, we study interactions between polyclonal activated T cells that are mediated by IL-2 extracellular feedback as a model system. Results Using mathematical modeling we show that extracellular feedback can give rise to opposite outcomes: competition or cooperation between interacting T cells, depending on their relative levels of activation. Furthermore, the outcome of the interaction also depends on the relative timing of activation of the cells. A critical time window exists after which a cell that has been more strongly activated nevertheless cannot exclude an inferior competitor. Conclusions In a number of experimental studies of polyclonal T-cell systems, outcomes ranging from cooperation to competition as well as time dependent competition were observed. Our model suggests that extracellular feedback can contribute to these observed behaviors as it translates quantitative differences in T cells’ activation strength and in their relative activation time into qualitatively different outcomes. We propose extracellular feedback as a general mechanism that can balance speed and accuracy – choosing the most suitable responders out of a polyclonal population under the clock of an escalating threat.

  11. Virulence testing and extracellular subtilisin-like (Pr1) and trypsin-like (Pr2) activity during propagule production of Paecilomyces fumosoroseus isolates from whiteflies (Homoptera: Aleyrodidae).

    Science.gov (United States)

    Castellanos-Moguel, Judith; González-Barajas, Margarita; Mier, Teresa; Reyes-Montes, María Del Rocío; Aranda, Eduardo; Toriello, Conchita

    2007-03-01

    To properly characterize several isolates of Paecilomyces fumosoroseus, a fungal entomopathogen of whiteflies (Homoptera: Aleyrodidae) and other insect pests for biocontrol purposes, virulence towards Trialeurodes vaporariorum, and subtilisin-like (Pr1) and trypsin-like (Pr2) protease activity during propagule production were investigated in monospore cultures (MCs). The virulence of three MCs towards second instar whiteflies was measured and expressed as lethal median concentration (LC50). Number and widthlength ratio of propagules (blastospores, hyphal bodies, short hyphae, submerged conidia) and extracellular proteolytic activity was determined simultaneously in liquid medium. Total protease activity was assayed with azocasein, Pr1 and Pr2 activity was determined with the substrates N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-Benzoyl-Phe-Val-Arg-pnitroanilide, respectively. Natural variability in virulence, propagule production and cuticle-degrading proteases among isolates was observed. Bioassays showed a LC50 of 1.1 x 1,000, 2.5 x 10,000 and 7.6 x 10,000 conidia/ml for MCs EH-506/3, EH-503/3 and EH-520/3, respectively, EH-506/3 being the most virulent isolate. Isolate EH-503/3 produced the highest yield of propagules (7.7 x 10000000 propagules/ml), followed by EH-520/3 with 6.4 x 10000000 and EH-506/3 with 1.0 x 10000000 propagules/ml. Subtilisin-like (Pr1) and trypsin-like (Pr2) activity was present in the three MCs. Subtilisin-like (Pr1) activity was highest (745.7 UPr1/ml at 120 h) in the most virulent isolate, EH-506/3, pointing at Pr1 as a phenotypic marker of virulence for P. fumosoroseus. EH-506/3 appears to be a good candidate for whitefly biocontrol due to its high virulence, Pr1 concentration and rapid transition to blastospores in submerged liquid medium.

  12. Extracellular potassium dynamics in the hyperexcitable state of the neuronal ictal activity

    Science.gov (United States)

    Florence, Gerson; Pereira, Tiago; Kurths, Jürgen

    2012-12-01

    An enduring question in epilepsy research concerns with the mechanisms responsible for the neuronal hyperexcitability. This theme is under debate and different hypotheses have been put forward. One hypothesis relates to extracellular ionic variations, especially the increase of the extracellular potassium concentration ([K+]o). During the epileptiform bursting, an increase of [K+]o is observed which raises the cellular excitability. It remains unclear, however, how the extracellular potassium variation could affect the generation and persistence of epileptiform bursting within the ictal phase, that is, during the epileptic seizure. The neuronal mechanisms responsible for this cellular hyperexcitability are not yet fully understood, hindering the development of more efficient therapies to control epilepsy. Mathematical models with biological plausibility have provided considerable insights into the mechanisms underlying epileptiform pattern. This paper reviews experimental evidences and computational studies concerning effects of the extracellular potassium dynamics on the cellular excitability within the neuronal ictal activity.

  13. Receptor-like activity evoked by extracellular ADP in Arabidopsis root epidermal plasma membrane.

    Science.gov (United States)

    Demidchik, Vadim; Shang, Zhonglin; Shin, Ryoung; Colaço, Renato; Laohavisit, Anuphon; Shabala, Sergey; Davies, Julia M

    2011-07-01

    Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca(2+) influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca(2+) in root epidermal protoplasts. An inward Ca(2+)-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca(2+) efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.

  14. Receptor-Like Activity Evoked by Extracellular ADP in Arabidopsis Root Epidermal Plasma Membrane1

    Science.gov (United States)

    Demidchik, Vadim; Shang, Zhonglin; Shin, Ryoung; Colaço, Renato; Laohavisit, Anuphon; Shabala, Sergey; Davies, Julia M.

    2011-01-01

    Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca2+ influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca2+ in root epidermal protoplasts. An inward Ca2+-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca2+ efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM. PMID:21562328

  15. Production and Characterization of Alkaline Protease from a High Yielding and Moderately Halophilic Strain of SD11 Marine Bacteria

    Directory of Open Access Journals (Sweden)

    Hongxia Cui

    2015-01-01

    Full Text Available A marine bacterium SD11, which was isolated from sea muds (Geziwo Qinhuangdao Sea area, China, was used to produce thermostable alkaline serine nonmetal protease in the skim milk agar plate medium with 10% NaCl. The optimal temperature about the manufacture of the extracellular protease was ~60°C. The crude enzyme was stable at 20–50°C. The activity was retained to 60% and 45% after heating for 1 h at 60 and 70°C, respectively. The protease was highly active in a wide pH scope (8.0–10.0 and maximum protease activity exhibited at pH 10.0. The activity was restrained by phenylmethylsulfonyl fluoride (PMSF but mildly increased (~107% in the presence of ethylenediaminetetraacetic acid (EDTA, indicating that the production contains serine-protease(s and nonmetal protease(s. Moreover, the crude alkaline protease was active with the 5 mM Ca2+, Mn2+, Zn2+, Cu2+, Na+, and K+ that existed separately. In addition, the protease showed superduper stability when exposed to an anionic surfactant (5 mM SDS, an oxidizing agent (1% H2O2, and several organic solvents (methanol, isopropanol, and acetone. These results suggest that the marine bacterium SD11 is significant in the industry from the prospects of its ability to produce thermally stable alkaline protease.

  16. Extracellular enzyme activities during cassava fermentation for 'fufu' production.

    Science.gov (United States)

    Oyewole, O B; Odunfa, S A

    1992-01-01

    Amylase and pectin methyl esterase activities increased rapidly during the early period of the fermentation of cassava for 'fufu' production, attaining their peak activities after 12 and 24h, respectively. Cellulase activity was lower and approximately constant for most of the fermentation period.

  17. The HhoA protease from Synechocystis sp. PCC 6803 - Novel insights into structure and activity regulation.

    Science.gov (United States)

    Hall, Michael; Wagner, Raik; Lam, Xuan Tam; Funk, Christiane; Persson, Karina

    2017-06-01

    Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca(2+) or Mg(2+), binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed. Copyright © 2016. Published by Elsevier Inc.

  18. A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.

    Science.gov (United States)

    Sun, Jian; Zhao, Yongchang; Chai, Hongmei; Wang, Hexiang; Ng, Tzi Bun

    2011-01-01

    A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.

  19. MALT1 Protease Activation Triggers Acute Disruption of Endothelial Barrier Integrity via CYLD Cleavage.

    Science.gov (United States)

    Klei, Linda R; Hu, Dong; Panek, Robert; Alfano, Danielle N; Bridwell, Rachel E; Bailey, Kelly M; Oravecz-Wilson, Katherine I; Concel, Vincent J; Hess, Emily M; Van Beek, Matthew; Delekta, Phillip C; Gu, Shufang; Watkins, Simon C; Ting, Adrian T; Gough, Peter J; Foley, Kevin P; Bertin, John; McAllister-Lucas, Linda M; Lucas, Peter C

    2016-09-27

    Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.

  20. MALT1 Protease Activation Triggers Acute Disruption of Endothelial Barrier Integrity via CYLD Cleavage

    Directory of Open Access Journals (Sweden)

    Linda R. Klei

    2016-09-01

    Full Text Available Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment, yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation, but the mechanisms responsible are still incompletely understood. Here, we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD. Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.

  1. Regulation of protease-activated receptor-1 expression in human buccal fibroblasts stimulated with arecoline.

    Science.gov (United States)

    Tsai, Chung-Hung; Lee, Shiuan-Shinn; Huang, Fu-Mei; Chang, Yu-Chao

    2013-09-01

    The purpose of this study was to compare the major thrombin receptor protease-activated receptor-1 (PAR-1) expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanisms that may lead to induce PAR-1 expression. Thirty OSF and 10 normal buccal mucosa specimens were examined by immunohistochemistry. Buccal mucosal fibroblasts (BMFs) were challenged with arecoline by using Western blot analysis. N-acetyl-L-cysteine (NAC), LY294002, herbimycin A, NS-398, and PD98059 were added to find the possible regulatory mechanisms. PAR-1 expression was significantly higher in OSF specimens (p Arecoline was found to elevate PAR-1 expression in a dose-dependent and time-dependent manner (p arecoline-induced PAR-1 expression (p Arecoline-induced PAR-1 expression was downregulated by NAC, LY294002, herbimycin A, NS398, and PD98059. Copyright © 2012 Wiley Periodicals, Inc.

  2. Detection of Extracellular Enzyme Activity in Penicillium using Chromogenic Media.

    Science.gov (United States)

    Yoon, Ji Hwan; Hong, Seung Beom; Ko, Seung Ju; Kim, Seong Hwan

    2007-09-01

    A total of 106 Penicillium species were tested to examine their ability of degrading cellobiose, pectin and xylan. The activity of β-glucosidase was generally strong in all the Penicillium species tested. P. citrinum, P. charlesii, P. manginii and P. aurantiacum showed the higher ability of producing β-glucosidase than other tested species. Pectinase activity was detected in 24 Penicillium species. P. paracanescens, P. sizovae, P. sartoryi, P. chrysogenum, and P. claviforme showed strong pectinase activity. In xylanase assay, 84 Penicillium species showed activity. Strong xylanase activity was detected from P. megasporum, P. sartoryi, P. chrysogenum, P. glandicola, P. discolor, and P. coprophilum. Overall, most of the Penicillium species tested showed strong β-glucosidase activity. The degree of pectinase and xylanase activity varied depending on Penicillium species.

  3. Preparation, Characterization and Activity of a Peptide-Cellulosic Aerogel Protease Sensor from Cotton

    Directory of Open Access Journals (Sweden)

    J. Vincent Edwards

    2016-10-01

    Full Text Available Nanocellulosic aerogels (NA provide a lightweight biocompatible material with structural properties, like interconnected high porosity and specific surface area, suitable for biosensor design. We report here the preparation, characterization and activity of peptide-nanocellulose aerogels (PepNA made from unprocessed cotton and designed with protease detection activity. Low-density cellulosic aerogels were prepared from greige cotton by employing calcium thiocyanate octahydrate/lithium chloride as a direct cellulose dissolving medium. Subsequent casting, coagulation, solvent exchange and supercritical carbon dioxide drying afforded homogeneous cellulose II aerogels of fibrous morphology. The cotton-based aerogel had a porosity of 99% largely dominated by mesopores (2–50 nm and an internal surface of 163 m2·g−1. A fluorescent tripeptide-substrate (succinyl-alanine-proline-alanine-4-amino-7-methyl-coumarin was tethered to NA by (1 esterification of cellulose C6 surface hydroxyl groups with glycidyl-fluorenylmethyloxycarbonyl (FMOC, (2 deprotection and (3 coupling of the immobilized glycine with the tripeptide. Characterization of the NA and PepNA included techniques, such as elemental analysis, mass spectral analysis, attenuated total reflectance infrared imaging, nitrogen adsorption, scanning electron microscopy and bioactivity studies. The degree of substitution of the peptide analog attached to the anhydroglucose units of PepNA was 0.015. The findings from mass spectral analysis and attenuated total reflectance infrared imaging indicated that the peptide substrate was immobilized on to the surface of the NA. Nitrogen adsorption revealed a high specific surface area and a highly porous system, which supports the open porous structure observed from scanning electron microscopy images. Bioactivity studies of PepNA revealed a detection sensitivity of 0.13 units/milliliter for human neutrophil elastase, a diagnostic biomarker for inflammatory

  4. Characterization and stability of extracellular alkaline proteases from halophilic and alkaliphilic bacteria isolated from saline habitat of coastal Gujarat, India Caracterização e estabilidade de proteases alcalinas extracelulares de bactérias halofílicas e alcalifílicas isoladas de habitat salino de Gujarat, Índia

    Directory of Open Access Journals (Sweden)

    Mital S. Dodia

    2006-09-01

    Full Text Available The present study deals with the isolation and characterization of the moderately halophilic-alkaliphilic bacteria from a saline habitat in western India. Eight different bacterial strains were isolated using enrichment techniques at 20% (w/v NaCl and pH 10. The isolates exhibited diversity towards gram's reaction, colony and cell morphology. They were able to grow and produce alkaline protease over a broad range of NaCl, 5-20% (w/v and pH, 8-10. None of the isolates could grow at pH 7, and one could not grow even at pH 8. Crude and partially purified proteases from strain S5 were subjected to characterization with reference to pH, salt stability and protein folding. Optimum protease activity and stability was recorded at 10% salt and pH 9-9.5. Denaturation kinetics of S5 alkaline protease along with a reference protease was studied at 8M urea followed by renaturation. The S5 alkaline protease could be partially renatured up to 32% of the original activity. Despite of the fact that all the 8 isolates were from the same site, they displayed significant diversity with respect to their salt requirement for growth and enzyme secretion. While the effect of pH was less demarcated on growth, the protease production was significantly affected. Isolate S5 produced substantial amount of halotolerant and alkaline protease. The activity and stability of the alkaline protease in a broader range of pH and salt would definitely make this enzyme an important candidate for various industrial applications.O presente estudo relata o isolamento e caracterização de bactérias moderadamente halofilicas e alcalífilicas de um habitat salino no oeste da Índia. Oito cepas diferentes de bactérias foram isoladas empregando técnicas de enriquecimento em NaCl a 20% (p/v e pH 10. As cepas apresentaram diversidade em relação à coloração de Gram e à morfologia das colônias e células. As cepas foram capazes de multiplicar e produzir protease alcalina em uma ampla

  5. Methylene blue not ferrocene: Optimal reporters for electrochemical detection of protease activity.

    Science.gov (United States)

    González-Fernández, Eva; Avlonitis, Nicolaos; Murray, Alan F; Mount, Andrew R; Bradley, Mark

    2016-10-15

    Electrochemical peptide-based biosensors are attracting significant attention for the detection and analysis of proteins. Here we report the optimisation and evaluation of an electrochemical biosensor for the detection of protease activity using self-assembled monolayers (SAMs) on gold surfaces, using trypsin as a model protease. The principle of detection was the specific proteolytic cleavage of redox-tagged peptides by trypsin, which causes the release of the redox reporter, resulting in a decrease of the peak current as measured by square wave voltammetry. A systematic enhancement of detection was achieved through optimisation of the properties of the redox-tagged peptide; this included for the first time a side-by-side study of the applicability of two of the most commonly applied redox reporters used for developing electrochemical biosensors, ferrocene and methylene blue, along with the effect of changing both the nature of the spacer and the composition of the SAM. Methylene blue-tagged peptides combined with a polyethylene-glycol (PEG) based spacer were shown to be the best platform for trypsin detection, leading to the highest fidelity signals (characterised by the highest sensitivity (signal gain) and a much more stable background than that registered when using ferrocene as a reporter). A ternary SAM (T-SAM) configuration, which included a PEG-based dithiol, minimised the non-specific adsorption of other proteins and was sensitive towards trypsin in the clinically relevant range, with a Limit of Detection (LoD) of 250pM. Kinetic analysis of the electrochemical response with time showed a good fit to a Michaelis-Menten surface cleavage model, enabling the extraction of values for kcat and KM. Fitting to this model enabled quantitative determination of the solution concentration of trypsin across the entire measurement range. Studies using an enzyme inhibitor and a range of real world possible interferents demonstrated a selective response to trypsin

  6. Bacterial Versus Archaeal Origin of Extracellular Enzymatic Activity in the Northeast Atlantic Deep Waters

    NARCIS (Netherlands)

    Baltar, F.; Arístegui, J.; Gasol, J.M.; Yokokawa, T.; Herndl, G.J.

    2013-01-01

    We determined the total and dissolved extracellular enzymatic activity (EEA) of alpha-glucosidase and beta-glucosidase (AGase and BGase), alkaline phosphatase (APase) and leucine aminopeptidase (LAPase) activities in the epi-, meso- and bathypelagic waters of the subtropical Northeast Atlantic. EEA

  7. PATHOGEN IMPACT ON THE ACTIVITY DYNAMICS OF POTATO SUSPENSION CELLS EXTRA-CELLULAR PEROXIDASE

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2005-08-01

    Full Text Available Changes in the activity of extracellular peroxidases were measured in cell suspension cultures of potato infected by Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. The total extracellular peroxidases activity of the resistant potato variety was higher than that of the sensitive variety both before and after infection. The enzyme of the resistant variety had a рН optimum of 6.2, while that of the sensitive variety was 5.4. Extracellular peroxidases of the sensitive potato variety were activated 10 minutes after infection, and displayed highest activity 1.5-2 hours later. In the resistant variety, peroxidase activity rose sharply in the first minutes of infection, and second peak of activity occurred 1.5-2 hours later. The increase of extracellular peroxidases activity of the sensitive potato variety under pathogenesis is connected with the change of genome expression and synthesis of proteins. The increase of enzyme activity of resistant potato variety in the first moments of infection is not related to proteins synthesis and is apparently conditioned by the change of kinetic parameters.

  8. Fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus aspartic protease, an Aspergillus aspartic protease per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a protease of the aspartic proteinase-type,

  9. The Effect of pH on protease activity on Atlantic salmon%PH对大西洋鲑蛋白酶活性的影响

    Institute of Scientific and Technical Information of China (English)

    杨春桥; 张瑞雪; Ma Yue

    2013-01-01

    In order to study pH of protease activity of Atlantic salmon.The impact of PH on Protease Activity in Atlantic salmon stomach,liver,foregut,midgut,hindgut with theLowry method.The results show that during set-ting pH range,Atlantic salmon allorganization protease activity were increased with increasing temperature and pH first increased and then decreased trends.Among them,pepsin optimum pH of 3.0,liver 7.0,the other is 7.5,the protease activity of Atlantic salmon exists organ differences.Organizations in their optimum pH ,pro-tease activity order stomach>liver>foregut>midgut>hindgut.Significant differences between thevarious parts (P肝脏>前肠>中肠>后肠。各部位间差异显著(P<0.05)。

  10. Protease-specific nanosensors for magnetic resonance imaging.

    Science.gov (United States)

    Schellenberger, Eyk; Rudloff, Franziska; Warmuth, Carsten; Taupitz, Matthias; Hamm, Bernd; Schnorr, Jörg

    2008-12-01

    Imaging of enzyme activity is a central goal of molecular imaging. With the introduction of fluorescent smart probes, optical imaging has become the modality of choice for experimental in vivo detection of enzyme activity. Here, we present a novel high-relaxivity nanosensor that is suitable for in vivo imaging of protease activity by magnetic resonance imaging. Upon specific protease cleavage, the nanoparticles rapidly switch from a stable low-relaxivity stealth state to become adhesive, aggregating high-relaxivity particles. To demonstrate the principle, we chose a cleavage motif of matrix metalloproteinase 9 (MMP-9), an enzyme important in inflammation, atherosclerosis, tumor progression, and many other diseases with alterations of the extracellular matrix. On the basis of clinically tested very small iron oxide particles (VSOP), the MMP-9-activatable protease-specific iron oxide particles (PSOP) have a hydrodynamic diameter of only 25 nm. PSOP are rapidly activated, resulting in aggregation and increased T2*-relaxivity.

  11. Protease activated receptors 1 and 4 sensitize TRPV1 in nociceptive neurones

    Directory of Open Access Journals (Sweden)

    Magherini Pier C

    2010-09-01

    Full Text Available Abstract Protease-activated receptors (PAR1-4 are activated by proteases released by cell damage or blood clotting, and are known to be involved in promoting pain and hyperalgesia. Previous studies have shown that PAR2 receptors enhance activation of TRPV1 but the role of other PARs is less clear. In this paper we investigate the expression and function of the PAR1, 3 and 4 thrombin-activated receptors in sensory neurones. Immunocytochemistry and in situ hybridization show that PAR1 and PAR4 are expressed in 10 - 15% of neurons, distributed across all size classes. Thrombin or a specific PAR1 or PAR4 activating peptide (PAR1/4-AP caused functional effects characteristic of activation of the PLCβ/PKC pathway: intracellular calcium release, sensitisation of TRPV1, and translocation of the epsilon isoform of PKC (PKCε to the neuronal cell membrane. Sensitisation of TRPV1 was significantly reduced by PKC inhibitors. Neurons responding to thrombin or PAR1-AP were either small nociceptive neurones of the peptidergic subclass, or larger neurones which expressed markers for myelinated fibres. Sequential application of PAR1-AP and PAR4-AP showed that PAR4 is expressed in a subset of the PAR1-expressing neurons. Calcium responses to PAR2-AP were by contrast seen in a distinct population of small IB4+ nociceptive neurones. PAR3 appears to be non-functional in sensory neurones. In a skin-nerve preparation the release of the neuropeptide CGRP by heat was potentiated by PAR1-AP. Culture with nerve growth factor (NGF increased the proportion of thrombin-responsive neurons in the IB4- population, while glial-derived neurotropic factor (GDNF and neurturin upregulated the proportion of thrombin-responsive neurons in the IB4+ population. We conclude that PAR1 and PAR4 are functionally expressed in large myelinated fibre neurons, and are also expressed in small nociceptors of the peptidergic subclass, where they are able to potentiate TRPV1 activity.

  12. The human immunodeficiency virus protease inhibitor ritonavir is potentially active against urological malignancies

    Directory of Open Access Journals (Sweden)

    Sato A

    2015-04-01

    Full Text Available Akinori Sato Department of Urology, National Defense Medical College, Tokorozawa, Japan Abstract: The human immunodeficiency virus protease inhibitor ritonavir has recently been shown to have antineoplastic activity, and its use in urological malignancies is under investigation with an eye toward drug repositioning. Ritonavir is thought to exert its antineoplastic activity by inhibiting multiple signaling pathways, including the Akt and nuclear factor-kappaB pathways. It can increase the amount of unfolded proteins in the cell by inhibiting both the proteasome and heat shock protein 90. Combinations of ritonavir with agents that increase the amount of unfolded proteins, such as proteasome inhibitors, histone deacetylase inhibitors, or heat shock protein 90 inhibitors, therefore, induce endoplasmic reticulum stress cooperatively and thereby kill cancer cells effectively. Ritonavir is also a potent cytochrome P450 3A4 and P-glycoprotein inhibitor, increasing the intracellular concentration of combined drugs by inhibiting their degradation and efflux from cancer cells and thereby enhancing their antineoplastic activity. Furthermore, riotnavir’s antineoplastic activity includes modulation of immune system activity. Therapies using ritonavir are thus an attractive new approach to cancer treatment and, due to their novel mechanisms of action, are expected to be effective against malignancies that are refractory to current treatment strategies. Further investigations using ritonavir are expected to find new uses for clinically available drugs in the treatment of urological malignancies as well as many other types of cancer. Keywords: drug repositioning, novel treatment

  13. Profiling gene expression induced by protease-activated receptor 2 (PAR2 activation in human kidney cells.

    Directory of Open Access Journals (Sweden)

    Jacky Y Suen

    Full Text Available Protease-Activated Receptor-2 (PAR2 has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD and key events in tumor progression (angiogenesis, metastasis, but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293, a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2 and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2. Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes, the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2 and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15. Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4 known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.

  14. The Enzymatic Activity and Molecular Characterization of a Secreted Subtilisin-Like Protease in Microsporum gypseum and Trichophyton vanbreuseghemii

    Directory of Open Access Journals (Sweden)

    H Moallaei

    2009-03-01

    Full Text Available "nBackground: Subtilisin -like proteases are the group of proteases including keratinases found in dermatophytes which de­graded keratin. Determination of the proteases activity of Trichophyton vanbreuseghemii isolates which were obtained from soil and clinical and soil isolates of Microsporum gypseum in Iran and characterization of their genome were aim of present study."nMethods: Ezymatic activity was determined by use of chromogenic substrates. The genes, which coded subtilisin-like pro­teases in above-mentioned dermatophytes, was identified and amplified by using specific primers in PCR."nResults:  The highest yield of enzyme production was observed in only one isolate of T. vanbreuseghemii Ir-84 whereas low enzyme activity was observed in M. gypseum isolates. Homology study of obtained nucleotide as well as amino acid sequences indicated different rates of homology with other subtilisin-like proteases genes in other pathogenic dermato­phytes."nConclusion: Intra-strain differences were observed in production of serine proteinases and molecular characterization of genes encoding such enzymes could be of great interest for studies on pathogenicity and other purposes.  

  15. A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity.

    Science.gov (United States)

    Counts, Christopher J; Ho, P Shing; Donlin, Maureen J; Tavis, John E; Chen, Chaoping

    2015-01-01

    HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

  16. A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity.

    Directory of Open Access Journals (Sweden)

    Christopher J Counts

    Full Text Available HIV-1 protease (PR is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93 revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

  17. The Cladosporium fulvum virulence protein Avr2 inhibits host proteases required for basal defense.

    Science.gov (United States)

    van Esse, H Peter; Van't Klooster, John W; Bolton, Melvin D; Yadeta, Koste A; van Baarlen, Peter; Boeren, Sjef; Vervoort, Jacques; de Wit, Pierre J G M; Thomma, Bart P H J

    2008-07-01

    Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mold of tomato (Solanum lycopersicum). During growth in the apoplast, the fungus establishes disease by secreting effector proteins, 10 of which have been characterized. We have previously shown that the Avr2 effector interacts with the apoplastic tomato Cys protease Rcr3, which is required for Cf-2-mediated immunity. We now show that Avr2 is a genuine virulence factor of C. fulvum. Heterologous expression of Avr2 in Arabidopsis thaliana causes enhanced susceptibility toward extracellular fungal pathogens, including Botrytis cinerea and Verticillium dahliae, and microarray analysis showed that Avr2 expression triggers a global transcriptome reflecting pathogen challenge. Cys protease activity profiling showed that Avr2 inhibits multiple extracellular Arabidopsis Cys proteases. In tomato, Avr2 expression caused enhanced susceptibility toward Avr2-defective C. fulvum strains and also toward B. cinerea and V. dahliae. Cys protease activity profiling in tomato revealed that, in this plant also, Avr2 inhibits multiple extracellular Cys proteases, including Rcr3 and its close relative Pip1. Finally, silencing of Avr2 significantly compromised C. fulvum virulence on tomato. We conclude that Avr2 is a genuine virulence factor of C. fulvum that inhibits several Cys proteases required for plant basal defense.

  18. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    DEFF Research Database (Denmark)

    Rossol, Manuela; Pierer, Matthias; Raulien, Nora;

    2012-01-01

    Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1ß during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular cal......, and this effect was inhibited in GPRC6A(-/-) mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation....

  19. Localized serine protease activity and the establishment of Drosophila embryonic dorsoventral polarity.

    Science.gov (United States)

    Stein, David; Cho, Yong Suk; Stevens, Leslie M

    2013-01-01

    Drosophila embryo dorsoventral polarity is established by a maternally encoded signal transduction pathway in which three sequentially acting serine proteases, Gastrulation Defective, Snake and Easter, generate the ligand that activates the Toll receptor on the ventral side of the embryo. The spatial regulation of this pathway depends upon ventrally restricted expression of the Pipe sulfotransferase in the ovarian follicle during egg formation. Several recent observations have advanced our understanding of the mechanism regulating the spatially restricted activation of Toll. First, several protein components of the vitelline membrane layer of the eggshell have been determined to be targets of Pipe-mediated sulfation. Second, the processing of Easter by Snake has been identified as the first Pipe-dependent, ventrally-restricted processing event in the pathway. Finally, Gastrulation Defective has been shown to undergo Pipe-dependent, ventral localization within the perivitelline space and to facilitate Snake-mediated processing of Easter. Together, these observations suggest that Gastrulation Defective, localized on the interior ventral surface of the eggshell in association with Pipe-sulfated eggshell proteins, recruits and mediates an interaction between Snake and Easter. This event leads to ventrally-restricted processing and activation of Easter and consequently, localized formation of the Toll ligand, and Toll activation.

  20. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    NARCIS (Netherlands)

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M

    2014-01-01

    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  1. Evaluation of von Willebrand factor-cleaving protease activity in patients with thrombotic thrombocytopenic purpura

    Institute of Scientific and Technical Information of China (English)

    高维强; 苏健; 白霞; 王兆钺; 阮长耿

    2004-01-01

    Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.Methods The plasma or serum vWF-cp activity was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) by detecting the residual collagen binding activity (R-CBA) of von Willebrand factor (vWF) before and after digestion by vWF-cp. Multimers of vWF in plasma of patients with TTP were also analyzed by SDS-agarose electrophoresis. Moreover, the serum vWF-cp activities were compared between the patients with TTP and those with tumors.Results The coefficient of variation for intra-batch and inter-batch of the assay were 3.60% and 8.35%. The plasma and serum vWF-cp activity in healthy individuals were (78.79±9.17)% (n=30) and (79.47±10.78)% (n=53), respectively, while the plasma vWF-cp activity in 5 patients with TTP was markedly decreased [(21.83±19.98)%, P<0.001]. The unusually large vWF multimers were observed in two plasma samples of the patients with TTP. Although the vWF-cp activities in patients with benign and malignant tumors were also decreased (P<0.03 and P<0.001, respectively), they were relatively high in comparison with that of TTP patients (P<0.001).Conclusion Measurement of the vWF-cp activity using R-CBA is a simple and rapid method for diagnosing TTP. The vWF-cp activity in patients with TTP was markedly lower than those of patients with tumors.

  2. Plasma factor VII-activating protease is increased by oral contraceptives and induces factor VII activation in-vivo

    DEFF Research Database (Denmark)

    Sidelmann, Johannes J; Skouby, Sven O; Kluft, Cornelis

    2011-01-01

    Oral contraceptive (OC) use influences the hemostatic system significantly and is a risk factor for development of cardiovascular disease. Factor VII-activating protease (FSAP) has potential effects on hemostasis. The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene expresses a FSAP...... alloenzyme with reduced pro-fibrinolytic activity. Presently, we address whether OC use and OC formulation affect FSAP measures in human blood. Healthy women (n=588) were allocated to six cycles of OCs with estrogen contents of 20µg (n=158), 30µg (n=284), 35µg (n=79) or 50µg (n=67) combined with various...... progestins. FSAP genotypes, FSAP and factor VII (FVII) plasma measures were assessed at baseline and after 6 cycles of OC. The 1601GA genotype was present in 49 (8.3%) of the women and was associated with significantly reduced levels of FSAP (P=0.001). OC use increased FSAP antigen by 25% and FSAP activity...

  3. Suppression of granzyme B activity and caspase-3 activation in leukaemia cells constitutively expressing the protease inhibitor 9.

    Science.gov (United States)

    Fritsch, Kristina; Finke, Jürgen; Grüllich, Carsten

    2013-12-01

    Immune surveillance against malignant cells is mediated by cytotoxic T-lymphocytes and NK-cells (CTL/NK) that induce apoptosis through the granzyme-B-dependent pathway. The serine protease inhibitor serpinB9/protease inhibitor-9 (PI-9) is a known inhibitor of granzyme B. Ectopic expression of PI-9 in tumour cells has been reported. However, the impact of PI-9 on granzyme-B-induced apoptosis in tumour cells remains unclear. The aim of this study was to investigate the influence of constitutive PI-9 expression in leukaemia cell lines on the activity of granzyme B and apoptosis induction. PI-9 negative (lymphoblastic Jurkat cells; myeloblastic U937 cells) and PI-9-expressing cell lines (myeloblastic K562 cells, EBV-transformed LCL-1 and LCL-2 B-cells, lymphoblastic Daudi cells, AML-R cells f leukaemia and the U937 subclone U937PI-9(+)). For accurate granzyme B activity determination a quantitative substrate (Ac-IEPD-pNA) cleavage assay was established and caspase-3 activation measured for apoptosis assessment. Cells were treated with a cytotoxic granule isolate that has previously been shown to induce apoptosis through granzyme B signalling. We found a robust correlation between constitutive PI-9 expression levels and the suppression of granzyme B activity. Further, inhibition of granzyme B translated into reduced caspase-3 activation. We conclude, suppression of granzyme B initiated apoptosis in PI-9-expressing cells could contribute to immune evasion and the measurement of granzyme B activity with our assay might be a useful predictive marker in immune-therapeutic approaches against cancer.

  4. Mite and cockroach proteases activate p44/p42 MAP kinases in human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Kong Xiaoyuan

    2003-10-01

    Full Text Available Abstract Background The mechanisms underlying epithelial cell activation by indoor inhaled antigens are poorly understood. Methods In this study, we investigated the role of mitogen-activated protein kinases (MAPKs in A549 epithelial cells upon exposure to antigens of house dust mite (HDMA, German cockroach (GCA, and American cockroach (ACA. Results Each of these antigens induced a significant increase in IL-8 levels compared to the medium control. Exposure of A549 cells to these antigens induced the phosphorylation of p44/42 MAPKs within 5 minutes, which reached a peak at 25 minutes later and reached baseline levels at 1 hour after exposure. PD98059, a MEK1 inhibitor, significantly decreased phosphorylation of p44/p42 MAPKs and IL-8 production. Exposure of A549 cells with antigens, which had been preincubated with different protease inhibitors, also resulted in a reduction of both MAPK phosphorylation and IL-8 production. Conclusion Thus, proteolytic antigens present in HDMA, GCA and ACA activate the p44/42 MAPKs airway epithelial cells, which lead to elevated IL-8 production and initiation of the inflammatory cascade.

  5. Factor VII activating protease (FSAP) promotes the proteolysis and inhibition of tissue factor pathway inhibitor (TFPI)

    Science.gov (United States)

    Kanse, Sandip M.; Declerck, Paul J.; Ruf, Wolfram; Broze, George; Etscheid, Michael

    2013-01-01

    Objectives Factor VII activating protease (FSAP) activates FVII as well as pro-urokinase and inhibits platelet-derived growth factor-BB, thus regulating haemostasis- and remodeling-associated processes in the vasculature. A genetic variant of FSAP (Marburg I polymorphism) results in low enzymatic activity and is associated with an enhanced risk for carotid stenosis and stroke. We postulate that there are additional substrates for FSAP that will help to explain its role in vascular biology and have searched for such a substrate. Results and Methods Using screening procedures to determine the influence of FSAP on various haemostasis-related processes on endothelial cells we discovered that FSAP inhibited tissue factor pathway inhibitor (TFPI), a major anti-coagulant secreted by these cells. Proteolytic degradation of TFPI by FSAP could also be demonstrated by Western blotting and the exact cleavage sites were determined by N-terminal sequencing. The Marburg I variant of FSAP had a diminished ability to inhibit TFPI. A monoclonal antibody to FSAP, that specifically inhibited FSAP binding to TFPI, reversed the inhibitory effect of FSAP on TFPI. Conclusions The identification of TFPI as a sensitive substrate for FSAP increases our understanding of its role in regulating haemostasis and proliferative remodeling events in the vasculature. PMID:22116096

  6. Effects of nanosuspension and inclusion complex techniques on the in vitro protease inhibitory activity of naproxen

    Energy Technology Data Exchange (ETDEWEB)

    Dharmalingam, Senthil Rajan; Chidambaram, Kumarappan; Srinivasan, Ramamurthy; Nadaraju, Shamala, E-mail: dsenthilrajan@yahoo.co.in [School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur (Malaysia)

    2014-01-15

    This study investigated the effects of nanosuspension and inclusion complex techniques on in vitro trypsin inhibitory activity of naproxen—a member of the propionic acid derivatives, which are a group of antipyretic, analgesic, and non-steroidal anti-inflammatory drugs. Nanosuspension and inclusion complex techniques were used to increase the solubility and anti-inflammatory efficacy of naproxen. The evaporative precipitation into aqueous solution (EPAS) technique and the kneading methods were used to prepare the nanosuspension and inclusion complex of naproxen, respectively. We also used an in vitro protease inhibitory assay to investigate the anti-inflammatory effect of modified naproxen formulations. Physiochemical properties of modified naproxen formulations were analyzed using UV, IR spectra, and solubility studies. Beta-cyclodextrin inclusion complex of naproxen was found to have a lower percentage of antitryptic activity than a pure nanosuspension of naproxen did. In conclusion, nanosuspension of naproxen has a greater anti-inflammatory effect than the other two tested formulations. This is because the nanosuspension formulation reduces the particle size of naproxen. Based on these results, the antitryptic activity of naproxen nanosuspension was noteworthy; therefore, this formulation can be used for the management of inflammatory disorders. (author)

  7. A new method for monitoring the extracellular proteolytic activity of wine yeasts during alcoholic fermentation of grape must.

    Science.gov (United States)

    Chasseriaud, Laura; Miot-Sertier, Cécile; Coulon, Joana; Iturmendi, Nerea; Moine, Virginie; Albertin, Warren; Bely, Marina

    2015-12-01

    The existing methods for testing proteolytic activity are time consuming, quite difficult to perform, and do not allow real-time monitoring. Proteases have attracted considerable interest in winemaking and some yeast species naturally present in grape must, such as Metschnikowia pulcherrima, are capable of expressing this activity. In this study, a new test is proposed for measuring proteolytic activity directly in fermenting grape must, using azocasein, a chromogenic substrate. Several yeast strains were tested and differences in proteolytic activity were observed. Moreover, analysis of grape must proteins in wines revealed that protease secreted by Metschnikowia strains may be active against wine proteins.

  8. Purification,characterization and gene cloning of the extracellular protease EYHⅠ from Salinivibrio sp.YH4%Salinivibrio sp.YH4胞外蛋白酶EYHⅠ的分离纯化、酶学性质及基因克隆

    Institute of Scientific and Technical Information of China (English)

    武翠玲; 刘丹; 杨兴昊; 吴日帮; 黄嘉丰; 张姜; 伦梓丰; 何海伦

    2016-01-01

    The extracellular proteases from moderate halophilic strain,Salinivibrio sp.YH4 was isolated,purified, identified and characterized to provide data for enzyme system of Salinivibrio sp..Strain was cultured in liquid fermen-tation medium,the extracellular proteases was purified by using HiTrap Capto DEAE column chromatography and Gel filtration chromatography.The protease was identified by using mass spectrometry (MS)and designated as EYHⅠ. The metalloprotease EYHⅠ had optimal activity at 55 ℃ and pH 9. 0,stabilized at 60℃ and pH 7. 0~1 1. 0.EYHⅠshowed higher proteolytic activity at 4 mol/L NaCl.The nucleotide sequence contains 1836 bp,encoding EYHⅠof 611 amino acid residues,which is highly conserved to zinc metalloprotease precursor in Salinivibrio sp.AF-2004 with 96%sequence similarity.Analysis of the structure showed that EYHⅠcontained an FTP domain,a PepSY domain,M4 neu-tral protease and a PPC domain.These results may provide important data for the study of Salinivibrio sp.and the appli-cation of protease EYHⅠ.%从中度嗜盐菌Salinivibrio sp.YH4发酵的粗酶液中分离纯化出蛋白酶EYHⅠ,对其进行酶学性质分析、串联质谱鉴定及全基因克隆。结果表明,EYHⅠ属于金属蛋白酶,最适温度55℃,热稳定性较好。最适pH为9.0,碱性条件下较稳定;在4 mol/L的NaCl溶液中EYHⅠ仍保持较高活性,EYHⅠ全基因序列共1836 bp,蛋白序列共611个氨基酸。比对发现EYHⅠ氨基酸序列与Salinivibrio sp.AF-2004所产Zn金属蛋白酶前体(ABI93183)同源性最高(96%)。结构分析表明,EYHⅠ由一个FTP结构域,一个PepSY结构域,一个M4中性蛋白酶和一个PPC结构域组成。本研究为嗜盐菌及其胞外蛋白酶的生产和应用奠定了理论基础。

  9. Lon protease affects the RdxA nitroreductase activity and metronidazole susceptibility in Helicobacter pylori.

    Science.gov (United States)

    Tu, I-Fan; Liao, Jiahn-Haur; Yang, Feng-Ling; Lin, Nien-Tsung; Chan, Hong-Lin; Wu, Shih-Hsiung

    2014-10-01

    The lon gene of Helicobacter pylori strains is constitutively expressed during growth. However, virtually nothing is understood concerning the role of Lon in H. pylori. This study examined the function and physiological role of Lon in H. pylori (HpLon) using a trapping approach to identify putative Lon binding partners in the bacterium. Protease-deficient Lon was expressed and served as the bait in trapping approach to capture the interacting partners in H. pylori. The antibiotic susceptibility of wild-type and lon derivative mutants was determined by the E test trips and the disc diffusion assay. The effect of HpLon on RdxA activity was detected the change in NADPH oxidation and metronidazole reduction by spectrophotometer. Lon in Helicobacter pylori (HpLon) interacting partners are mostly associated with metronidazole activation. lon mutant presents more susceptible to metronidazole than that of the wild type, and this phenotype is recovered by complementation of the wild-type Lon. We found that the ATPases associated with a variety of cellular activities (AAA(+) ) module of HpLon causes a decrease in both NADPH oxidase and Mtz reductase activity in RdxA, a major Mtz-activating enzyme in H. pylori. Metronidazole resistance of H. pylori causes the serious medical problem worldwide. In this study, HpLon is involved in metronidazole susceptibility among H. pylori strains. We provide the evidence that HpLon alters RdxA activity in vitro. The decrease in metronidazole activation caused by HpLon is possibly prior to accumulate mutation in rdxA gene before the metronidazole-resistant strains to be occurred. © 2014 John Wiley & Sons Ltd.

  10. Interdomain Contacts and the Stability of Serralysin Protease from Serratia marcescens.

    Directory of Open Access Journals (Sweden)

    Liang Zhang

    Full Text Available The serralysin family of bacterial metalloproteases is associated with virulence in multiple modes of infection. These extracellular proteases are members of the Repeats-in-ToXin (RTX family of toxins and virulence factors, which mediated virulence in E. coli, B. pertussis, and P. aeruginosa, as well as other animal and plant pathogens. The serralysin proteases are structurally dynamic and their folding is regulated by calcium binding to a C-terminal domain that defines the RTX family of proteins. Previous studies have suggested that interactions between N-terminal sequences and this C-terminal domain are important for the high thermal and chemical stabilities of the RTX proteases. Extending from this, stabilization of these interactions in the native structure may lead to hyperstabilization of the folded protein. To test this hypothesis, cysteine pairs were introduced into the N-terminal helix and the RTX domain and protease folding and activity were assessed. Under stringent pH and temperature conditions, the disulfide-bonded mutant showed increased protease activity and stability. This activity was dependent on the redox environment of the refolding reaction and could be blocked by selective modification of the cysteine residues before protease refolding. These data demonstrate that the thermal and chemical stability of these proteases is, in part, mediated by binding between the RTX domain and the N-terminal helix and demonstrate that stabilization of this interaction can further stabilize the active protease, leading to additional pH and thermal tolerance.

  11. Antibacterial and anticancer activity of extracellular synthesized silver nanoparticles from marine Streptomyces rochei MHM13

    Directory of Open Access Journals (Sweden)

    Hanan M. Abd-Elnaby

    2016-09-01

    Full Text Available The study investigated silver nanoparticles (AgNPs synthesized extracellularly using an actinomycete isolated from sediment of the Suez Gulf, Red Sea, Egypt. Screening for biosynthesis of AgNPs revealed that among the forty one actinomycetes tested, only two exhibited the ability to synthesize AgNPs with antibacterial activity

  12. Microbial respiration and extracellular enzyme activity in sediments from the Gulf of Mexico hypoxic zone

    Science.gov (United States)

    This study explores the relationship between sediment chemistry (TC, TN, TP) and microbial respiration (DHA) and extracellular enzyme activity (EEA) across the Gulf of Mexico (GOM) hypoxic zone. TC, TN, and TP were all positively correlated with each other (r=0.19-0.68). DHA was ...

  13. Microbial respiration and extracellular enzyme activity in sediments from the Gulf of Mexico hypoxic zone

    Science.gov (United States)

    This study explores the relationship between sediment chemistry (TC, TN, TP) and microbial respiration (DHA) and extracellular enzyme activity (EEA) across the Gulf of Mexico (GOM) hypoxic zone. TC, TN, and TP were all positively correlated with each other (r=0.19-0.68). DHA was ...

  14. Na+ -K+ pump activity in rat peritoneal mast cells: inhibition by extracellular calcium

    DEFF Research Database (Denmark)

    Knudsen, Torben; Johansen, Torben

    1989-01-01

    of an enzyme, and it is mediated by the Na+ -K+ pump located in the plasma membrane. It is demonstrated that the activity of the Na+ -K+ pump mechanism is inhibited by low concentrations of extracellular calcium (0.1-1.2 mmol l-1). The possibility is discussed that calcium-deprivation may increase the pump...

  15. Large lateral movement of transmembrane helix S5 is not required for substrate access to the active site of rhomboid intramembrane protease.

    Science.gov (United States)

    Xue, Yi; Ha, Ya

    2013-06-07

    Rhomboids represent an evolutionarily ancient protease family. Unlike most other proteases, they are polytopic membrane proteins and specialize in cleaving transmembrane protein substrates. The polar active site of rhomboid protease is embedded in the membrane and normally closed. For the bacterial rhomboid GlpG, it has been proposed that one of the transmembrane helices (S5) of the protease can rotate to open a lateral gate, enabling substrate to enter the protease from inside the membrane. Here, we studied the conformational change in GlpG by solving the cocrystal structure of the protease with a mechanism-based inhibitor. We also examined the lateral gating model by cross-linking S5 to a neighboring helix (S2). The crystal structure shows that inhibitor binding displaces a capping loop (L5) from the active site but causes only minor shifts in the transmembrane helices. Cross-linking S5 and S2, which not only restricts the lateral movement of S5 but also prevents substrate from passing between the two helices, does not hinder the ability of the protease to cleave a membrane protein substrate in detergent solution and in reconstituted membrane vesicles. Taken together, these data suggest that a large lateral movement of the S5 helix is not required for substrate access to the active site of rhomboid protease.

  16. Role of mast cells and protease-activated receptor-2 in cyclooxygenase-2 expression in urothelial cells

    OpenAIRE

    Wang, Zun-Yi; Wang, Peiqing; Bjorling, Dale E.

    2009-01-01

    Mast cells have been shown to play a role in development and persistence of various inflammatory bladder disorders. Mast cell-derived tryptase specifically activates protease-activated receptor-2 (PAR-2), and PAR-2 is known to be involved in inflammation. We investigated whether mast cells participate in increase of cyclooxygenase-2 (COX-2) protein abundance in urothelium/suburothelium of bladders of mice subsequent to cyclophosphamide (CYP)-induced bladder inflammation. We also used primary ...

  17. Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

    OpenAIRE

    Côté Claude H; Tremblay Marie-Hélène; Duchesne Elise

    2011-01-01

    Abstract Background Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2). The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade. Methods Proliferation of L6 rat skeletal m...

  18. Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases.

    Science.gov (United States)

    Jahanbani, Raheleh; Ghaffari, S Mahmood; Salami, Maryam; Vahdati, Kourosh; Sepehri, Houri; Sarvestani, Nazanin Namazi; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

    2016-12-01

    Walnut (Juglans regia L.) contains approximately 20-25 % protein with abundant essential amino acids. The enzymatic hydrolysate of Persian walnut (Chandler) seed proteins was prepared by incubation with three different proteases, including pancreatic chymotrypsin and trypsin, and a microbial enzyme proteinase K. The hydrolysates were found to possess excellent antioxidant capacities. The peptide fractions scavenged the 2, 2'-anizo-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and inhibited the activity of reactive oxygen species. Walnut protein hydrolysates were also tested, for the first time, against the viability of human breast (MDA-MB231) and colon (HT-29) cancer cell lines. MTT, [3-(4, 5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide], assay was used to assess in vitro cancer cell viability upon treatment with the peptide fractions. The peptide fractions showed cell growth inhibition of 63 ± 1.73 % for breast cancer and 51 ± 1.45 % for colon cancer cells. Thus, a direct correlation between antioxidant and anticancer activities of walnut peptide fractions exists and supports their potential therapeutic benefit.

  19. Purification and characterization of a serine protease (CPM-2) with fibrinolytic activity from the dung beetles.

    Science.gov (United States)

    Ahn, Mi Young; Hahn, Bum-Soo; Ryu, Kang Sun; Hwang, Jae Sam; Kim, Yeong Shik

    2005-07-01

    Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gln Asp Phe Val Glu Glu Ile Leu. CPM-2 was inactivated by Cu2+ and Zn2+ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and alpha1-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved Aalpha- and Bbeta-chains of fibrinogen and fibrin, and gamma-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

  20. Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities.

    Science.gov (United States)

    Imamura, Takahisa; Murakami, Yoji; Nitta, Hidetoshi

    2017-09-26

    Aeromonas sobria serine protease (ASP) is secreted from Aeromonas sobria, a pathogen causing gastroenteritis and sepsis. ASP resembles Saccharomyces cerevisiae Kex2, a member of the subtilisin family, and preferentially cleaves peptide bonds at the C-terminal side of paired basic amino acid residues; also accepting unpaired arginine at the P1 site. Unlike Kex2, however, ASP lacks an intramolecular chaperone N-terminal propeptide, instead utilizes the external chaperone ORF2 for proper folding, therefore, ASP and its homologues constitute a new subfamily in the subtilisin family. Through activation of the kallikrein/kinin system, ASP induces vascular leakage, and presumably causes edema and septic shock. ASP accelerates plasma clotting by α-thrombin generation from prothrombin, whereas it impairs plasma clottability by fibrinogen degradation, together bringing about blood coagulation disorder that occurs in disseminated intravascular coagulation, a major complication of sepsis. From complement C5 ASP liberates C5a that induces neutrophil recruitment and superoxide release, and mast cell degranulation, which are associated with pus formation, tissue injury and diarrhea, respectively. Nicked two-chain ASP also secreted from A. sobria is more resistant to inactivation by α2-macroglobulin than single-chain ASP, thereby raising virulence activities. Thus, ASP is a potent virulence factor and may participate in the pathogenesis of A. sobria infection.

  1. Strategy for improving extracellular lipolytic activities by a novel thermotolerant Staphylococcus sp. strain

    Directory of Open Access Journals (Sweden)

    Cherif Slim

    2011-11-01

    Full Text Available Abstract Background Extracellular bacterial lipases received much attention for their substrate specificity and their ability to function under extreme environments (pH, temperature.... Many staphylococci produced lipases which were released into the culture medium. Reports of extracellular thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results This study focused on novel strategies to increase extracellular lipolytic enzyme production by a novel Staphylococcus sp. strain ESW. The microorganism needed neutral or alkaline pH values between 7.0 and 12.0 for growth. For pH values outside this range, cell growth seemed to be significantly inhibited. Staphylococcus sp. culture was able to grow within a wide temperature range (from 30 to 55°C. The presence of oils in the culture medium leaded to improvements in cells growth and lipolytic enzyme activity. On the other hand, although chemical surfactants leaded to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. In addition, our results showed that this novel Staphylococcus sp. strain produced biosurfactants simultaneously with lipolytic activity, when soapstock (The main co-product of the vegetable oil refining industry, was used as the sole carbon source. Conclusion A simultaneous biosurfactant and extracellular lipolytic enzymes produced bacterial strain with potential application in soap stock treatment

  2. The differential expression of protease activated receptors contributes to functional differences between dark and fair keratinocytes.

    Science.gov (United States)

    Xue, Meilang; Lin, Haiyan; Zhao, Ruilong; Liang, Hai Po Helena; Jackson, Christopher

    2017-03-01

    Dark skin has different properties in comparison to fair skin. Melanocytes have been shown to partly contribute to these differences, however, the involvement of keratinocytes from dark or fair skin is not well demonstrated. This study investigated the proliferation and barrier function of dark keratinocytes (DK) and fair keratinocytes (FK), and the role of protease activated receptor (PAR)1 and PAR2. DK and FK were isolated from human neonatal foreskins. Cells were treated with PAR1/PAR2 agonists or antagonists, proliferation was measured by BrdU assay; permeability by the flux of FITC-dextran; protein expression by immunostaining or western blot. When compared to FK, DK proliferated significantly slower; had higher cell permeability; expressed less phosphorylated (P)-ERK/ERK, caspase-14, E-cadherin, tissue growth factor (TGF)-β3 and PAR1; and expressed more PAR2, and matrix metalloproteinase (MMP)-9. Activation of PAR1 or inhibition of PAR2 stimulated cell proliferation and ERK activation, and in concordance inhibition of PAR1 or activation of PAR2 suppressed cell proliferation and ERK activation in both DK and FK. Inhibition of PAR2 decreased and inhibition of PAR1 increased cell permeability. In foreskin sections, the epidermis of dark foreskin expressed less caspase-14 and the same level but different distribution of E-cadherin, when compared to fair foreskin. These data highlight functional differences in proliferation and barrier integrity between HK and FK that are partly associated with their differential expression of PAR1 and PAR2. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  3. Influence of hydrophobic mismatch on the catalytic activity of Escherichia coli GlpG rhomboid protease.

    Science.gov (United States)

    Foo, Alexander C Y; Harvey, Brandon G R; Metz, Jeff J; Goto, Natalie K

    2015-04-01

    Rhomboids comprise a broad family of intramembrane serine proteases that are found in a wide range of organisms and participate in a diverse array of biological processes. High-resolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid have provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. Key to this are observations that GlpG hydrophobic domain dimensions may not be sufficient to completely span the native lipid bilayer. This formed the basis for a model where hydrophobic mismatch Induces thinning of the local membrane environment to promote access to transmembrane substrates. However, hydrophobic mismatch also has the potential to alter the functional properties of the rhomboid, a possibility we explore in the current work. For this purpose, we purified the catalytic transmembrane domain of GlpG into phosphocholine or maltoside detergent micelles of varying alkyl chain lengths, and assessed proteolytic function with a model water-soluble substrate. Catalytic turnover numbers were found to depend on detergent alkyl chain length, with saturated chains containing 10-12 carbon atoms supporting maximal activity. Similar results were obtained in phospholipid bicelles, with no proteolytic activity being detected in longer-chain lipids. Although differences in thermal stability and GlpG oligomerization could not explain these activity differences, circular dichroism spectra suggest that mismatch gives rise to a small change in structure. Overall, these results demonstrate that hydrophobic mismatch can exert an inhibitory effect on rhomboid activity, with the potential for changes in local membrane environment to regulate activity in vivo.

  4. Proteases, neutrophils, and periodontitis: the NET effect.

    Science.gov (United States)

    Nauseef, William M

    2014-10-01

    Neutrophils exert potent antimicrobial activities in their role as first-line cellular defenders against infection. The synergistic and collective actions of oxidants and granule proteins, including serine proteases, support the microbial killing in phagosomes, where most neutrophil-mediated antimicrobial action occurs. In addition to phagocytosis, specific stimuli prompt neutrophils to extrude a matrix of DNA, histones, and granule proteins to produce neutrophil extracellular traps (NETs), which can trap microbes. Mice lacking the serine proteases necessary for NET production are more susceptible to infection, an observation suggesting that functional NETs are required for host protection. In this issue of the JCI, Sørensen and colleagues characterize neutrophils from a patient with Papillon-Lefèvre syndrome. The patient has an inactivating mutation in the gene encoding dipeptidyl peptidase I, resulting in neutrophils lacking elastase, a serine protease required for NET production. Despite the inability to form NETS, neutrophils from this patient killed pathogens in vitro, and the patient did not exhibit evidence of an increased propensity toward bacterial infections. Together, these results suggest that proteases in human neutrophils are dispensable for protection against bacterial infection and that the ability to generate NETs in vitro does not compromise host defense.

  5. A fibrinolytic protease AfeE from Streptomyces sp. CC5, with potent thrombolytic activity in a mouse model.

    Science.gov (United States)

    Sun, Zhibin; Liu, Pingping; Cheng, Guangyan; Zhang, Biying; Dong, Weiliang; Su, Xingli; Huang, Yan; Cui, Zhongli; Kong, Yi

    2016-04-01

    Fibrinolytic proteases have potential applications in cardiovascular disease therapy. A novel fibrinolytic protease, AfeE, with strong thrombolytic activity was purified from Streptomyces sp. CC5. AfeE displayed maximum activity at 40°C in the pH range of 7.0-12.0. It was strongly inhibited by serine protease inhibitor phenylmethanesulfonylfluoride, soybean trypsin inhibitor, tosyl-l-lysine chloromethyl ketone and tosyl-l-phenylalanine chloromethyl ketone. The activity of the enzyme was partially inhibited by Cu(2+), Co(2+) and Zn(2+). AfeE exhibited higher substrate specificity for fibrin than fibrinogen, which has rarely been reported in fibrinolytic enzymes. AfeE also showed high thrombolytic activity in a carrageenan-induced mouse tail thrombosis model. AfeE prolonged prothrombin time, activated partial thromboplastin time, and thrombin time in rat blood. A bleeding time assay revealed that AfeE did not prolong bleeding time in mice at a dose of 1mg/kg. No acute cytotoxicity was observed for AfeE at 320μg/well in human umbilical vein endothelial cells. The afeE gene was cloned from the genome of Streptomyces sp. CC5. Full-length AFE-CC5E contained 434 amino acids and was processed into a mature form consisting 284 amino acids by posttranslational modification, as revealed by high-resolution mass spectrometry analysis. These results indicate that AfeE is a prospective candidate for antithrombotic drug development.

  6. A specific cathepsin-L-like protease purified from an insect midgut shows antibacterial activity against gut symbiotic bacteria.

    Science.gov (United States)

    Byeon, Jin Hee; Seo, Eun Sil; Lee, Jun Beom; Lee, Min Ja; Kim, Jiyeun Kate; Yoo, Jin Wook; Jung, Yunjin; Lee, Bok Luel

    2015-11-01

    Because gut symbiotic bacteria affect host biology, host insects are expected to evolve some mechanisms for regulating symbiont population. The bean bug, Riptortus pedestris, harbors the Burkholderia genus as a gut symbiont in the midgut organ, designated as the M4 region. Recently, we demonstrated that the lysate of M4B, the region adjacent to M4, harbors potent antibacterial activity against symbiotic Burkholderia but not to cultured Burkholderia. However, the bona fide substance responsible for observed antibacterial activity was not identified in the previous study. Here, we report that cathepsin-L-like protease purified from the lysate of M4B showed strong antibacterial activity against symbiotic Burkholderia but not the cultured Burkholderia. To further confirm this activity, recombinant cathepsin-L-like protease expressed in Escherichia coli also showed antibacterial activity against symbiotic Burkholderia. These results suggest that cathepsin-L-like protease purified from the M4B region plays a critical role in controlling the population of the Burkholderia gut symbiont. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

    Science.gov (United States)

    Kong, Lulu; Lu, Anrui; Guan, Jingmin; Yang, Bing; Li, Muwang; Hillyer, Julián F; Ramarao, Nalini; Söderhäll, Kenneth; Liu, Chaoliang; Ling, Erjun

    2015-01-01

    Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.

  8. Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.

    Science.gov (United States)

    Qian, Ye-Qing; Li, Ye; Yang, Fan; Yu, Yan-Qin; Yang, Jin-Shu; Yang, Wei-Jun

    2012-03-01

    Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system.

  9. PROGNOSTIC VALUE OF PROTEASE ACTIVATED RECEPTOR-1 IN EGYPTIAN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA

    Directory of Open Access Journals (Sweden)

    Adel Abd Elhaleim Hagag

    2014-04-01

    Full Text Available Background: Acute Lymphoblastic leukemia (ALL is a malignant disorder of lymphoid progenitor cells that proliferate and replace the normal hematopoietic cells of the bone marrow. Protease-activated receptor 1 (PAR-1, is atypical member of this family of receptors that mediate cellular responses to thrombin and related proteases. PAR1 is expressed by a wide range of tumor cells and can promote tumor growth, invasion and metastasis. The aim of this work was to study the role of PAR-1 expression in newly diagnosed ALL patients. Patients and methods: This study was conducted on 44 children with newly diagnosed ALL who were admitted to Hematology Unit, Pediatric department, Tanta University Hospital including 24 males and 20 females with their age ranged from 4-17 years and their mean age value of 9.06±3.26 who were divided into two groups; PAR-1 positive group (18 patients and PAR-1 negative group (26 patients. All patients were subjected to complete history taking, thorough clinical examination, bone marrow aspiration and flow cytometric analysis for detection of PAR-1 expression by malignant cells. Results: PAR-1 was positive in 18 cases (41% and negative in 26 cases (59% of studied patients. This study showed no significant relation between PAR-1 expression and age, sex and most of the clinical data including hepatomegaly, splenomegaly and purpura while generalized lymphadenopathy was significantly higher in PAR-1 positive group. PAR-1 positive expression was associated with some bad prognostic laboratory parameters including higher hemoglobin, higher white blood cells, higher peripheral blood and bone marrow blast cells, higher serum LDH and lower platelets count. No significant association was detected between PAR-1 expression and immunophenotyping. There were significantly higher remission rates in PAR-1 negative group and significantly higher relapse and death rates in PAR-1 positive group. Conclusion: From this study, it could be concluded

  10. Plasminogen activation and cancer

    DEFF Research Database (Denmark)

    Danø, Keld; Behrendt, N.; Hoyer-Hansen, G.

    2005-01-01

    Breakdown of the extracellular matrix is crucial for cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease plasmin and a number of matrix metalloproteases.The activity of each of these proteases is regulated by an array......, the regulation of extracellular proteolysis in cancer involves a complex interplay between cancer cells and non-malignant stromal cells in the expression of the molecular components involved. For some types of cancer, this cellular interplay mimics that observed in the tissue of ori- gin during non......-neoplastic tissue remodelling processes.We propose that cancer invasion can be considered as uncontrolled tissue remodelling. Inhibition of extracellular proteases is an attractive approach to cancer therapy. Because proteases have many different functions in the normal organism, efficient inhibition will have...

  11. Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.

    OpenAIRE

    Sours, Kevin M.; Xiao,Yao; Ahn, Natalie G.

    2014-01-01

    Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flex...

  12. Crystal structure of the caseinolytic protease gene regulator, a transcriptional activator in actinomycetes.

    Science.gov (United States)

    Russo, Santina; Schweitzer, Jens-Eric; Polen, Tino; Bott, Michael; Pohl, Ehmke

    2009-02-20

    Human pathogens of the genera Corynebacterium and Mycobacterium possess the transcriptional activator ClgR (clp gene regulator) which in Corynebacterium glutamicum has been shown to regulate the expression of the ClpCP protease genes. ClgR specifically binds to pseudo-palindromic operator regions upstream of clpC and clpP1P2. Here, we present the first crystal structure of a ClgR protein from C. glutamicum. The structure was determined from two different crystal forms to resolutions of 1.75 and 2.05 A, respectively. ClgR folds into a five-helix bundle with a helix-turn-helix motif typical for DNA-binding proteins. Upon dimerization the two DNA-recognition helices are arranged opposite to each other at the protein surface in a distance of approximately 30 A, which suggests that they bind into two adjacent major grooves of B-DNA in an anti-parallel manner. A binding pocket is situated at a strategic position in the dimer interface and could possess a regulatory role altering the positions of the DNA-binding helices.

  13. Quantitation of fibroblast activation protein (FAP-specific protease activity in mouse, baboon and human fluids and organs

    Directory of Open Access Journals (Sweden)

    Fiona M. Keane

    2014-01-01

    Full Text Available The protease fibroblast activation protein (FAP is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

  14. Direct visualization of protease action on collagen triple helical structure.

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    Gabriel Rosenblum

    Full Text Available Enzymatic processing of extracellular matrix (ECM macromolecules by matrix metalloproteases (MMPs is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen (3/4 fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.

  15. CHANGES IN LEVELS OF ACTIVITY OF SERINE PROTEASES ACCOMPANY THE EXPOSURE OF COMMON BEAN (PHASEOLUS VULGARIS L. TO WATER DEFICIT

    Directory of Open Access Journals (Sweden)

    M. Budič

    2008-09-01

    Full Text Available A wide variety of proteolytic enzymes exist in plants. On their levels depends protein turnover, a fundamental component in plant development and adaptation to environmental conditions. Cysteine proteases have frequently been reported to be influenced by drought, but only a few serine proteases (SP, among them the trypsin-like enzyme and two aminopeptidases from bean leaves (Bartels and Sunkar, 2005; Hieng et al., 2004. Our starting point was to identify proteolytic activities assigned to SPs that change with drought and then to characterize the corresponding proteases. A quantitative, analytical one-step method was used to separate endopeptidases and aminopeptidases active against a range of substrates in leaf extracts of plants grown in the field (FC. The influence of drought was determined for those of these activities which were confirmed as SPs, based on their inhibition by specific inhibitors. Under water deficit in plants grown under controlled conditions (CC their levels changed in different ways. The levels of SP activities in FC plants, observed during a period of relative drought, were similar to those measured in mildly stressed CC plants. The partial characterisations of some of these SPs will be presented. Our results point to a number of roles for different SPs in the plant response to water stress, which could range from enhanced protein turnover to limited proteolysis at specific sites.

  16. Inhibition of Protease-activated Receptor 1 Ameliorates Intestinal Radiation Mucositis in a Preclinical Rat Model

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Junru; Kulkarni, Ashwini [Division of Radiation Health, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Chintala, Madhu [Schering-Plough Research Institute, Kenilworth, New Jersey (United States); Fink, Louis M. [Nevada Cancer Institute, Las Vegas, Nevada (United States); Hauer-Jensen, Martin, E-mail: mhjensen@life.uams.edu [Division of Radiation Health, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Surgery Service, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas (United States)

    2013-01-01

    Purpose: To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects. Methods and Materials: Rats underwent fractionated X-irradiation (5 Gy Multiplication-Sign 9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation. Results: Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups. Conclusion: Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

  17. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

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    Jarroll Edward L

    2006-05-01

    Full Text Available Abstract Background Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. Results Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM, which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM, elastin (elastic fibrils of ECM, plasminogen (involved in proteolytic degradation of ECM, as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC. Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like suggesting a role in blood-brain barrier perturbations. Conclusion Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s in invasion of the brain tissue by amoebae.

  18. Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury.

    Science.gov (United States)

    Indyk, J A; Chen, Z L; Tsirka, S E; Strickland, S

    2003-01-01

    Laminins are important components of the extracellular matrix, and participate in neuronal development, survival and regeneration. The tissue plasminogen activator/plasmin extracellular protease cascade and downstream laminin degradation are implicated in excitotoxin-induced neuronal degeneration. To determine which specific laminin chains are involved, we investigated the expression of laminins in the hippocampus, and the cell types expressing them. Reverse transcription-PCR demonstrated that the messenger RNAs for all laminin chains could be detected in the hippocampus. To determine the localization of laminin chain expression, immunostaining was used. This method showed that alpha5, beta1 and gamma1 are most highly expressed in the neuronal cell layers. Immunoblotting confirmed the hippocampal expression of the chains alpha5, beta1 and gamma1, and RNA in situ hybridization showed a neuronal expression pattern of alpha5, beta1 and gamma1. At early time points following intrahippocampal injection of kainate, alpha5, beta1 and gamma1 chain immunoreactivities were lost. In addition, tissue plasminogen activator-deficient mice, which are resistant to kainate-induced neuronal death, show no significant change in laminins alpha5, beta1 and gamma1 after intrahippocampal kainate injection. Taken together, these results suggest that laminin-10 (alpha5-beta1-gamma1) comprises a major neuronal laminin in the mouse hippocampus, and is degraded before neuronal death during excitotoxic injury by the tissue plasminogen activator/plasmin protease cascade. By identifying a neuronal laminin (laminin-10) that participates in neuronal degeneration after excitotoxic injury, this study clarifies the molecular definition of the extracellular matrix in the hippocampus and further defines a pathway for mechanisms of neuronal death.

  19. Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

    Directory of Open Access Journals (Sweden)

    Wagner A S Judice

    Full Text Available Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold, and also decrease in k 3 (3.5-fold. The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25-S(-/(His(163-Im(+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

  20. Biochemical analysis and investigation on the prospective applications of alkaline protease from a Bacillus cereus strain.

    Science.gov (United States)

    Saleem, Mahjabeen; Rehman, Atiqa; Yasmin, Riffat; Munir, Bushra

    2012-06-01

    Proteases have prospective financial and environment-friendly applications; hence attention is focused currently on the finding of new protease producing microorganism so as to meet the requirements of industry. A thermophilic bacterial strain producing extracellular protease activity was isolated from soil and identified as Bacillus cereus by analysis of 16S rRNA. Protease production by the microorganism was improved by studying the impact of the type of nitrogen and carbon source, fermentation period, growth temperature and initial pH of the culture medium in cultivation optimization experiments. The enzyme was purified to homogeneity in two step procedure involving Sephadex G-75 and Q-Sepharose chromatography. The molecular weight of purified enzyme was found to be 58 kDa by SDS-PAGE. Protease exhibited a pH and temperature optima of 7.5 and 60°, respectively. The enzyme was active in the pH range of 6.0-9.0 and stable up to 70°C. Histological analysis of protease treated goat and cow skin pelts showed complete removal of non leather forming structures such as hair shaft, hair follicles and glandular structures. The protease showed the stain removing property from blood stained cotton cloth and found to be compatible with six commercially available detergents. The protease could release peptides from natural proteins after digestion of coagulated egg albumin and blood clot.

  1. Production, partial purification and characterization of protease from a phytopathogenic fungi Alternaria solani (Ell. and Mart.) Sorauer.

    Science.gov (United States)

    Chandrasekaran, Murugesan; Sathiyabama, Muthukrishnan

    2014-08-01

    An alkaline serine protease producing strain Alternaria solani was optimized for its enzyme production under submerged conditions. The maximum production of protease by A. solani was achieved by using sodium nitrate at the optimum concentration of 0.2% w/v. A. solani produced higher quantities (3.75 [unit/mg of protein]) of an inducible extracellular proteases on day 9 after incubation in czapek's dox broth medium amended with 1% casein as an inducer at pH 8.5, temperature 27 °C and 3% sucrose as carbon source. Extracellular proteases were precipitated by ammonium sulphate saturation (80%) method and purified on Sephadex G-100 column chromatography. The molecular mass of SDS-PAGE and Sephadex G-100 Column Gel permeation chromatography purified protease was estimated to 42 kDa. In addition, trypsin digestion of 42 kDa protein band was carried out and analyzed by MALDI-TOF for the identification of protease. The sequence IKELATNGVVTNVK (378-391) segment of the alkaline serine protease was found by using MS/MS spectrum at 1485 m/z from the purified fraction. It showed optimal activity at 50 °C and pH 9-10 and broad pH stability between pH 6-12. The protease activity was inhibited by phenyl methyl sulfonyl fluoride (PMSF), all the results indicated that the presence of a serine residue in the active site and is thus most likely a member of the serine protease family. This may function as a virulence protein during pathogenesis by A. solani. The results suggested that the presence of appreciable extracellular proteolytic activity in filamentous fungi may serve as a marker of their phytopathogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Activity-dependent release of precursor nerve growth factor, conversion to mature nerve growth factor, and its degradation by a protease cascade.

    Science.gov (United States)

    Bruno, Martin A; Cuello, A Claudio

    2006-04-25

    In this report, we provide direct demonstration that the neurotrophin nerve growth factor (NGF) is released in the extracellular space in an activity-dependent manner in its precursor form (proNGF) and that it is in this compartment that its maturation and degradation takes place because of the coordinated release and the action of proenzymes and enzyme regulators. This converting protease cascade and its endogenous regulators (including tissue plasminogen activator, plasminogen, neuroserpin, precursor matrix metalloproteinase 9, and tissue inhibitor metalloproteinase 1) are colocalized in neurons of the cerebral cortex and released upon neuronal stimulation. We also provide evidence that this mechanism operates in in vivo conditions, as the CNS application of inhibitors of converting and degrading enzymes lead to dramatic alterations in the tissue levels of either precursor NGF or mature NGF. Pathological alterations of this cascade in the CNS might cause or contribute to a lack of proper neuronal trophic support in conditions such as cerebral ischemia, seizure and Alzheimer's disease or, conversely, to excessive local production of neurotrophins as reported in inflammatory arthritis pain.

  3. A new tagged-TEV protease: construction, optimisation of production, purification and test activity.

    Science.gov (United States)

    Miladi, Baligh; Bouallagui, Hassib; Dridi, Cyrine; El Marjou, Ahmed; Boeuf, Guilhem; Di Martino, Patrick; Dufour, Florence; Elm'Selmi, Abdellatif

    2011-01-01

    The Tobacco Etch Virus (TEV) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity. In this work, we present a new recombinant form of TEV termed Streptag II-TEV for high-level production and purification of TEV protease from Escherichia coli and compare it to the hexahistidine (6xHis) tagged version of TEV. The effects of varying the host strain, the bacterial induction temperature (25, 30 and 37°C) and the IPTG inducer concentration on production and solubility of the two recombinant TEV proteases have been examined. Optimal Streptag II-TEV protein expression were obtained in the E. coli KRX strain under an induction temperature of 25°C in the presence of IPTG at 0.5 mM. In these conditions, soluble Streptag II-TEV and 6xHis-TEV proteases accounted for about 25% and 18% of total soluble proteins, respectively. About 70% of Streptag II-TEV and 60% of 6xHis-TEV were detected in the supernatant. Streptag II-TEV protease purifies to near homogeneity (approximately 99%) via a simple, single step Strep-Tactin chromatography purification protocol based on the presence of Streptag II. The higher production of Streptag II-TEV coupled to its purification and cleavage efficiencies make it an attractive alternate to 6xHis-TEV.

  4. Reprint of: A new tagged-TEV protease: Construction, optimisation of production, purification and test activity.

    Science.gov (United States)

    Miladi, Baligh; Bouallagui, Hassib; Dridi, Cyrine; El Marjou, Ahmed; Boeuf, Guilhem; Di Martino, Patrick; Dufour, Florence; Elm'selmi, Abdellatif

    2011-09-02

    The Tobacco Etch Virus (TEV) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity. In this work, we present a new recombinant form of TEV termed Streptag II-TEV for high-level production and purification of TEV protease from Escherichia coli and compare it to the hexahistidine (6xHis) tagged version of TEV. The effects of varying the host strain, the bacterial induction temperature (25, 30 and 37°C) and the IPTG inducer concentration on production and solubility of the two recombinant TEV proteases have been examined. Optimal Streptag II-TEV protein expression were obtained in the E. coli KRX strain under an induction temperature of 25°C in the presence of IPTG at 0.5mM. In these conditions, soluble Streptag II-TEV and 6xHis-TEV proteases accounted for about 25% and 18% of total soluble proteins, respectively. About 70% of Streptag II-TEV and 60% of 6xHis-TEV were detected in the supernatant. Streptag II-TEV protease purifies to near homogeneity (approximately 99%) via a simple, single step Strep-Tactin chromatography purification protocol based on the presence of Streptag II. The higher production of Streptag II-TEV coupled to its purification and cleavage efficiencies make it an attractive alternate to 6xHis-TEV.

  5. A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L. syn Senna tora (L. Roxb. seed extract

    Directory of Open Access Journals (Sweden)

    Garg Satyendra K

    2011-07-01

    Full Text Available Abstract Background Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases. Methods The crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90% followed by dialysis and size exclusion chromatography (SEC. The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60% and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD and ANOVA were employed as statistical tools. Results The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein extract for 60 min. The inhibitory activity was evident in gelatin SDS-PAGE where a major band (~17-19 kD of protease

  6. Protease activated receptor 4 limits bacterial growth and lung pathology during late stage Streptococcus pneumoniae induced pneumonia in mice.

    Science.gov (United States)

    de Stoppelaar, S F; Van't Veer, C; van den Boogaard, F E; Nieuwland, R; Hoogendijk, A J; de Boer, O J; Roelofs, J J T H; van der Poll, T

    2013-09-01

    Streptococcus pneumoniae is a common causative pathogen of pneumonia and sepsis. Pneumonia and sepsis are associated with enhanced activation of coagulation, resulting in the production of several host-derived proteases at the primary site of infection and in the circulation. Serine proteases cleave protease activated receptors (PARs), which form a molecular link between coagulation and inflammation. PAR4 is one of four subtypes of PARs and is widely expressed by multiple cell types in the respiratory tract implicated in pulmonary inflammation, by immune cells and by platelets. In mice, mouse (m)PAR4 is the only thrombin receptor expressed by platelets. We here sought to determine the contribution of mPAR4 to the host response during pneumococcal pneumonia. Pneumonia was induced by intranasal inoculation with S. pneumoniae in mPAR4-deficient (par4-/-) and wild-type mice. Mice were sacrificed after 6, 24 or 48 hours (h). Blood, lungs, liver and spleen were collected for analyses. Ex vivo stimulation assays were performed with S. pneumoniae and mPAR4 activating peptides. At 48 h after infection, higher bacterial loads were found in the lungs and blood of par4-/- mice (p pneumoniae. Thrombin inhibition resulted in decreased cytokine release after S. pneumoniae stimulation in human whole blood. Our findings suggest that mPAR4 contributes to antibacterial defence during murine pneumococcal pneumonia.

  7. Antioxidant and Anti-Protease Activities of Diazepinomicin from the Sponge-Associated Micromonospora Strain RV115

    Directory of Open Access Journals (Sweden)

    Helga Stopper

    2012-10-01

    Full Text Available Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2 and human promyelocytic (HL-60 cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70–90 µM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 µM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate.

  8. Comparison of clinical and environmental isolates of Acanthamoeba based on morphology, protease and gelatinase activity, and the cysteine proteinase gene

    Directory of Open Access Journals (Sweden)

    Gil M. Penuliar

    2010-06-01

    Full Text Available Acanthamoeba spp. are opportunistic pathogens that cause amebic keratitis and granulomatous amebic encephalitis in man. Recent attempts to correlate pathogenicity with species have been proven difficult due to inconsistencies in morphology-based classification. The objectives of this study were: (1 to compare clinical and environmental isolates based on morphology, protease and gelatinase activity, and the cysteine proteinase (CP gene, and (2 to determine whether these features can be used to differentiate the isolates. Results show some degree of variation in trophozoite and cyst morphology. Zymography, demonstrated gross differences in banding patterns, and the protease activity of clinical isolates was greater than the environmental isolates (p-value < 0.01. Amplification of the CP gene yielded two bands in the environmental isolates, approximately 755 bp and 440 bp in length. In contrast, only one band, either the 755 bp or 440 bp band was amplified in the clinical isolates. The results confirmed the limitations of morphology in differentiating Acanthamoeba species, and suggest that zymography, protease activity, and detection of the CP gene are useful reference tests to distinguish pathogenic from non-pathogenic isolates.

  9. Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

    Energy Technology Data Exchange (ETDEWEB)

    Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.; Fairlie, W. Douglas; Colman, Peter M.; Crabb, Brendan S.; Smith, Brian J.; (WEHIMR); (Melbourne)

    2009-08-28

    The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

  10. Alkaline serine protease AprE plays an essential role in poly-γ-glutamate production during natto fermentation.

    Science.gov (United States)

    Kada, Shigeki; Ishikawa, Atsushi; Ohshima, Yoshifumi; Yoshida, Ken-ichi

    2013-01-01

    Natto is a traditional Japanese food made from soybeans fermented by natto starter strains of Bacillus subtilis natto. It has been suggested that extracellular protease activity released by the bacteria are involved in the production of poly-γ-glutamate (γ-PGA) during natto fermentation. One of the natto starters, strain r22, possesses at least seven genes, each of which encoded an extracellular protease orthologous to its counterpart in B. subtilis 168, aprE, bpr, epr, mpr, nprE, vpr, and wprA, but it was found to lack nprB. Inactivating the aprE ortholog alone resulted in a severe decrease in γ-PGA production and in the total extracellular protease activity. The defect in γ-PGA production of the mutant lacking the aprE ortholog was complemented when the medium was supplemented with sufficient glutamate. These results suggest that the alkaline serine protease encoded by aprE plays an indispensable role in supplying materials to produce γ-PGA. On the other hand, simultaneous inactivation of all the protease genes except for aprE did not significantly affect either γ-PGA production or total protease activity.

  11. Enhancement of proteolytic activity of a thermostable papain-like protease by structure-based rational design.

    Directory of Open Access Journals (Sweden)

    Sruti Dutta

    Full Text Available Ervatamins (A, B and C are papain-like cysteine proteases from the plant Ervatamia coronaria. Among Ervatamins, Ervatamin-C is a thermostable protease, but it shows lower catalytic efficiency. In contrast, Ervatamin-A which has a high amino acid sequence identity (∼90% and structural homology (Cα rmsd 0.4 Å with Ervatamin-C, has much higher catalytic efficiency (∼57 times. From the structural comparison of Ervatamin-A and -C, two residues Thr32 and Tyr67 in the catalytic cleft of Ervatamin-A have been identified whose contributions for higher activity of Ervatamin-A are established in our earlier studies. In this study, these two residues have been introduced in Ervatamin-C by site directed mutagenesis to enhance the catalytic efficiency of the thermostable protease. Two single mutants (S32T and A67Y and one double mutant (S32T/A67Y of Ervatamin-C have been generated and characterized. All the three mutants show ∼ 8 times higher catalytic efficiency (k cat/K m than the wild-type. The thermostability of all the three mutant enzymes remained unchanged. The double mutant does not achieve the catalytic efficiency of the template enzyme Ervatamin-A. By modeling the structure of the double mutant and probing the role of active site residues by docking a substrate, the mechanistic insights of higher activity of the mutant protease have been addressed. The in-silico study demonstrates that the residues beyond the catalytic cleft also influence the substrate binding and positioning of the substrate at the catalytic centre, thus controlling the catalytic efficiency of an enzyme.

  12. Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

    Science.gov (United States)

    Matthews, D A; Dragovich, P S; Webber, S E; Fuhrman, S A; Patick, A K; Zalman, L S; Hendrickson, T F; Love, R A; Prins, T J; Marakovits, J T; Zhou, R; Tikhe, J; Ford, C E; Meador, J W; Ferre, R A; Brown, E L; Binford, S L; Brothers, M A; DeLisle, D M; Worland, S T

    1999-09-28

    Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

  13. Modification of the secretion pattern of proteases, inflammatory mediators, and extracellular matrix proteins by human aortic valve is key in severe aortic stenosis.

    Science.gov (United States)

    Alvarez-Llamas, Gloria; Martín-Rojas, Tatiana; de la Cuesta, Fernando; Calvo, Enrique; Gil-Dones, Felix; Dardé, Veronica M; Lopez-Almodovar, Luis F; Padial, Luis R; Lopez, Juan-Antonio; Vivanco, Fernando; Barderas, Maria G

    2013-09-01

    One of the major challenges in cardiovascular medicine is to identify candidate biomarker proteins. Secretome analysis is particularly relevant in this search as it focuses on a subset of proteins released by a cell or tissue under certain conditions. The sample can be considered as a plasma subproteome and it provides a more direct approximation to the in vivo situation. Degenerative aortic stenosis is the most common worldwide cause of valve replacement. Using a proteomic analysis of the secretome from aortic stenosis valves we could identify candidate markers related to this pathology, which may facilitate early diagnosis and treatment. For this purpose, we have designed a method to validate the origin of secreted proteins, demonstrating their synthesis and release by the tissue and ruling out blood origin. The nLC-MS/MS analysis showed the labeling of 61 proteins, 82% of which incorporated the label in only one group. Western blot and selective reaction monitoring differential analysis, revealed a notable role of the extracellular matrix. Variation in particular proteins such as PEDF, cystatin and clusterin emphasizes the link between aortic stenosis and atherosclerosis. In particular, certain proteins variation in secretome levels correlates well, not only with label incorporation trend (only labeled in aortic stenosis group) but, more importantly, with alterations found in plasma from an independent cohort of samples, pointing to specific candidate markers to follow up in diagnosis, prognosis, and therapeutic intervention.

  14. Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2

    Energy Technology Data Exchange (ETDEWEB)

    Kalayarasan, Srinivasan, E-mail: kalaivasanbio@gmail.com; Sriram, Narayanan; Soumyakrishnan, Syamala; Sudhandiran, Ganapasam, E-mail: sudhandiran@yahoo.com

    2013-09-01

    Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis. Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF. - Highlights: • DAS inhibits PAR-2 activity; bleomycin stimulates PAR-2 activity. • Increase in PAR-2 activity is correlated with pulmonary fibrosis • DAS reduces pro-inflammatory activity linked to facilitating pulmonary fibrosis. • DAS inhibits apoptosis of alveolar epithelial cells.

  15. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    Science.gov (United States)

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S-activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. PMID:21435673

  16. HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-β type I receptor

    Science.gov (United States)

    Sakata, Kotaro; Hara, Mitsuko; Terada, Takaho; Watanabe, Noriyuki; Takaya, Daisuke; Yaguchi, So-Ichi; Matsumoto, Takehisa; Matsuura, Tomokazu; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamaguchi, Tokio; Miyazawa, Keiji; Aizaki, Hideki; Suzuki, Tetsuro; Wakita, Takaji; Imoto, Masaya; Kojima, Soichi

    2013-11-01

    Viruses sometimes mimic host proteins and hijack the host cell machinery. Hepatitis C virus (HCV) causes liver fibrosis, a process largely mediated by the overexpression of transforming growth factor (TGF)-β and collagen, although the precise underlying mechanism is unknown. Here, we report that HCV non-structural protein 3 (NS3) protease affects the antigenicity and bioactivity of TGF-β2 in (CAGA)9-Luc CCL64 cells and in human hepatic cell lines via binding to TGF-β type I receptor (TβRI). Tumor necrosis factor (TNF)-α facilitates this mechanism by increasing the colocalization of TβRI with NS3 protease on the surface of HCV-infected cells. An anti-NS3 antibody against computationally predicted binding sites for TβRI blocked the TGF-β mimetic activities of NS3 in vitro and attenuated liver fibrosis in HCV-infected chimeric mice. These data suggest that HCV NS3 protease mimics TGF-β2 and functions, at least in part, via directly binding to and activating TβRI, thereby enhancing liver fibrosis.

  17. Crimean-Congo hemorrhagic fever virus-encoded ovarian tumor protease activity is dispensable for virus RNA polymerase function.

    Science.gov (United States)

    Bergeron, Eric; Albariño, César G; Khristova, Marina L; Nichol, Stuart T

    2010-01-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus (genus Nairovirus, family Bunyaviridae) associated with high case fatality disease outbreaks in regions of Africa, Europe, and Asia. The CCHFV genome consists of three negative-strand RNA segments, S, M, and L. The unusually large virus L polymerase protein and the need for biosafety level 4 (BSL-4) containment conditions for work with infectious virus have hampered the study of CCHFV replication. The L protein has an ovarian tumor (OTU) protease domain located in the N terminus, which has led to speculation that the protein may be autoproteolytically cleaved to generate the active virus L polymerase and additional functions. We report the successful development of efficient CCHFV helper virus-independent S, M, and L segment minigenome systems for analysis of virus RNA and protein features involved in replication. The virus RNA segment S, M, and L untranslated regions were found to be similar in support of replication of the respective minigenomes. In addition, the OTU domain located in the N terminus of the expressed virus L protein was shown to be a functional protease. However, no evidence of L protein autoproteolytic processing was found, and the OTU protease activity was dispensable for virus RNA replication. Finally, physiologically relevant doses of ribavirin inhibited CCHFV minigenome replication. These results demonstrated the utility of the minigenome system for use in BSL-2 laboratory settings to analyze CCHFV biology and in antiviral drug discovery programs for this important public health and bioterrorism threat.

  18. Bacterial community composition and extracellular enzyme activity in temperate streambed sediment during drying and rewetting.

    Directory of Open Access Journals (Sweden)

    Elisabeth Pohlon

    Full Text Available Droughts are among the most important disturbance events for stream ecosystems; they not only affect stream hydrology but also the stream biota. Although desiccation of streams is common in Mediterranean regions, phases of dryness in headwaters have been observed more often and for longer periods in extended temperate regions, including Central Europe, reflecting global climate change and enhanced water withdrawal. The effects of desiccation and rewetting on the bacterial community composition and extracellular enzyme activity, a key process in the carbon flow of streams and rivers, were investigated in a typical Central European stream, the Breitenbach (Hesse, Germany. Wet streambed sediment is an important habitat in streams. It was sampled and exposed in the laboratory to different drying scenarios (fast, intermediate, slow for 13 weeks, followed by rewetting of the sediment from the fast drying scenario via a sediment core perfusion technique for 2 weeks. Bacterial community structure was analyzed using CARD-FISH and TGGE, and extracellular enzyme activity was assessed using fluorogenic model substrates. During desiccation the bacterial community composition shifted toward composition in soil, exhibiting increasing proportions of Actinobacteria and Alphaproteobacteria and decreasing proportions of Bacteroidetes and Betaproteobacteria. Simultaneously the activities of extracellular enzymes decreased, most pronounced with aminopeptidases and less pronounced with enzymes involved in the degradation of polymeric carbohydrates. After rewetting, the general ecosystem functioning, with respect to extracellular enzyme activity, recovered after 10 to 14 days. However, the bacterial community composition had not yet achieved its original composition as in unaffected sediments within this time. Thus, whether the bacterial community eventually recovers completely after these events remains unknown. Perhaps this community undergoes permanent changes

  19. Bone mineral density changes in protease inhibitor-sparing vs. nucleoside reverse transcriptase inhibitor-sparing highly active antiretroviral therapy: data from a randomized trial

    DEFF Research Database (Denmark)

    Hansen, Ann-Brit Eg; Obel, N; Nielsen, H

    2011-01-01

    The aim of the study was to compare changes in bone mineral density (BMD) over 144 weeks in HIV-infected patients initiating nucleoside reverse transcriptase inhibitor (NRTI)-sparing or protease inhibitor-sparing highly active antiretroviral therapy (HAART).......The aim of the study was to compare changes in bone mineral density (BMD) over 144 weeks in HIV-infected patients initiating nucleoside reverse transcriptase inhibitor (NRTI)-sparing or protease inhibitor-sparing highly active antiretroviral therapy (HAART)....

  20. The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate.

    Science.gov (United States)

    Wu, Y P; Siao, C J; Lu, W; Sung, T C; Frohman, M A; Milev, P; Bugge, T H; Degen, J L; Levine, J M; Margolis, R U; Tsirka, S E

    2000-03-20

    Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

  1. Characterization and biological activities of extracellular melanin produced by Schizophyllum commune (Fries).

    Science.gov (United States)

    Arun, G; Eyini, M; Gunasekaran, P

    2015-06-01

    Melanins are enigmatic pigments produced by a wide variety of microorganisms including bacteria and fungi. Here, we have isolated and characterized extracellular melanin from mushroom fungus, Schizophyllum commune. The extracellular dark pigment produced by the broth culture of S. commune, after 21 days of incubation was recovered by hot acid-alkali treatment. The melanin nature of the pigment was characterized by biochemical tests and further, confirmed by UV, IR, EPR, NMR and MALDI-TOF Mass Spectra. Extracellular melanin, at 100 μg/ml, showed significant antibacterial activity against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas fluorescens and antifungal activity against Trichophyton simii and T. rubrum. At a concentration of 50 μg/ml, melanin showed high free radical scavenging activity of DPPH (2,2-diphenyl-1-picrylhydrazyl) indicating its antioxidant potential. It showed concentration dependent inhibition of cell proliferation of Human Epidermoid Larynx Carcinoma Cell Line (HEP-2). This study has demonstrated characterization of melanin from basidiomycetes mushroom fungus, Schizophyllum commune and its applications.

  2. Role of Protease-Activated Receptor 2 in Regulating Focal Segmental Glomerulosclerosis

    Directory of Open Access Journals (Sweden)

    Yongjun Wang

    2017-02-01

    Full Text Available Background /Aims: The underlying mechanisms leading to focal segmental glomerulosclerosis (FSGS are lacking. In this report, we examined the role of protease-activated receptors (PARs subtype PAR2 and its downstream signals in regulating the pathophysiological process of FSGS. Methods: Nephropathy was induced by intravenous injections of adriamycin (ADR in rats to study FSGS. Western Blot analysis and ELISA were employed to determine the protein expression levels of PAR2 and its downstream signal pathways as well as the levels of PICs. Results: In ADR rats, expression of PAR2, PKCε and PKA was amplified and this was accompanied with increases of pro-inflammatory cytokines (PICs including IL-1β, IL-6 and TNF-α. Inhibition of PAR2 signal by systemic administration of FSLLRY-NH2 (FSL attenuated amplification of PICs. Notably, FSL further influenced key molecular mediators during development of FSGS. i.e., it specifically restored the impaired nephrin and attenuated the exaggerated transforming growth factor beta 1 (TGF-β1, caspase-9 and desmin thereby improving worsened renal functions and glomerular injury. Consistent with this, in cultured podocytes FSL also largely restored downregulation of nephrin and attenuated amplifications of caspase-9 and desmin induced by TGF-β1. Conclusions: Results of this study suggest that PAR2 plays an important role in mediating renal injury induced by glomerulosclerosis. Inhibition of PAR2 signal pathway has a protective effect on FSGS mainly via PIC and TGF-β1 mechanisms. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of FSGS observed in patients.

  3. Modulation of protease activated receptor 1 influences human metapneumovirus disease severity in a mouse model.

    Directory of Open Access Journals (Sweden)

    Laetitia Aerts

    Full Text Available Human metapneumovirus (hMPV infection causes acute respiratory tract infections (RTI which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1 and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.

  4. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F

    2011-04-01

    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  5. [Aspergillus ochraceus myxomycetes produce extracellular proteinases--protein C activators of blood plasma].

    Science.gov (United States)

    Osmolovskiĭ, A A; Kreĭer, V G; Kurakov, A V; Baranova, N A; Egorov, N S

    2012-01-01

    Natural isolates of Aspergillus ochraceus myxomycetes from soil and plant remains from various regions have been screened. The isolated strains were characterized by similar cultural and morphological features and an identical nucleotide sequence in the ITS1-5,8S-ITS2 region of rDNA. The ability of the extracellular proteinases of A. ochraceus myxomycetes to activate protein C of blood plasma has been established. Differences are revealed in the accumulation of proteinases activating protein C and proteinases with thrombin- and plasmin-like activities in the growth dynamics of producers.

  6. Plasma factor VII-activating protease is increased by oral contraceptives and induces factor VII activation in-vivo

    DEFF Research Database (Denmark)

    Sidelmann, Johannes Jakobsen; Skouby, Sven O.; Kluft, Cornelis

    2011-01-01

    Oral contraceptive (OC) use influences the hemostatic system significantly and is a risk factor for development of cardiovascular disease. Factor VII-activating protease (FSAP) has potential effects on hemostasis. The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene expresses a FSAP...... alloenzyme with reduced pro-fibrinolytic activity. Presently, we address whether OC use and OC formulation affect FSAP measures in human blood. Healthy women (n=588) were allocated to six cycles of OCs with estrogen contents of 20μg (n=158), 30μg (n=284), 35μg (n=79) or 50μg (n=67) combined with various...... progestins. FSAP genotypes, FSAP and factor VII (FVII) plasma measures were assessed at baseline and after 6 cycles of OC. The 1601GA genotype was present in 49 (8.3%) of the women and was associated with significantly reduced levels of FSAP (P≤0.001). OC use increased FSAP antigen by 25% and FSAP activity...

  7. Staphylococcus aureus Manipulates Innate Immunity through Own and Host-Expressed Proteases

    Science.gov (United States)

    Pietrocola, Giampiero; Nobile, Giulia; Rindi, Simonetta; Speziale, Pietro

    2017-01-01

    Neutrophils, complement system and skin collectively represent the main elements of the innate immune system, the first line of defense of the host against many common microorganisms. Bacterial pathogens have evolved strategies to counteract all these defense activities. Specifically, Staphylococcus aureus, a major human pathogen, secretes a variety of immune evasion molecules including proteases, which cleave components of the innate immune system or disrupt the integrity of extracellular matrix and intercellular connections of tissues. Additionally, S. aureus secretes proteins that can activate host zymogens which, in turn, target specific defense components. Secreted proteins can also inhibit the anti-bacterial function of neutrophils or complement system proteases, potentiating S. aureus chances of survival. Here, we review the current understanding of these proteases and modulators of host proteases in the functioning of innate immunity and describe the importance of these mechanisms in the pathology of staphylococcal diseases. PMID:28529927

  8. The Inflammatory response induced by aspartic proteases of Candida albicans is independent of proteolytic activity.

    NARCIS (Netherlands)

    Pietrella, D.; Rachini, A.; Pandey, N.; Schild, L.; Netea, M.G.; Bistoni, F.; Hube, B.; Vecchiarelli, A.

    2010-01-01

    The secretion of aspartic proteases (Saps) has long been recognized as a virulence-associated trait of the pathogenic yeast Candida albicans. In this study, we report that different recombinant Saps, including Sap1, Sap2, Sap3, and Sap6, have differing abilities to induce secretion of proinflammator

  9. Central domain of IL-33 is cleaved by mast cell proteases for potent activation of group-2 innate lymphoid cells.

    Science.gov (United States)

    Lefrançais, Emma; Duval, Anais; Mirey, Emilie; Roga, Stéphane; Espinosa, Eric; Cayrol, Corinne; Girard, Jean-Philippe

    2014-10-28

    Interleukin-33 (IL-33) is an alarmin cytokine from the IL-1 family. IL-33 activates many immune cell types expressing the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2, including group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes), the major producers of IL-5 and IL-13 during type-2 innate immune responses and allergic airway inflammation. IL-33 is likely to play a critical role in asthma because the IL33 and ST2/IL1RL1 genes have been reproducibly identified as major susceptibility loci in large-scale genome-wide association studies. A better understanding of the mechanisms regulating IL-33 activity is thus urgently needed. Here, we investigated the role of mast cells, critical effector cells in allergic disorders, known to interact with ILC2s in vivo. We found that serine proteases secreted by activated mast cells (chymase and tryptase) generate mature forms of IL-33 with potent activity on ILC2s. The major forms produced by mast cell proteases, IL-33(95-270), IL-33(107-270), and IL-33(109-270), were 30-fold more potent than full-length human IL-33(1-270) for activation of ILC2s ex vivo. They induced a strong expansion of ILC2s and eosinophils in vivo, associated with elevated concentrations of IL-5 and IL-13. Murine IL-33 is also cleaved by mast cell tryptase, and a tryptase inhibitor reduced IL-33-dependent allergic airway inflammation in vivo. Our study identifies the central cleavage/activation domain of IL-33 (amino acids 66-111) as an important functional domain of the protein and suggests that interference with IL-33 cleavage and activation by mast cell and other inflammatory proteases could be useful to reduce IL-33-mediated responses in allergic asthma and other inflammatory diseases.

  10. The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity.

    Science.gov (United States)

    Matthews, Krystal; Schäfer, Alexandra; Pham, Alissa; Frieman, Matthew

    2014-12-07

    The outcome of a viral infection is regulated by complex interactions of viral and host factors. SARS coronavirus (SARS-CoV) engages and regulates several innate immune response pathways during infection. We have previously shown that the SARS-CoV Papain-like Protease (PLpro) inhibits type I interferon (IFN) by inhibiting IRF3 phosphorylation thereby blocking downstream Interferon induction. This finding prompted us to identify other potential mechanisms of inhibition of PLpro on IFN induction. We have used plasmids expressing PLpro and IRF3 including an IRF3 mutant that is constitutively active, called IRF3(5D). In these experiments we utilize transfections, chromatin immunoprecipitation, Electro-mobility Shift Assays (EMSA) and protein localization to identify where IRF3 and IRF3(5D) are inhibited by PLpro. Here we show that PLpro also inhibits IRF3 activation at a step after phosphorylation and that this inhibition is dependent on the de-ubiquitination (DUB) activity of PLpro. We found that PLpro is able to block the type I IFN induction of a constitutively active IRF3, but does not inhibit IRF3 dimerization, nuclear localization or DNA binding. However, inhibition of PLpro's DUB activity by mutagenesis blocked the IRF3 inhibition activity of PLpro, suggesting a role for IRF3 ubiquitination in induction of a type I IFN innate immune response. These results demonstrate an additional mechanism that PLpro is able to inhibit IRF3 signaling. These data suggest novel innate immune antagonism activities of PLpro that may contribute to SARS-CoV pathogenesis.

  11. HIV protease inhibitors disrupt lipid metabolism by activating endoplasmic reticulum stress and inhibiting autophagy activity in adipocytes.

    Directory of Open Access Journals (Sweden)

    Beth S Zha

    Full Text Available BACKGROUND: HIV protease inhibitors (PI are core components of Highly Active Antiretroviral Therapy (HAART, the most effective treatment for HIV infection currently available. However, HIV PIs have now been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease and metabolic syndrome. Our previous studies have shown that HIV PIs activate endoplasmic reticulum (ER stress and disrupt lipid metabolism in hepatocytes and macrophages. Yet, little is known on how HIV PIs disrupt lipid metabolism in adipocytes, a major cell type involved in the pathogenesis of metabolic syndrome. METHODOLOGY AND PRINCIPAL FINDINGS: Cultured and primary mouse adipocytes and human adipocytes were used to examine the effect of frequently used HIV PIs in the clinic, lopinavir/ritonavir, on adipocyte differentiation and further identify the underlying molecular mechanism of HIV PI-induced dysregulation of lipid metabolism in adipocytes. The results indicated that lopinavir alone or in combination with ritonavir, significantly activated the ER stress response, inhibited cell differentiation, and induced cell apoptosis in adipocytes. In addition, HIV PI-induced ER stress was closely linked to inhibition of autophagy activity. We also identified through the use of primary adipocytes of CHOP(-/- mice that CHOP, the major transcriptional factor of the ER stress signaling pathway, is involved in lopinavir/ritonavir-induced inhibition of cell differentiation in adipocytes. In addition, lopinavir/ritonavir-induced ER stress appears to be associated with inhibition of autophagy activity in adipocytes. CONCLUSION AND SIGNIFICANCE: Activation of ER stress and impairment of autophagy activity are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV patients.

  12. Increased expression of protease-activated receptor 4 and Trefoil factor 2 in human colorectal cancer.

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    Guoyu Yu

    Full Text Available Protease-activated receptor 4 (PAR4, a member of G-protein coupled receptors family, was recently reported to exhibit decreased expression in gastric cancer and esophageal squamous cancer, yet increased expression during the progression of prostate cancer. Trefoil factor 2 (TFF2, a small peptide constitutively expressed in the gastric mucosa, plays a protective role in restitution of gastric mucosa. Altered TFF2 expression was also related to the development of gastrointestinal cancer. TFF2 has been verified to promote cell migration via PAR4, but the roles of PAR4 and TFF2 in the progress of colorectal cancer are still unknown. In this study, the expression level of PAR4 and TFF2 in colorectal cancer tissues was measured using real-time PCR (n = 38, western blotting (n=38 and tissue microarrays (n = 66. The mRNA and protein expression levels of PAR4 and TFF2 were remarkably increased in colorectal cancer compared with matched noncancerous tissues, especially in positive lymph node and poorly differentiated cancers. The colorectal carcinoma cell LoVo showed an increased response to TFF2 as assessed by cell invasion upon PAR4 expression. However, after intervention of PAR4 expression, PAR4 positive colorectal carcinoma cell HT-29 was less responsive to TFF2 in cell invasion. Genomic bisulfite sequencing showed the hypomethylation of PAR4 promoter in colorectal cancer tissues and the hypermethylation in the normal mucosa that suggested the low methylation of promoter was correlated to the increased PAR4 expression. Taken together, the results demonstrated that the up-regulated expression of PAR4 and TFF2 frequently occurs in colorectal cancer tissues, and that overexpression of PAR4 may be resulted from promoter hypomethylation. While TFF2 promotes invasion activity of LoVo cells overexpressing PAR4, and this effect was significantly decreased when PAR4 was knockdowned in HT-29 cells. Our findings will be helpful in further investigations into the

  13. A Novel Aspartic Protease with HIV-1 Reverse Transcriptase Inhibitory Activity from Fresh Fruiting Bodies of the Wild Mushroom Xylaria hypoxylon

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    Qing-Xiu Hu

    2012-01-01

    Full Text Available A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession, and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6–8 and in the temperature range 35–60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

  14. Nano-TiO2 affects Cu speciation, extracellular enzyme activity, and bacterial communities in sediments.

    Science.gov (United States)

    Fan, Wenhong; Liu, Tong; Li, Xiaomin; Peng, Ruishuang; Zhang, Yilin

    2016-11-01

    In aquatic ecosystems, titanium dioxide nanoparticles (nano-TiO2) coexist with heavy metals and influence the existing forms and toxicities of the metal in water. However, limited information is available regarding the ecological risk of this coexistence in sediments. In this study, the effect of nano-TiO2 on Cu speciation in sediments was investigated using sequential extraction. The microcosm approach was also employed to analyze the effects of the coexistence of nano-TiO2 and Cu on extracellular enzyme activity and bacterial communities in sediments. Results showed that nano-TiO2 decreased the organic matter-bound fraction of Cu and increased the corresponding residual fraction Cu. As a result, speciation of exogenous Cu in sediments changed. During the course of the 30-day experiment, the presence of nano-TiO2 did not affect Cu-induced changes in bacterial community structure. However, the coexistence of nano-TiO2 and Cu restrained the activity of bacterial extracellular enzymes, such as alkaline phosphatase and β-glucosidase. The degree of inhibition also varied because of the different properties of extracellular enzymes. This research highlighted the importance of understanding and predicting the effects of the coexistence of nanomaterials and other pollutants in sediments.

  15. The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

    Science.gov (United States)

    Knopf, Julia D; Tholen, Stefan; Koczorowska, Maria M; De Wever, Olivier; Biniossek, Martin L; Schilling, Oliver

    2015-10-01

    Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

  16. Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Science.gov (United States)

    Tillery, Lakeisha C; Epperson, Tenille A; Eguchi, Satoru; Motley, Evangeline D

    2016-03-01

    Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632

  17. Antimicrobial activity of mast cells: Role and relevance of extracellular DNA traps

    Directory of Open Access Journals (Sweden)

    Helene Möllerherm

    2016-07-01

    Full Text Available Mast cells (MCs have been shown to release their nuclear DNA and subsequently form mast cell extracellular traps (MCETs comparable to neutrophil extracellular traps, which are able to entrap and kill various microbes. The formation of extracellular traps is associated with the disruption of the nuclear membrane, leads to mixing of nuclear compounds with granule components and causes the death of the cell, a process called ETosis. The question arises why do MCs release MCETs although they are very well known as multifunctional long-living sentinel cells? In the first place, MCs are known to play a role during allergic reactions and certain parasitic infections. Nonetheless, they are also critical components of the early host innate immune response to bacterial and fungal pathogens: MCs contribute to the initiation of the early immune response by recruiting effector cells including neutrophils and macrophages by locally releasing inflammatory mediators such as TNF-α. Moreover, various studies exhibit that MCs are able to eliminate microbes through intracellular as well as extracellular antimicrobial mechanisms including MCET-formation similar to that of professional phagocytes. The actual literature leads to the suggestion that MCET-formation is not the result of passive release of DNA and granule proteins during cellular disintegration, but rather an active and controlled process in response to specific stimulation which contributes to the innate host defense. This review will discuss the different known aspects of the antimicrobial activities of MCs with special focus on MCETs and their role and relevance during infection and inflammation.

  18. Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

    Science.gov (United States)

    Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

    1998-01-01

    Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; Pmuscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

  19. Seleção de leveduras da Região Amazônica para produção de protease extracelular Screening of yeasts from Amazon Region for extracellular protease production

    Directory of Open Access Journals (Sweden)

    Kilma Cristiane Silva Neves

    2006-01-01

    Full Text Available O objetivo deste trabalho foi identificar 50 leveduras isoladas a partir de diferentes substratos da Região Amazônica e selecionar uma espécie de maior atividade proteolítica. Entre as 26 espécies identificadas predominaram Candida aquatica (12% e Candida tropicalis (10%. A fermentação submersa foi realizada em Extrato de Malte suplementado com gelatina 1%, Candida intermedia foi a que expressou maior atividade proteolítica, halo = 25 mm e 273 U/mL, crescimento máximo a 30 ºC, viabilidade celular 6,2 x 10(6 UFC, em 48 horas, não demonstrou características de patogenicidade e a melhor densidade do inóculo foi 3%, utilizando-se culturas com 24 horas de crescimento em ágar Malte.The objective of this work was to identify 50 yeasts isolates obtained from differents substracts from Amazon Region and to select one species of higher proteolytic activity. Among the 26 identified species it had predominance of Candida aquatica (12% and Candida tropicalis (10%. The submerged fermentation was carried out in Malt Extract supplemented with gelatin 1%, Candida intermedia exhibited higher proteolytic activity, halo = 25 mm and 273U/mL, the maximum growth 30 ºC, the cellular viability 6.2 x 10(6 UFC, in 48 hours, did not demonstrate pathogenicity characteristics and the best inoculum density tested was 3%, using 24 hours Malt agar cultures.

  20. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Jon [University of Southampton, England; Coates, Leighton [ORNL; Hussey, Robert [University of Southampton, England

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  1. Extracellular K+ activates a K(+)- and H(+)-permeable conductance in frog taste receptor cells.

    Science.gov (United States)

    Kolesnikov, S S; Margolskee, R F

    1998-03-01

    1. The effect of extracellular K+ on membrane currents of bull frog (Rana catesbeiana) taste receptor cells (TRCs) was investigated by the patch clamp and fast perfusion techniques. Extracellular K+ (2.5-90 mM) increased a TRC resting conductance and enhanced both inward and outward whole-cell currents. 2. To isolate the inward current activated by external potassium (PA current), TRCs were dialysed with 110 mM NMGCl while extracellular NaCl was replaced with NMGCl. Under these conditions, the PA current displayed an S-shaped current-voltage (I-V) curve in the -100 to 100 mV range. Extracellular Rb+ and NH4+, but not Li+, Na+ or Cs+, evoked similar currents. 3. The PA current reversal potential (Vr) did not follow the equilibrium K+ potential under experimental conditions. Therefore, K+ ions were not the only current carriers. The influence of other ions on the PA current Vr indicated that the channels involved are permeable to K+ and H+ and much less so to Na+, Ca2+ and Mg2+. Relative permeabilities were estimated on the basis of the Goldman-Hodgkin-Katz equation as follows: PH:PK:PNa = 4000:1:0.04. 4. All I-V curves of the PA current were nearly linear at low negative potentials. The slope conductance at these voltages was used to characterize the dependence of the PA current on external K+ and H+. The slope conductance versus K+ concentration was fitted by the Hill equation. The data yielded a half-maximal concentration, K1/2 = 19 +/- 3 mM and a Hill coefficient, nH = 1.53 +/- 0.36 (means +/- S.E.M.). 5. The dependence of the mean PA current and the current variance on the K+ concentration indicated a rise in the open probability of the corresponding channels as extracellular K+ was increased. With 110 mM KCl in the bath, the single channel conductance was estimated at about 6 pS. Taken together, the data suggest that extracellular K+ may serve as a ligand to activate specific small-conductance cation channels (PA channels). The mean number of the PA channels

  2. The influence of repeated administration of poloxamer 407 on serum lipoproteins and protease activity in mouse liver and heart.

    Science.gov (United States)

    Korolenko, Tatyana A; Tuzikov, Fedor V; Johnston, Thomas P; Tuzikova, Natalia A; Kisarova, Yana A; Zhanaeva, Svetlana Ya; Alexeenko, Tatyana V; Zhukova, Natalia A; Brak, Ivan V; Spiridonov, Victor K; Filjushina, Elena E; Cherkanova, Marina S; Monoszon, Anna A

    2012-11-01

    The effects of repeated administration of poloxamer 407 (P-407) on lipoprotein-cholesterol (LP-C) and lipoprotein-triglyceride (LP-TG) fractions and subfractions, as well as the effect on liver and heart proteases, were studied. Repeated administration of P-407 to male CBA mice resulted in a model of atherosclerosis with increased diastolic blood pressure; there was a drastic increase in total serum cholesterol and especially TG. A novel small-angle X-ray scattering method for the determination of the fractional and subfractional composition of LP-C and LP-TG was used. In chronically P-407-treated mice, P-407 significantly increased atherogenic low-density lipoprotein C (LDL-C) fractions, as well as intermediate-density lipoprotein C (IDL-C), and LDL₁₋₃-C subfractions, and very-low-density lipoprotein-C (VLDL-C) fractions, as well as VLDL₁₋₂-C and VLDL₃₋₅-C subfractions), to a lesser extent, the total anti-atherogenic high-density lipoprotein C (HDL-C) fraction, as well as HDL₂-C and HDL₃-C subfractions. Additionally, we demonstrated an increase in the serum chitotriosidase activity, without significant changes in serum matrix metalloprotease (MMP) activity. Morphological changes observed in P-407-treated mice included atherosclerosis in the heart and storage syndrome in the liver macrophages. P-407 significantly increased the activity of cysteine, aspartate proteases, and MMPs in the heart, and only the activity of cathepsin B and MMPs in the liver of mice. Thus, repeated administration of P-407 to mice induced atherosclerosis secondary to sustained dyslipidemia and formation of foamy macrophages in liver, and also modulated the activity of heart and liver proteases.

  3. Botryosphaeriales fungi produce extracellular enzymes with biotechnological potential.

    Science.gov (United States)

    Esteves, Ana Cristina; Saraiva, Márcia; Correia, António; Alves, Artur

    2014-05-01

    Phytopathogenic fungi are known for producing an arsenal of extracellular enzymes whose involvement in the infection mechanism has been suggested. However, these enzymes are largely unknown and their biotechnological potential also remains poorly understood. In this study, the production and thermostability of extracellular enzymes produced by phytopathogenic Botryosphaeriaceae was investigated. Hydrolytic and oxidative activities were detected and quantified at different temperatures. Most strains (70%; 37/53) were able to produce simultaneously cellulases, laccases, xylanases, pectinases, pectin lyases, amylases, lipases, and proteases. Surprisingly for mesophilic filamentous fungi, several enzymes proved to be thermostable: cellulases from Neofusicoccum mediterraneum CAA 001 and from Dothiorella prunicola CBS 124723, lipases from Diplodia pinea (CAA 015 and CBS 109726), and proteases from Melanops tulasnei CBS 116806 were more active at 70 °C than at any of the other temperatures tested. In addition, lipases produced by Diplodia pinea were found to be significantly more active than any other known lipase from Botryosphaeriales. The thermal activity profile and the wide array of activities secreted by these fungi make them optimal producers of biotechnologically relevant enzymes that may be applied in the food and the health industries (proteases), the pulp-and-paper and biofuel industries (cellulases), or even in the detergent industry (lipases, proteases, amylases, and cellulases).

  4. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

    -specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers......, 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...

  5. Up-regulation of interleukin-8 expressions induced by mast cell tryptase via protease activated receptor-2 in endothelial cell line

    Institute of Scientific and Technical Information of China (English)

    LU Chao; ZHAO Feng-di; LI Xiao-bo; YIN Lian-hua

    2005-01-01

    Background Protease activated receptor-2 is cleaved and activated by trypsin or mast cell tryptase and may play an important role in inflammation. However, it is unknown whetehr PAR-2 can mediate tryptase-induced inflammatory reaction. This study was conduct to investigate wheter PAR-2 could be the activated by mast cell tryptase and medicated the tryptase induced interleukin-8 expression in endothelial cells.Methods Protease activated receptor-2 expression was found in endothelial cell lines ECV304 cell by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Interleukin-8 stimulated by purified human mast cell tryptase was determined by RT-PCR and enzyme linked immunosorbent assay (ELISA). Data were analysed by the S-N-K one-way ANOVA test.Results The present study shows that mRNA and protein of protease activated receptor-2 could be expressed in ECV304 cells, and tryptase upregulated the expression levels of both interleukin-8 mRNA and protein. The increased expression of interleukin-8 was inhibited by an antiprotease activated receptor-2 monoclonal antibody, SAM11. An additional band was observed by Western blotting after the incubation of ECV304 cells with tryptase for 2 hours, which suggested that protease activated receptor-2 was activated. Conclusion Protease activated receptor-2 can mediate the mast cell tryptase stimulated expression of interleukin-8 in ECV304 cell.

  6. A preliminary study on estimating extra-cellular nitrate reductase activities in estuarine systems

    Directory of Open Access Journals (Sweden)

    Pant H. K.

    2009-07-01

    Full Text Available Enzymes catalyzing ammonium (NH4+/nitrate (NO3– into nitrous oxide (N2O/molecular nitrogen (N2, play critical roles in water quality management. The objective of this paper was to investigate the role of extra-cellular enzymes in cycling of nitrogen (N in aquatic systems. It appears that N in estuaries, salt marshes, etc., does not stay long enough to be available for uptake, thus, creating N limited conditions. This study showed that indigenous extra-cellular nitrate reductase along with others involved in N transformations in the waters/sediments of estuarine systems can cause complete removal of NH4+ and NO3– from the waters and available NH4+ and NO3– from the sediments. These results indicate that due to high extra-cellular nitrate reductase and other enzymes associated with N transformations in sediments/waters, substantial amounts of NH4+ and NO3– can be quickly lost from the systems as N2O and/or nitric oxide (NO, in turn, creating N limited conditions in estuarine systems. Such high activities of indigenous nitrate reductase and others are useful in removing readily bioavailable N from the systems, thereby avoidance of eutrophic conditions. However, they might contribute in increasing the N2O, a potent greenhouse gas with global warming potential (GWP of 296, in the atmosphere.

  7. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein.

    Science.gov (United States)

    Kamphorst, Jurre J; Nofal, Michel; Commisso, Cosimo; Hackett, Sean R; Lu, Wenyun; Grabocka, Elda; Vander Heiden, Matthew G; Miller, George; Drebin, Jeffrey A; Bar-Sagi, Dafna; Thompson, Craig B; Rabinowitz, Joshua D

    2015-02-01

    Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate, and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiologic albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC.

  8. Coagulation Factor Xa inhibits cancer cell migration via Protease-activated receptor-1 activation

    NARCIS (Netherlands)

    Borensztajn, Keren; Bijlsma, Maarten F.; Reitsma, Pieter H.; Peppelenbosch, Maikel R.; Spek, C. Arnold

    2009-01-01

    Cell migration is critically important in (patho) physiological processes. The metastatic potential of cancer cells partly depends on activation of the coagulation cascade. The aim of the present study was to determine whether coagulation factor X (FXa) can regulate the migration and invasion of can

  9. Characterisation of faecal protease activity in irritable bowel syndrome with diarrhoea: origin and effect of gut transit.

    Science.gov (United States)

    Tooth, David; Garsed, Klara; Singh, Gulzar; Marciani, Luca; Lam, Ching; Fordham, Imogen; Fields, Annie; Banwait, Rawinder; Lingaya, Melanie; Layfield, Robert; Hastings, Maggie; Whorwell, Peter; Spiller, Robin

    2014-05-01

    Faecal serine proteases (FSPs) may play a role in irritable bowel syndrome with diarrhoea (IBS-D), but their origin is unclear. We aimed to structurally characterise them and define the impact of colonic cleansing and transit time. Faecal samples were obtained from 30 healthy volunteers (HV) and 79 patients with IBS-D participating in a trial of ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Samples were also obtained from 24 HV before and after colonic cleansing with the osmotic laxative MoviPrep. FSPs were purified from faecal extracts using benzamidine-Sepharose affinity chromatography. SDS-PAGE profiled components were identified using trypsinolysis and tandem mass spectrometry. Functional protease activity in faecal extracts was measured using a colorimetric assay based on the proteolysis of azo-casein. Protein analysis identified the most abundant FSPs as being of human origin and probably derived from pancreatic juice. Functional assays showed increased faecal protease (FP) and amylase in patients with IBS-D compared with HV. Those with higher amylase had significantly higher FP and greater anxiety. FP activity correlated negatively with whole gut transit in patients with IBS-D (Spearman r=-0.32, p=0.005) and HV (r=-0.55, p=0.014). Colon cleansing caused a significant rise in FP activity in HV from a baseline of median (IQR) 253 (140-426) to 1031 (435-2296), levels similar to those seen in patients with IBS-D. FSP activity correlated positively with days/week with urgency. The most abundant FSPs are of human origin. Rapid transit through the colon and/or decreased (possibly bacterial) proteolytic degradation increases their faecal concentration and could contribute to visceral hypersensitivity in patients with IBS-D. NCT00745004.

  10. The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

    Science.gov (United States)

    Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

    2011-10-01

    Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

  11. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

    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...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  12. Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: differential role of metal ions in stability and activity.

    Science.gov (United States)

    Sinha, Rajeshwari; Khare, S K

    2013-10-01

    A moderately halophilic protease producer, Bacillus sp. strain isolated from sea water is described. The protease is purified to homogeneity by ammonium sulphate precipitation and CM cellulose chromatography. The serine protease has a molecular mass of 29 kDa. Enzymatic characterization of protease revealed K(m) 2.22 mg mL(-1), Vmax 1111.11 U mL(-1), pH optimum 9.0, t1/2 190 min at 60°C and salt optima 1% (w/v) NaCl. The protease is remarkably stable in hydrophilic and hydrophobic solvents at high concentrations. The purified preparation is unstable at room temperature. Ca(2+) ions are required for preventing this loss of activity. Interestingly, the activity and stability are modulated differentially. Whereas, divalent cation Ca(2+) are involved in maintaining stability in solution at room temperature by preventing unfolding, monovalent Na(+) and K(+) ions participate in regulating the activity and assist in refolding of the enzyme. Application of the protease is shown in efficient removal of blood stain.

  13. Activity-Dependent Changes in Extracellular Ca2+ and K+ Reveal Pacemakers in the Spinal Locomotor-Related Network

    OpenAIRE

    Brocard, Frédéric; Shevtsova, Natalia A.; Bouhadfane, Mouloud; Tazerart, Sabrina; Heinemann, Uwe; Rybak, Ilya A.; Vinay, Laurent

    2013-01-01

    Changes in the extracellular ionic concentrations occur as a natural consequence of firing activity in large populations of neurons. The extent to which these changes alter the properties of individual neurons and the operation of neuronal networks remains unknown. Here, we show that the locomotor-like activity in the isolated neonatal rodent spinal cord reduces the extracellular calcium ([Ca2+]o) to 0.9 mM and increases the extracellular potassium ([K+]o) to 6 mM. Such changes in [Ca2+]o and...

  14. Bioproduction, characterization, anticancer and antioxidant activities of extracellular melanin pigment produced by newly isolated microbial cell factories Streptomyces glaucescens NEAE-H

    Science.gov (United States)

    El-Naggar, Noura El-Ahmady; El-Ewasy, Sara M.

    2017-01-01

    In this present study, a newly isolated strain, Streptomyces sp. NEAE-H, capable of producing high amount of black extracellular melanin pigment on peptone-yeast extract iron agar and identified as Streptomyces glaucescens NEAE-H. Plackett–Burman statistical design was conducted for initial screening of 17 independent (assigned) variables for their significances on melanin pigment production by Streptomyces glaucescens NEAE-H. The most significant factors affecting melanin production are incubation period, protease-peptone and ferric ammonium citrate. The levels of these significant variables and their interaction effects were optimized by using face-centered central composite design. The maximum melanin production (31.650 μg/0.1 ml) and tyrosinase activity (6089.10 U/ml) were achieved in the central point runs under the conditions of incubation period (6 days), protease-peptone (5 g/L) and ferric ammonium citrate (0.5 g/L). Melanin pigment was recovered by acid-treatment. Higher absorption of the purified melanin pigment was observed in the UV region at 250 nm. It appeared to have defined small spheres by scanning electron microscopy imaging. The maximum melanin yield was 350 mg dry wt/L of production medium. In vitro anticancer activity of melanin pigment was assayed against skin cancer cell line using MTT assay. The IC50 value was 16.34 ± 1.31 μg/ml for melanin and 8.8 ± 0.5 μg/ml for standard 5-fluorouracil. PMID:28195138

  15. In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

    Directory of Open Access Journals (Sweden)

    Thais Batista de Carvalho

    2014-01-01

    Full Text Available The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105 were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.

  16. Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA.

    Directory of Open Access Journals (Sweden)

    Cristina Antonella Nadalutti

    Full Text Available PURPOSE: To investigate the role of thioredoxin (TRX, a novel regulator of extracellular transglutaminase 2 (TG2, in celiac patients IgA (CD IgA mediated TG2 enzymatic activation. METHODS: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. RESULTS: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. CONCLUSIONS: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.

  17. Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Jeffrey C Lee

    2006-12-01

    Full Text Available BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132 of glioblastomas and 12.5% (1/8 of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

  18. Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1β.

    Science.gov (United States)

    Lee, Paul R; Johnson, Tory P; Gnanapavan, Sharmilee; Giovannoni, Gavin; Wang, Tongguang; Steiner, Joseph P; Medynets, Marie; Vaal, Mark J; Gartner, Valerie; Nath, Avindra

    2017-06-27

    The cause of neurodegeneration in progressive forms of multiple sclerosis is unknown. We investigated the impact of specific neuroinflammatory markers on human neurons to identify potential therapeutic targets for neuroprotection against chronic inflammation. Surface immunocytochemistry directly visualized protease-activated receptor-1 (PAR1) and interleukin-1 (IL-1) receptors on neurons in human postmortem cortex in patients with and without neuroinflammatory lesions. Viability of cultured neurons was determined after exposure to cerebrospinal fluid from patients with progressive multiple sclerosis or purified granzyme B and IL-1β. Inhibitors of PAR1 activation and of PAR1-associated second messenger signaling were used to elucidate a mechanism of neurotoxicity. Immunohistochemistry of human post-mortem brain tissue demonstrated cells expressing higher amounts of PAR1 near and within subcortical lesions in patients with multiple sclerosis compared to control tissue. Human cerebrospinal fluid samples containing granzyme B and IL-1β were toxic to human neuronal cultures. Granzyme B was neurotoxic through activation of PAR1 and subsequently the phospholipase Cβ-IP3 second messenger system. Inhibition of PAR1 or IP3 prevented granzyme B toxicity. IL-1β enhanced granzyme B-mediated neurotoxicity by increasing PAR1 expression. Neurons within the inflamed central nervous system are imperiled because they express more PAR1 and are exposed to a neurotoxic combination of both granzyme B and IL-1β. The effects of these inflammatory mediators may be a contributing factor in the progressive brain atrophy associated with neuroinflammatory diseases. Knowledge of how exposure to IL-1β and granzyme B act synergistically to cause neuronal death yields potential novel neuroprotective treatments for neuroinflammatory diseases.

  19. Mitochondrial cereblon functions as a Lon-type protease.

    Science.gov (United States)

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-07-15

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria.

  20. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    Directory of Open Access Journals (Sweden)

    Aline B. M Vaz

    2011-09-01

    Full Text Available The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia, Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4ºC and 20ºC, indicating that they could be metabolically active in the sampled substrates.

  1. The selection of alkaline protease-producing yeasts from marine environments and evaluation of their bioactive peptide production

    Institute of Scientific and Technical Information of China (English)

    LI Jing; CHI Zhenming; WANG Xianghong; PENG Ying; CHI Zhe

    2009-01-01

    A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains (HN3.11, N11b, YF04C and HN4.9) capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, Issatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N11b was 10.0. The optimal temperature for protease activity was 45°C for strains HN3.11, N11b, and YF04C, and 50°C for strain HN4.9. After digestion of shrimp (Penaeus vannamei) protein and spirulina (Arthospira platensis) protein with the four crude alkaline proteases, the filtrate from spirulina (Arthrospira platensis) powder digested by the crude alkaline protease of strain HN3.11 was found to have the highest antioxidant activity (61.4%) and the highest angiotensin I converting enzyme (ACE)-inhibitory activities (68.4%). The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.

  2. Phytophthora infestans has a plethora of phospholipase D enzymes including a subclass that has extracellular activity.

    Directory of Open Access Journals (Sweden)

    Harold J G Meijer

    Full Text Available In eukaryotes phospholipase D (PLD is involved in many cellular processes. Currently little is known about PLDs in oomycetes. Here we report that the oomycete plant pathogen Phytophthora infestans has a large repertoire of PLDs divided over six subfamilies: PXPH-PLD, PXTM-PLD, TM-PLD, PLD-likes, and type A and B sPLD-likes. Since the latter have signal peptides we developed a method using metabolically labelled phospholipids to monitor if P. infestans secretes PLD. In extracellular medium of ten P. infestans strains PLD activity was detected as demonstrated by the production of phosphatidic acid and the PLD specific marker phosphatidylalcohol.

  3. Comparison of the active sites of atropinesterase and some serine proteases by spin-labeling.

    Science.gov (United States)

    van der Drift, A C; Moes, G W; van der Drift, E; Rousseeuw, B A

    1985-09-24

    going from label I to label II, the relative contribution of the latter state increases at the expense of that of the former. This is ascribed to an equilibrium between a relatively free state of the aromatic cores and a firmly bound position in the specificity pocket of these proteases. The apparently more rigid embedding of the spin-labels in the enzyme structure of AtrE suggests that the size of the nonpolar binding pocket in the active center region of this esterase allows a deeper penetration of the aromatic portions of the labels than is possible for the specificity pocket of Chymo or Sub.

  4. Functions and behaviors of activated sludge extracellular polymeric substances (EPS): a promising environmental interest

    Institute of Scientific and Technical Information of China (English)

    TIAN Yu; ZHENG Lei; SUN De-zhi

    2006-01-01

    Extracellular polymeric substances (EPS) are the predominant constituents of activated sludge and represent up to 80% of the mass of activated sludge. They play a crucial role in the flocculation, settling and dewatering of activated sludge. Furthermore,EPS also show great efficiency in binding heavy metals. So EPS are key factors influencing reduction in sludge volume and mass, as well as activity and utilization of sludge. EPS are of considerable environmental interest and hundreds of articles on EPS have been published abroad, while information on EPS in China is limited. In this paper, results of over 60 publications related to constituents and characteristics of EPS and their influences on flocculation, settling and dewatering of sludge are compiled and analyzed.Metal-binding ability of EPS is also discussed, together with a brief consideration of possible research interests in the future.

  5. 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.

  6. Extracellular Mipp1 Activity Confers Migratory Advantage to Epithelial Cells during Collective Migration

    Directory of Open Access Journals (Sweden)

    Yim Ling Cheng

    2015-12-01

    Full Text Available Multiple inositol polyphosphate phosphatase (Mipp, a highly conserved but poorly understood histidine phosphatase, dephosphorylates higher-order IPs (IP4–IP6 to IP3. To gain insight into the biological roles of these enzymes, we have characterized Drosophila mipp1. mipp1 is dynamically expressed in the embryonic trachea, specifically in the leading cells of migrating branches at late stages, where Mipp1 localizes to the plasma membrane and filopodia. FGF signaling activates mipp1 expression in these cells, where extensive filopodia form to drive migration and elongation by cell intercalation. We show that Mipp1 facilitates formation and/or stabilization of filopodia in leading cells through its extracellular activity. mipp1 loss decreases filopodia number, whereas mipp1 overexpression increases filopodia number in a phosphatase-activity-dependent manner. Importantly, expression of Mipp1 gives cells a migratory advantage for the lead position in elongating tracheal branches. Altogether, these findings suggest that extracellular pools of inositol polyphosphates affect cell behavior during development.

  7. Potential antimicrobial and antiproliferative activities of autochthonous starter cultures and protease EPg222 in dry-fermented sausages.

    Science.gov (United States)

    Fernández, Margarita; Ruiz-Moyano, Santiago; Benito, María José; Martín, Alberto; Hernández, Alejandro; Córdoba, María de Guía

    2016-05-18

    This work studied the presence of nitrogen compounds with bioactive properties in Iberian pork sausages that were manufactured using different autochthonous starter cultures (Pediococcus acidilactici MS200 and Staphylococcus vitulus RS34) and protease EPg222. Nitrogen compounds were extracted and evaluated for their antimicrobial effect against spoilage and pathogenic bacteria, such as Bacillus cereus, Escherichia coli, Salmonella choleraesuis, Staphylococcus aureus and Listeria monocytogenes, and antiproliferative activity on the HT-29 colon adenocarcinoma cell line. Dry-fermented sausages elaborated with starter cultures P200S34 and protease EPg222 generate extracts that cause inhibition of the growth of pathogens reaching 25% inhibition of Bacillus cereus, making this a promising tool for biocontrol in the meat industry. On the other hand, the inoculation of well-adapted starter cultures with high proteolytic activity also increased the antiproliferative activity of these extracts, around 45% inhibition at 72 h, mainly due to an increase in free amino acids, such as Lys and Pro, but also small peptides.

  8. The cysteine protease α-clostripain is not essential for the pathogenesis of Clostridium perfringens-mediated myonecrosis.

    Directory of Open Access Journals (Sweden)

    Anjana Chakravorty

    Full Text Available Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.

  9. Comparison in effect of different metal ions, pH and reducing agent on the protease activity in human hyper mature and mature cataract

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study was undertaken to isolate and characterize the protease activity of human eye lens sample of mature and hyper mature cataract. Samples were collected just after surgery of the cataract lens and were stored at -20 ℃. The total protein extract was isolated from 5 samples in each case (mature and hyper mature cataract) and clear supernatant obtained after centrifugation was used as an enzyme source. The optimum pH for the proteases of mature cataract was 7.5 while the proteases of hyper mature cataract were recorded for maximum activity at pH 5.5 and 7.5. The optimum temperature for both enzyme sources was 50 ℃. Effect of different metal ions such as potassium, lead, silver, zinc and borate was studied. In each case protease activity was increased. Reducing agent e.g. β mercaptoethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates.

  10. Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

    DEFF Research Database (Denmark)

    Petersen, R H; Knudsen, T; Johansen, Torben

    1991-01-01

    The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat...... and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in erythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation...

  11. Enhanced activity of meprin-α, a pro-migratory and pro-angiogenic protease, in colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Daniel Lottaz

    Full Text Available Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, protein and proteolytic activity in primary tumors at progressive stages and in liver metastases of patients with colorectal cancer, as well as inhibitory activity towards meprin-α in sera of cancer patient as compared to healthy controls. We found that the hepatocyte growth factor (HGF-induced migratory response of meprin-transfected epithelial cells was increased compared to wild-type cells in the presence of plasminogen, and that the angiogenic response in organ-cultured rat aortic explants was enhanced in the presence of exogenous human meprin-α. In patients, meprin-α mRNA was expressed in colonic adenomas, primary tumors UICC (International Union Against Cancer stage I, II, III and IV, as well as in liver metastases. In contrast, the corresponding protein accumulated only in primary tumors and liver metastases, but not in adenomas. However, liver metastases lacked meprin-α activity despite increased expression of the corresponding protein, which correlated with inefficient zymogen activation. Sera from cancer patients exhibited reduced meprin-α inhibition compared to healthy controls. In conclusion, meprin-α activity is regulated differently in primary tumors and metastases, leading to high proteolytic activity in primary tumors and low activity in liver metastases. By virtue of its pro-migratory and pro-angiogenic activity, meprin-α may promote tumor progression in colorectal cancer.

  12. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation.

    Science.gov (United States)

    Rossaint, Jan; Herter, Jan M; Van Aken, Hugo; Napirei, Markus; Döring, Yvonne; Weber, Christian; Soehnlein, Oliver; Zarbock, Alexander

    2014-04-17

    There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in- and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute lung injury (ALI), which is associated with a high mortality rate in critically ill patients. NETs consist of decondensed chromatin decorated with granular and cytosolic proteins and they can trap extracellular pathogens. The prerequisite for NET formation is the activation of neutrophils and the release of their DNA. In a neutrophil- and platelet-dependent mouse model of ventilator-induced lung injury (VILI), NETs were found in the lung microvasculature, and circulating NET components increased in the plasma. In this model, blocking integrin-mediated outside-in or either GPCR-signaling or heteromerization of platelet chemokines decreased NET formation and lung injury. Targeting NET components by DNAse1 application or neutrophil elastase-deficient mice protected mice from ALI, whereas DNase1(-/-)/Trap1(m/m) mice had an aggravated ALI, suggesting that NETs directly influence the severity of ALI. These data suggest that NETs form in the lungs during VILI, contribute to the disease process, and thus may be a promising new direction for the treatment of ALI.

  13. Glutathione-dependent extracellular ferric reductase activities in dimorphic zoopathogenic fungi

    Science.gov (United States)

    Zarnowski, Robert; Woods, Jon P.

    2009-01-01

    In this study, extracellular glutathione-dependent ferric reductase (GSH-FeR) activities in different dimorphic zoopathogenic fungal species were characterized. Supernatants from Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis and Sporothrix schenckii strains grown in their yeast form were able to reduce iron enzymically with glutathione as a cofactor. Some variations in the level of reduction were noted amongst the strains. This activity was stable in acidic, neutral and slightly alkaline environments and was inhibited when trivalent aluminium and gallium ions were present. Using zymography, single bands of GSH-FeRs with apparent molecular masses varying from 430 to 460 kDa were identified in all strains. The same molecular mass range was determined by size exclusion chromatography. These data demonstrate that dimorphic zoopathogenic fungi produce and secrete a family of similar GSH-FeRs that may be involved in the acquisition and utilization of iron. Siderophore production by these and other fungi has sometimes been considered to provide a full explanation of iron acquisition in these organisms. Our work reveals an additional common mechanism that may be biologically and pathogenically important. Furthermore, while some characteristics of these enzymes such as extracellular location, cofactor utilization and large size are not individually unique, when considered together and shared across a range of fungi, they represent an important novel physiological feature. PMID:16000713

  14. Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

    Science.gov (United States)

    Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

    2017-01-01

    Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

  15. Diabetic nephropathy is associated with increased urine excretion of proteases plasmin, prostasin and urokinase and activation of amiloride-sensitive current in collecting duct cells

    DEFF Research Database (Denmark)

    Andersen, Henrik; Friis, Ulla G; Hansen, Pernille B L;

    2015-01-01

    by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of γENaC by a monoclonal antibody directed against the 'inhibitory' peptide tract. RESULTS: Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion...... with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact γENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin...

  16. Enzymatic dehairing of goat skins using alkaline protease from Bacillus sp. SB12.

    Science.gov (United States)

    Briki, Selmen; Hamdi, Olfa; Landoulsi, Ahmed

    2016-05-01

    The present paper reports the production, purification and biochemical characterization of an extracellular alkaline protease from Bacillus sp. SB12. The enzyme has been used as an alternative to conventional chemicals treatment for dehairing of goat skins. The protease was optimally active at 37 °C and pH 9. Starch at 2% (w/v) was used as the best carbon source and the addition of yeast extract and peptone at 1% each supported the maximum level of protease production in the presence of 5 mM Ca(2+). Protease purification was performed with ammonium sulphate precipitation at 70% saturated fraction followed by dialysis and gel filtration chromatography using Sephadex G-100. The purified enzyme was homogeneous on non-denaturing PAGE and appeared as a single band with an apparent molecular weight of 41 kDa. This enzyme was moderately thermostable and has a wide pH stability range extending from pH 7 to 11. It showed high tolerance toward surfactants agents and organic solvents while it was completely inhibited by PMSF indicating the serine protease type. Purified protease was used to remove hair from goat skin proving its potential application in leather processing industry. The results revealed that the protease has enhanced the quality and physico-chemical properties of the skins while reducing the pollution.

  17. The expression of protease-acti