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Sample records for membrane protease ompt

  1. Structural studies of two outer membrane proteins: OmpT from Escherichia coli and NspA from Neisseria meningitidis

    NARCIS (Netherlands)

    Rutten, L.

    2003-01-01

    This Thesis describes the three-dimensional structures of two outer membrane proteins (OMPs), OmpT and NspA, from two pathogenic Gram-negative bacteria. These structures reveal information about the functioning of these proteins and can potentially be used for the design of antimicrobial drugs or

  2. Molecular dynamics simulations of outer-membrane protease T from E. coli based on a hybrid coarse-grained/atomistic potential

    International Nuclear Information System (INIS)

    Neri, Marilisa; Anselmi, Claudio; Carnevale, Vincenzo; Vargiu, Attilio V; Carloni, Paolo

    2006-01-01

    Outer-membrane proteases T (OmpT) are membrane enzymes used for defense by Gram-negative bacteria. Here we use hybrid molecular mechanics/coarse-grained simulations to investigate the role of large-scale motions of OmpT from Escherichia coli for its function. In this approach, the enzyme active site is treated at the all-atom level, whilst the rest of the protein is described at the coarse-grained level. Our calculations agree well with previously reported all-atom molecular dynamics simulations, suggesting that this approach is well suitable to investigate membrane proteins. In addition, our findings suggest that OmpT large-scale conformational fluctuations might play a role for its biological function, as found for another protease class, the aspartyl proteases

  3. Antimicrobial properties of arginine- and lysine-rich histones and involvement of bacterial outer membrane protease T in their differential mode of actions.

    Science.gov (United States)

    Tagai, Chihiro; Morita, Shuu; Shiraishi, Takayuki; Miyaji, Kazuyuki; Iwamuro, Shawichi

    2011-10-01

    There is growing evidence of the antimicrobial properties of histones and histone-derived peptides; however, most of them are specific to lysine (Lys)-rich histones (H1, H2A, and H2B). In the present study, we focused on arginine (Arg)-rich histones (H3 and H4) and investigated their antimicrobial properties in comparison with those of histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against the bacterial outer membrane protease T (OmpT) gene-expressing Escherichia coli strain JCM5491 with calculated 50% growth inhibitory concentrations of 3.8, 10, and 12.7 μM, respectively. A lysate prepared from the JCM5491 cells was capable of strongly, moderately, and slightly fragmenting histones H2B, H3, and H4, respectively. While the lysate prepared from the cells of the ompT-deleted E. coli strain BL21(DE3) did not digest these histones, the ompT-transformed BL21(DE3), termed BL21/OmpT(+), cell lysate digested the histones more strongly than the JCM5491 cell lysate. Laser confocal and scanning electron microscopic analyses demonstrated that while histone H2B penetrated the cell membrane of JCM5491 or BL21/OmpT(+) cells, histones H3 and H4 remained on the cell surface and subsequently disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. The BL21(DE3) cells treated with each histone showed no bleb formation, but cell integrity was affected and the cell surface was corrugated. Consequently, it is suggested that OmpT is involved in the antimicrobial properties of Arg- and Lys-rich histones and that the modes of antimicrobial action of these histones are different. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Structure Prediction of Outer Membrane Protease Protein of Salmonella typhimurium Using Computational Techniques

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    Rozina Tabassum

    2016-03-01

    Full Text Available Salmonella typhimurium, a facultative gram-negative intracellular pathogen belonging to family Enterobacteriaceae, is the most frequent cause of human gastroenteritis worldwide. PgtE gene product, outer membrane protease emerges important in the intracellular phases of salmonellosis. The pgtE gene product of S. typhimurium was predicted to be capable of proteolyzing T7 RNA polymerase and localize in the outer membrane of these gram negative bacteria. PgtE product of S. enterica and OmpT of E. coli, having high sequence similarity have been revealed to degrade macrophages, causing salmonellosis and other diseases. The three-dimensional structure of the protein was not available through Protein Data Bank (PDB creating lack of structural information about E protein. In our study, by performing Comparative model building, the three dimensional structure of outer membrane protease protein was generated using the backbone of the crystal structure of Pla of Yersinia pestis, retrieved from PDB, with MODELLER (9v8. Quality of the model was assessed by validation tool PROCHECK, web servers like ERRAT and ProSA are used to certify the reliability of the predicted model. This information might offer clues for better understanding of E protein and consequently for developmet of better therapeutic treatment against pathogenic role of this protein in salmonellosis and other diseases.

  5. The crystal structure of GXGD membrane protease FlaK

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Jian; Xue, Yi; Lee, Sangwon; Ha, Ya (Yale-MED)

    2011-09-20

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  6. The Crystal Structure of GXGD Membrane Protease FlaK

    Energy Technology Data Exchange (ETDEWEB)

    J Hu; Y Xue; S Lee; Y Ha

    2011-12-31

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  7. Characterization of a membrane-associated serine protease in Escherichia coli

    International Nuclear Information System (INIS)

    Palmer, S.M.; St John, A.C.

    1987-01-01

    Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [ 3 H]diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin

  8. 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.

  9. Promoting Tag Removal of a MBP-Fused Integral Membrane Protein by TEV Protease.

    Science.gov (United States)

    Chen, Yanke; Li, Qichang; Yang, Jun; Xie, Hao

    2017-03-01

    Tag removal is a prerequisite issue for structural and functional analysis of affinity-purified membrane proteins. The present study took a MBP-fused membrane protein, MrpF, as a model to investigate the tag removal by TEV protease. Influences of the linking sequence between TEV cleavage site and MrpF on protein expression and predicted secondary structure were investigated. The steric accessibility of TEV protease to cleavage site of MBP-fused MrpF was explored. It was found that reducing the size of hydrophilic group of detergents and/or extending the linking sequence between cleavage site and target protein can significantly improve the accessibility of the cleavage site and promote tag removal by TEV protease.

  10. Membrane-anchored Serine Protease Matriptase Is a Trigger of Pulmonary Fibrogenesis

    NARCIS (Netherlands)

    Bardou, Olivier; Menou, Awen; François, Charlène; Duitman, Jan Willem; von der Thüsen, Jan H.; Borie, Raphaël; Sales, Katiuchia Uzzun; Mutze, Kathrin; Castier, Yves; Sage, Edouard; Liu, Ligong; Bugge, Thomas H.; Fairlie, David P.; Königshoff, Mélanie; Crestani, Bruno; Borensztajn, Keren S.

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a devastating disease that remains refractory to current therapies. To characterize the expression and activity of the membrane-anchored serine protease matriptase in IPF in humans and unravel its potential role in human and experimental pulmonary fibrogenesis.

  11. Membrane-anchored serine protease matriptase is a trigger of pulmonary fibrogenesis

    NARCIS (Netherlands)

    Bardou, O. (Olivier); Menou, A. (Awen); François, C. (Charlène); J.W. Duitman (Jan Willem); J. von der Thusen (Jan); Borie, R. (Raphaël); Sales, K.U. (Katiuchia Uzzun); Mutze, K. (Kathrin); Y. Castier (Yves); Sage, E. (Edouard); Liu, L. (Ligong); Bugge, T.H. (Thomas H.); Fairlie, D.P. (David P.); Königshoff, M. (Mélanie); B. Crestani (Bruno); K. Borensztajn (Keren)

    2016-01-01

    textabstractRationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease that remains refractory to current therapies. Objectives: To characterize the expression and activity of the membrane-anchored serine protease matriptase in IPF in humans and unravel its potential role in human and

  12. A Deg-protease family protein in marine Synechococcus is involved in outer membrane protein organization

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    Rhona Kayra Stuart

    2014-06-01

    Full Text Available Deg-family proteases are a periplasm-associated group of proteins that are known to be involved in envelope stress responses and are found in most microorganisms. Orthologous genes SYNW2176 (in strain WH8102 and sync_2523 (strain CC9311 are predicted members of the Deg-protease family and are among the few genes induced by copper stress in both open ocean and coastal marine Synechococcus strains. In contrast to the lack of a phenotype in a similar knockout in Synechocystis PCC6803, a SYNW2176 knockout mutant in strain WH8102 was much more resistant to copper than the wild-type. The mutant also exhibited a significantly altered outer membrane protein composition which may contribute to copper resistance, longer lag phase after transfer, low-level consistent alkaline phosphatase activity, and an inability to induce high alkaline phosphatase activity in response to phosphate stress. This phenotype suggests a protein-quality-control role for SYNW2176, the absence of which leads to a constitutively activated stress response. Deg-protease family proteins in this ecologically important cyanobacterial group thus help to determine outer membrane responses to both nutrients and toxins.

  13. Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

    Science.gov (United States)

    Rompikuntal, Pramod K; Vdovikova, Svitlana; Duperthuy, Marylise; Johnson, Tanya L; Åhlund, Monika; Lundmark, Richard; Oscarsson, Jan; Sandkvist, Maria; Uhlin, Bernt Eric; Wai, Sun Nyunt

    2015-01-01

    Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

  14. Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla

    Science.gov (United States)

    2011-01-01

    Background Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. Results Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed. Conclusions We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens. PMID:21310089

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

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

    2017-08-01

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

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

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    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

  17. Membrane-associated transcription factor peptidase, site-2 protease, antagonizes ABA signaling in Arabidopsis.

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    Zhou, Shun-Fan; Sun, Le; Valdés, Ana Elisa; Engström, Peter; Song, Ze-Ting; Lu, Sun-Jie; Liu, Jian-Xiang

    2015-10-01

    Abscisic acid plays important roles in maintaining seed dormancy while gibberellins (GA) and other phytohormones antagonize ABA to promote germination. However, how ABA signaling is desensitized during the transition from dormancy to germination is still poorly understood. We functionally characterized the role of membrane-associated transcription factor peptidase, site-2 protease (S2P), in ABA signaling during seed germination in Arabidopsis. Genetic analysis showed that loss-of-function of S2P conferred high ABA sensitivity during seed germination, and expression of the activated form of membrane-associated transcription factor bZIP17, in which the transmembrane domain and endoplasmic reticulum (ER) lumen-facing C-terminus were deleted, in the S2P mutant rescued its ABA-sensitive phenotype. MYC and green fluorescent protein (GFP)-tagged bZIP17 were processed and translocated from the ER to the nucleus in response to ABA treatment. Furthermore, genes encoding negative regulators of ABA signaling, such as the transcription factor ATHB7 and its target genes HAB1, HAB2, HAI1 and AHG3, were up-regulated in seeds of the wild-type upon ABA treatment; this up-regulation was impaired in seeds of S2P mutants. Our results suggest that S2P desensitizes ABA signaling during seed germination through regulating the activation of the membrane-associated transcription factor bZIP17 and therefore controlling the expression level of genes encoding negative regulators of ABA signaling. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  18. Lack of the host membrane protease FtsH hinders release of the Lactococcus lactis bacteriophage TP712.

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    Roces, Clara; Wegmann, Udo; Campelo, Ana B; García, Pilar; Rodríguez, Ana; Martínez, Beatriz

    2013-12-01

    The temperate bacteriophage TP712 was unable to plaque on Lactococcus lactis ΔftsH lacking the membrane protease FtsH and complementation in trans restored the WT phenotype. Absence of ftsH did not hinder phage adsorption, phage DNA delivery or activation of the lytic cycle. Thin sections revealed that TP712 virions appeared to be correctly assembled inside the ΔftsH host, but were not released. These virions were infective, demonstrating that a functional host FtsH is required by TP712 to proceed effectively with lysis of the host.

  19. PathogenicLeptospiraSecreted Proteases Target the Membrane Attack Complex: A Potential Role for Thermolysin in Complement Inhibition.

    Science.gov (United States)

    Amamura, Thais A; Fraga, Tatiana R; Vasconcellos, Sílvio A; Barbosa, Angela S; Isaac, Lourdes

    2017-01-01

    Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira . This disease is common in tropical and subtropical areas, constituting a serious public health problem. Pathogenic Leptospira have the ability to escape the human Complement System, being able to survive when in contact with normal human serum. In a previous study, our group demonstrated that supernatants of pathogenic Leptospira (SPL) inhibit the three activation pathways of the Complement System. This inhibition can be directly correlated with the activity of secreted proteases, which cleave the Complement molecules C3, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Terminal Pathway of Complement, called the membrane attack complex (MAC). We observed that proteases present in SPL from different Leptospira strains were able to cleave the purified proteins C5, C6, C7, C8, and C9, while culture supernatant from non-pathogenic Leptospira strains (SNPL) had no significant proteolytic activity on these substrates. The cleavages occurred in a time-dependent and specificity manner. No cleavage was observed when we used whole serum as a source of C5-C9 proteins, probably because of the abundant presence of plasma protease inhibitors such as α 2 -macroglobulin. Complement protein cleavage by SPL was inhibited by 1,10-phenanthroline, indicating the involvement of metalloproteases. Furthermore, 1,10-phenanthroline- treated normal human serum diminished pathogenic leptospira survival. We also analyzed the proteolytic activity of thermolysin (LIC13322) a metalloprotease expressed exclusively by pathogenic Leptospira strains. Recombinant thermolysin was capable of cleaving the component C6, either purified or as part of the SC5b-9 complex. Furthermore, we found that the MAC proteins C6-C9 interact with thermolysin, indicating that this metalloprotease may have an additional inhibitory

  20. Pathogenic Leptospira Secreted Proteases Target the Membrane Attack Complex: A Potential Role for Thermolysin in Complement Inhibition

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    Thais A. Amamura

    2017-05-01

    Full Text Available Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. This disease is common in tropical and subtropical areas, constituting a serious public health problem. Pathogenic Leptospira have the ability to escape the human Complement System, being able to survive when in contact with normal human serum. In a previous study, our group demonstrated that supernatants of pathogenic Leptospira (SPL inhibit the three activation pathways of the Complement System. This inhibition can be directly correlated with the activity of secreted proteases, which cleave the Complement molecules C3, Factor B (Alternative Pathway, C4 and C2 (Classical and Lectin Pathways. In this work, we analyze the activity of the leptospiral proteases on the components of Terminal Pathway of Complement, called the membrane attack complex (MAC. We observed that proteases present in SPL from different Leptospira strains were able to cleave the purified proteins C5, C6, C7, C8, and C9, while culture supernatant from non-pathogenic Leptospira strains (SNPL had no significant proteolytic activity on these substrates. The cleavages occurred in a time-dependent and specificity manner. No cleavage was observed when we used whole serum as a source of C5–C9 proteins, probably because of the abundant presence of plasma protease inhibitors such as α2-macroglobulin. Complement protein cleavage by SPL was inhibited by 1,10-phenanthroline, indicating the involvement of metalloproteases. Furthermore, 1,10-phenanthroline- treated normal human serum diminished pathogenic leptospira survival. We also analyzed the proteolytic activity of thermolysin (LIC13322 a metalloprotease expressed exclusively by pathogenic Leptospira strains. Recombinant thermolysin was capable of cleaving the component C6, either purified or as part of the SC5b-9 complex. Furthermore, we found that the MAC proteins C6–C9 interact with thermolysin, indicating that this metalloprotease may have an

  1. Pathogenic Leptospira Secreted Proteases Target the Membrane Attack Complex: A Potential Role for Thermolysin in Complement Inhibition

    Science.gov (United States)

    Amamura, Thais A.; Fraga, Tatiana R.; Vasconcellos, Sílvio A.; Barbosa, Angela S.; Isaac, Lourdes

    2017-01-01

    Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira. This disease is common in tropical and subtropical areas, constituting a serious public health problem. Pathogenic Leptospira have the ability to escape the human Complement System, being able to survive when in contact with normal human serum. In a previous study, our group demonstrated that supernatants of pathogenic Leptospira (SPL) inhibit the three activation pathways of the Complement System. This inhibition can be directly correlated with the activity of secreted proteases, which cleave the Complement molecules C3, Factor B (Alternative Pathway), C4 and C2 (Classical and Lectin Pathways). In this work, we analyze the activity of the leptospiral proteases on the components of Terminal Pathway of Complement, called the membrane attack complex (MAC). We observed that proteases present in SPL from different Leptospira strains were able to cleave the purified proteins C5, C6, C7, C8, and C9, while culture supernatant from non-pathogenic Leptospira strains (SNPL) had no significant proteolytic activity on these substrates. The cleavages occurred in a time-dependent and specificity manner. No cleavage was observed when we used whole serum as a source of C5–C9 proteins, probably because of the abundant presence of plasma protease inhibitors such as α2-macroglobulin. Complement protein cleavage by SPL was inhibited by 1,10-phenanthroline, indicating the involvement of metalloproteases. Furthermore, 1,10-phenanthroline- treated normal human serum diminished pathogenic leptospira survival. We also analyzed the proteolytic activity of thermolysin (LIC13322) a metalloprotease expressed exclusively by pathogenic Leptospira strains. Recombinant thermolysin was capable of cleaving the component C6, either purified or as part of the SC5b-9 complex. Furthermore, we found that the MAC proteins C6–C9 interact with thermolysin, indicating that this metalloprotease may have an additional inhibitory

  2. Subcellular localization of a PhoE-LacZ fusion protein in E. coli by protease accessibility experiments reveals an inner-membrane-spanning form of the protein

    NARCIS (Netherlands)

    Tommassen, J.P.M.; Kroon, T. de

    1987-01-01

    Protease accessibility experiments were employed to localize a PhoE-LacZ hybrid protein, encompassing a large N-terminal fragment of the outer membrane PhoE protein of E. coli, fused to β-galactosidase, at the subcellular level. In previous studies, this protein was shown to co-fractionate with the

  3. Prevalence of Genes Encoding Outer Membrane Virulence Factors Among Fecal Escherichia coli Isolates

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    Ahmad Rashki

    2017-03-01

    Full Text Available Objective: Escherichia coli is commensal bacterium of human intestine. The gut is a common pool of E. coli isolates causing urinary tract infections (UTIs. Some of fecal E. coli (FeEC by the possession of certain virulence factors is able to cause diseases in human and other mammalian models. To evaluate the health threats coordinated with a given fecal source of E. coli strains, we determined the frequency of genes expressing virulence determinants in fecal E. coli isolates collected from human feces in Zabol, southeast of Iran. Methods: Escherichia coli isolates (n = 94 were separated from the feces of patients attending teaching hospitals, and screened for various virulence genes: fimH, his, hlyA, ompT, irp2, iucD, iroN, and cnf1 by using the multiplex polymerase chain reaction (PCR method. Results: The prevalence of virulence genes was as follows: adhesins (fimH, 98% and iha, 26%, alpha-hemolysins (hlyA, 10%, outer membrane protease (ompT, 67%, aerobactin (iucD, 67%, iron-repressible protein (irp2, 91% and salmochelin (iroN, 33% and cytotoxic necrotizing factor 1 (cnf1. According to the diversity of different virulence genes, the examined isolates exhibited 29 different patterns. Conclusion: Our results demonstrated that most of the assessed isolates harbored several virulence factors. Our findings propose possibility of human feces serving as a source for pathogenic organisms, supporting the notion that fecal materials of humans play a role in the epidemiological chain of extra-intestinal pathogenic E. coli. This is the first report of the frequency of virulence factors among E. coli isolates collected from human feces in Iran.

  4. 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.

  5. Structural Characterization and Determinants of Specificity of Single- Chain Antibody Inhibitors of Membrane-Type Serine Protease 1

    Science.gov (United States)

    2007-03-01

    protease involved in male chromatin remodeling blocks the development of sea urchin embryos at the initial cell cycle. J. Cell Biochem. 98, 335–342. 18...Macrophage Morphology Changes and Inhibition of Nitric Oxide Production by Macrophages. The cleavage of MSP-1 by MT-SP1 was then tested in primary cells in...inhibitor (Fig. 3) were studied. The morphology change in response to MSP-1 was independent of HAI-1 or anti-MT-SP1 antibody presence. Both inhibitors

  6. Sodium laurate, a novel protease- and mass spectrometry-compatible detergent for mass spectrometry-based membrane proteomics.

    Directory of Open Access Journals (Sweden)

    Yong Lin

    Full Text Available The hydrophobic nature of most membrane proteins severely complicates their extraction, proteolysis and identification. Although detergents can be used to enhance the solubility of the membrane proteins, it is often difficult for a detergent not only to have a strong ability to extract membrane proteins, but also to be compatible with the subsequent proteolysis and mass spectrometric analysis. In this study, we made evaluation on a novel application of sodium laurate (SL to the shotgun analysis of membrane proteomes. SL was found not only to lyse the membranes and solubilize membrane proteins as efficiently as SDS, but also to be well compatible with trypsin and chymotrypsin. Furthermore, SL could be efficiently removed by phase transfer method from samples after acidification, thus ensuring not to interfere with the subsequent CapLC-MS/MS analysis of the proteolytic peptides of proteins. When SL was applied to assist the digestion and identification of a standard protein mixture containing bacteriorhodoposin and the proteins in rat liver plasma membrane-enriched fractions, it was found that, compared with other two representative enzyme- and MS-compatible detergents RapiGest SF (RGS and sodium deoxycholate (SDC, SL exhibited obvious superiority in the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.

  7. The membrane-bound aspartyl protease BACE1:Molecular and functional properties in Alzheimer’s disease and beyond

    Directory of Open Access Journals (Sweden)

    Bastian eDislich

    2012-02-01

    Full Text Available The β-site APP cleaving enzyme 1 (BACE1 is a transmembrane aspartyl protease involved in Alzheimer’s disease (AD pathogenesis and in myelination. BACE1 initiates the generation of the pathogenic amyloid β-peptide, which makes BACE1 a major drug target for AD. BACE1 also cleaves and activates neuregulin 1, thereby contributing to postnatal myelination, in particular in the peripheral nervous system. Additional proteins are also cleaved by BACE1, but less is known about the physiological consequences of their cleavage. Recently, new phenotypes were described in BACE1-deficient mice. Although it remains unclear through which BACE1 substrates they are mediated, the phenotypes suggest a versatile role of this protease for diverse physiological processes. This review summarizes the enzymatic and cellular properties of BACE1 as well as its regulation by lipids, by transcriptional and by translational mechanisms. The main focus will be on the recent progress in understanding BACE1 function and its implication for potential mechanism-based side effects upon therapeutic inhibition.

  8. Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

    International Nuclear Information System (INIS)

    Lazarovici, P.; Yavin, E.

    1986-01-01

    The pharmacokinetic interaction of an affinity-purified 125 I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10 -9 M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37 0 C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4 0 C but not at 37 0 C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin

  9. Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

    Science.gov (United States)

    Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  10. Earthworm Protease

    Directory of Open Access Journals (Sweden)

    Rong Pan

    2010-01-01

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

  11. Earthworm Protease

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  12. An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains.

    Science.gov (United States)

    Fleetwood, Filippa; Andersson, Ken G; Ståhl, Stefan; Löfblom, John

    2014-12-30

    Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection

  13. The FtsH protease slr0228 is important for quality control of photosystem II in the thylakoid membrane of Synechocystis sp. PCC 6803

    Czech Academy of Sciences Publication Activity Database

    Komenda, Josef; Barker, M.; Kuviková, Stanislava; de Vries, R.; Mullineaux, C.W.; Tichý, Martin; Nixon, P.

    2006-01-01

    Roč. 281, č. 2 (2006), s. 1145-1151 ISSN 0021-9258 R&D Projects: GA ČR GA203/04/0800; GA MŠk LN00A141 Institutional research plan: CEZ:AV0Z50200510 Keywords : photosystem II * synechocystis sp. pcc 6803 * ftsh protease Subject RIV: EE - Microbiology, Virology Impact factor: 5.808, year: 2006

  14. Proteases and protease inhibitors in cancer

    NARCIS (Netherlands)

    van Noorden, C. J.

    1998-01-01

    The second conference on 'Proteases and protease inhibitors in cancer' was organized by the American Association for Cancer Research (AACR) and Acta Pathologica Microbiologica et Immunologica Scandinavica (APMIS). To understand the role of proteinases and to develop relevant synthetic inhibitors to

  15. Natural inhibitors of tumor-associated proteases

    International Nuclear Information System (INIS)

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

    2002-01-01

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

  16. HIV protease inhibitor resistance

    NARCIS (Netherlands)

    Wensing, Annemarie M.J.; Fun, Axel; Nijhuis, Monique

    2017-01-01

    HIV protease is pivotal in the viral replication cycle and directs the formation of mature infectious virus particles. The development of highly specific HIV protease inhibitors (PIs), based on thorough understanding of the structure of HIV protease and its substrate, serves as a prime example of

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    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 f...

  18. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell......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...... signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...

  19. 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.

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

    International Nuclear Information System (INIS)

    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

    2015-01-01

    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

  1. Why cells need intramembrane proteases - a mechanistic perspective

    Czech Academy of Sciences Publication Activity Database

    Stříšovský, Kvido

    2016-01-01

    Roč. 283, č. 10 (2016), s. 1837-1845 ISSN 1742-464X R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 EU Projects: European Commission(XE) 304154 - Rhomboid substrates Institutional support: RVO:61388963 Keywords : enzyme mechanism * intramembrane protease * membrane protein * rhomboid protease * substrate specificity Subject RIV: CE - Biochemistry Impact factor: 3.902, year: 2016

  2. Secreted proteases from pathogenic fungi.

    Science.gov (United States)

    Monod, Michel; Capoccia, Sabrina; Léchenne, Barbara; Zaugg, Christophe; Holdom, Mary; Jousson, Olivier

    2002-10-01

    Many species of human pathogenic fungi secrete proteases in vitro or during the infection process. Secreted endoproteases belong to the aspartic proteases of the pepsin family, serine proteases of the subtilisin family, and metalloproteases of two different families. To these proteases has to be added the non-pepsin-type aspartic protease from Aspergillus niger and a unique chymotrypsin-like protease from Coccidioides immitis. Pathogenic fungi also secrete aminopeptidases, carboxypeptidases and dipeptidyl-peptidases. The function of fungal secreted proteases and their importance in infections vary. It is evident that secreted proteases are important for the virulence of dermatophytes since these fungi grow exclusively in the stratum corneum, nails or hair, which constitutes their sole nitrogen and carbon sources. The aspartic proteases secreted by Candida albicans are involved in the adherence process and penetration of tissues, and in interactions with the immune system of the infected host. For Aspergillus fumigatus, the role of proteolytic activity has not yet been proved. Although the secreted proteases have been intensively investigated as potential virulence factors, knowledge on protease substrate specificities is rather poor and few studies have focused on the research of inhibitors. Knowledge of substrate specificities will increase our understanding about the action of each protease secreted by pathogenic fungi and will help to determine their contribution to virulence.

  3. Death proteases come alive

    NARCIS (Netherlands)

    Woltering, E.J.

    2004-01-01

    Cell death in plants exhibits morphological features comparable to caspase-mediated apoptosis in animals, suggesting that plant cell death is executed by (caspase-like) proteases. However, to date, no caspase homologues have been identified in plants and therefore the existence and nature of these

  4. Identification and network of outer membrane proteins regulating streptomysin resistance in Escherichia coli.

    Science.gov (United States)

    Li, Hui; Wang, Bao-Cheng; Xu, Wen-Jiao; Lin, Xiang-Min; Peng, Xuan-Xian

    2008-09-01

    Bacterial Outer membrane (OM) proteins involved in antibiotic resistance have been reported. However, little is known about the OM proteins and their interaction network regulating streptomycin (SM) resistance. In the present study, a subproteomic approach was utilized to characterize OM proteins of Escherichia coli with SM resistance. TolC, OmpT and LamB were found to be up-regulated, and FadL, OmpW and a location-unknown protein Dps were down-regulated in the SM-resistant E. coli strain. These changes at the level of protein expression were validated using Western blotting. The possible roles of the altered proteins involved in the SM resistance were investigated using genetic modified strains with the deletion of these altered genes. It is found that decreased and elevated minimum inhibitory concentrations and survival capabilities of the gene deleted strains and their resistant strains, Delta tolC, Delta ompT, Delta dps, Delta tolC-R, Delta ompT-R, Delta dps-R and Delta fadL-R, were correlated with the changes of TolC, OmpT, Dps and FadL at the protein expression levels detected by 2-DE gels, respectively. The results may suggest that these proteins are the key OM proteins and play important roles in the regulation of SM resistance in E. coli. Furthermore, an interaction network of altered OM proteins involved in the SM resistance was proposed in this report. Of the six altered proteins, TolC may play a central role in the network. These findings may provide novel insights into mechanisms of SM resistance in E. coli.

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

    Science.gov (United States)

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

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

  6. Comparative Detection of Alkaline Protease Production in Exiguobacterium acetylicum

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  7. Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases.

    Science.gov (United States)

    Nduaguibe, Chikodili C; Bentsi-Barnes, Kwamina; Mullen, Yoko; Kandeel, Fouad; Al-Abdullah, Ismail

    2010-01-01

    Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.

  8. Clp chaperone-proteases: structure and function.

    Science.gov (United States)

    Kress, Wolfgang; Maglica, Zeljka; Weber-Ban, Eilika

    2009-11-01

    Clp proteases are the most widespread energy-dependent proteases in bacteria. Their two-component architecture of protease core and ATPase rings results in an inventory of several Clp protease complexes that often coexist. Here, we present insights into Clp protease function, from their assembly to substrate recruitment and processing, and how this is coupled to the expense of energy.

  9. Protease-Sensitive Synthetic Prions

    OpenAIRE

    Colby, David W.; Wain, Rachel; Baskakov, Ilia V.; Legname, Giuseppe; Palmer, Christina G.; Nguyen, Hoang-Oanh B.; Lemus, Azucena; Cohen, Fred E.; DeArmond, Stephen J.; Prusiner, Stanley B.

    2010-01-01

    Prions arise when the cellular prion protein (PrPC) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrPSc. Frequently, PrPSc is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but no...

  10. Co-evolution of insect proteases and plant protease inhibitors.

    Science.gov (United States)

    Jongsma, Maarten A; Beekwilder, Jules

    2011-08-01

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

  11. Functional characterization of the mammalian iAAA protease subunit, YME1L

    OpenAIRE

    Majczak, Joanna

    2008-01-01

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

  12. Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

    Directory of Open Access Journals (Sweden)

    Lior Doron

    Full Text Available Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

  13. Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

    Science.gov (United States)

    Doron, Lior; Coppenhagen-Glazer, Shunit; Ibrahim, Yara; Eini, Amir; Naor, Ronit; Rosen, Graciela; Bachrach, Gilad

    2014-01-01

    Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

  14. Sulfur mustard-stimulated proteases and their inhibitors in a cultured normal human epidermal keratinocytes model: A potential approach for anti-vesicant drug development

    Directory of Open Access Journals (Sweden)

    Xiannu Jin

    Full Text Available Protease stimulation in cultured normal human epidermal keratinocytes (NHEK due to sulfur mustard (SM exposure is well documented. However, the specific protease(s stimulated by SM and the protease substrates remain to be determined. In this study, we observed that SM stimulates several proteases and the epidermal-dermal attachment protein laminin-5 is one of the substrates. We propose that following SM exposure of the skin, laminin-5 degradation causes the detachment of the epidermis from the dermis and, therefore, vesication. We utilized gelatin zymography, Western blotting, immuno-fluorescence staining, and real-time polymerase chain reaction (RT-PCR analyses to study the SM-stimulated proteases and laminin-5 degradation in NHEK. Two major protease bands (64 kDa and 72 kDa were observed by zymography in SM-exposed cells. Addition of serine protease inhibitor (aprotinin, 100 μM, or the metalloprotease inhibitor (amastatin, 100 μM to NHEK cultures prior to SM exposure decreased the SM-stimulated protease bands seen by zymography. These inhibitors completely or partially prevented SM-induced laminin-5 γ2 degradation as seen by Western blotting as well as immuno-fluorescence staining. Our results from Western blotting and RT-PCR studies also indicated that the membrane-type matrix metalloproteinase-1 (MT-MM-1 may be involved in SM-induced skin blistering.To summarize, our results in the NHEK model indicate the following: (a SM stimulates multiple proteases including serine protease(s, and metalloproteases; (b SM decreases the level of laminin-5 γ2, which is prevented by either a serine protease inhibitor or a metalloprotease inhibitor and (c MT-MMP-1 maybe one of the proteases that is involved in skin blistering due to SM exposure. Keywords: Sulfur mustard, Serine protease, Metalloprotease, Protease inhibiter, Zymography, Laminin-5 γ2

  15. Cytomegalovirus protease targeted prodrug development.

    Science.gov (United States)

    Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

    2013-04-01

    Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

  16. Cleavage of CD14 and LBP by a protease from Prevotella intermedia

    Science.gov (United States)

    Deschner, James; Singhal, Anuradha; Long, Ping; Liu, Chau-Ching; Piesco, Nicholas

    2016-01-01

    Periodontitis is an inflammatory disease caused by subgingival microorganisms and their components, such as lipopolysaccharide (LPS). Responses of the host to LPS are mediated by CD14 and LPS-binding protein (LBP). In this study, it was determined that proteases from a periodontal pathogen, Prevotella intermedia, cleave CD14 and LBP, and thereby modulate the virulence of LPS. Culture supernatants from two strains of P. intermedia (ATCC 25611 and 25261) cleaved CD14 and LBP in a concentration-dependent manner. Zymographic and molecular mass analysis revealed the presence of a membrane-associated, 170-kDa, monomeric protease. Class-specific inhibitors and stimulators demonstrated that this enzyme is a metal-requiring, thiol-activated, cysteine protease. The protease was stable over a wide range of temperatures (4–56 °C) and pH values (4.5–8.5). This enzyme also decreased the expression of interleukin-1β (IL-1β)-specific mRNA in the LPS-activated macrophage-like cell lines U937 and THP-1 in a concentration-dependent manner, indicating that it also cleaves membrane-associated CD14. Furthermore, addition of soluble CD14 abrogated protease-mediated inhibition of IL-1 mRNA expression induced by LPS. The observations suggest that proteolysis of CD14 and LBP by P. intermedia protease might modulate the virulence of LPS at sites of periodontal infections. PMID:12728301

  17. Contemporary protease inhibitors and cardiovascular risk

    DEFF Research Database (Denmark)

    Lundgren, Jens; Mocroft, Amanda; Ryom, Lene

    2018-01-01

    PURPOSE OF REVIEW: To review the evidence linking use of HIV protease inhibitors with excess risk of cardiovascular disease (CVD) in HIV+ populations. RECENT FINDINGS: For the two contemporary most frequently used protease inhibitors, darunavir and atazanavir [both pharmacologically boosted...

  18. tolerant alkaline protease from Bacillus coagulans PSB

    African Journals Online (AJOL)

    oyaide

    2013-05-22

    May 22, 2013 ... suggest the suitability of the enzyme for applications in peptide synthesis, detergent formulation and ... The cell free supernatant was recovered as crude enzyme preparation and used for further studies. Assay of protease activity. Protease activity was ... Effect of pH on growth and protease production.

  19. A genomic survey of proteases in Aspergilli

    NARCIS (Netherlands)

    Budak, Sebnem Ozturkoglu; Zhou, M.; Brouwer, Carlo; Wiebenga, A.; Benoit, Isabelle; Di Falco, Marcos; Tsang, Adrian; de Vries, Ronald P; van den Brink, J.

    2014-01-01

    BACKGROUND: Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide

  20. 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.

  1. Carbohydrase and protease supplementation increased ...

    African Journals Online (AJOL)

    User

    2014-09-15

    Sep 15, 2014 ... Department of Animal and Wildlife Sciences, Faculty of Natural and Agricultural Science ... control birds was 12% higher than that of the positive control, while diets supplemented with single enzyme ... The inclusion of exogenous proteases in maize-soya-based diets increases protein digestion by.

  2. Carbohydrase and protease supplementation increased ...

    African Journals Online (AJOL)

    A trial was conducted to evaluate whether the addition of commercial enzyme preparations containing carbohydrases and a protease would increase the available metabolizable energy (ME) of maize-soya-based broiler diets. Seven thousand five hundred and sixty (7560) day-old Ross 788 chicks were randomly allocated ...

  3. Intramembrane protease RasP boosts protein production in Bacillus.

    Science.gov (United States)

    Neef, Jolanda; Bongiorni, Cristina; Goosens, Vivianne J; Schmidt, Brian; van Dijl, Jan Maarten

    2017-04-04

    The microbial cell factory Bacillus subtilis is a popular industrial platform for high-level production of secreted technical enzymes. Nonetheless, the effective secretion of particular heterologous enzymes remains challenging. Over the past decades various studies have tackled this problem, and major improvements were achieved by optimizing signal peptides or removing proteases involved in product degradation. On the other hand, serious bottlenecks in the protein export process per se remained enigmatic, especially for protein secretion at commercially significant levels by cells grown to high density. The aim of our present study was to assess the relevance of the intramembrane protease RasP for high-level protein production in B. subtilis. Deletion of the rasP gene resulted in reduced precursor processing and extracellular levels of the overproduced α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis. Further, secretion of the overproduced serine protease BPN' from Bacillus amyloliquefaciens was severely impaired in the absence of RasP. Importantly, overexpression of rasP resulted in threefold increased production of a serine protease from Bacillus clausii, and 2.5- to 10-fold increased production of an AmyAc α-amylase from Paenibacillus curdlanolyticus, depending on the culture conditions. Of note, growth defects due to overproduction of the two latter enzymes were suppressed by rasP-overexpression. Here we show that an intramembrane protease, RasP, sets a limit to high-level production of two secreted heterologous enzymes that are difficult to produce in the B. subtilis cell factory. This finding was unexpected and suggests that proteolytic membrane sanitation is key to effective enzyme production in Bacillus.

  4. 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.

  5. Sulfur mustard-stimulated proteases and their inhibitors in a cultured normal human epidermal keratinocytes model: A potential approach for anti-vesicant drug development.

    Science.gov (United States)

    Jin, Xiannu; Ray, Radharaman; Ray, Prabhati

    2016-01-01

    Protease stimulation in cultured normal human epidermal keratinocytes (NHEK) due to sulfur mustard (SM) exposure is well documented. However, the specific protease(s) stimulated by SM and the protease substrates remain to be determined. In this study, we observed that SM stimulates several proteases and the epidermal-dermal attachment protein laminin-5 is one of the substrates. We propose that following SM exposure of the skin, laminin-5 degradation causes the detachment of the epidermis from the dermis and, therefore, vesication. We utilized gelatin zymography, Western blotting, immuno-fluorescence staining, and real-time polymerase chain reaction (RT-PCR) analyses to study the SM-stimulated proteases and laminin-5 degradation in NHEK. Two major protease bands (64 kDa and 72 kDa) were observed by zymography in SM-exposed cells. Addition of serine protease inhibitor (aprotinin, 100 μM), or the metalloprotease inhibitor (amastatin, 100 μM) to NHEK cultures prior to SM exposure decreased the SM-stimulated protease bands seen by zymography. These inhibitors completely or partially prevented SM-induced laminin-5 γ2 degradation as seen by Western blotting as well as immuno-fluorescence staining. Our results from Western blotting and RT-PCR studies also indicated that the membrane-type matrix metalloproteinase-1 (MT-MM-1) may be involved in SM-induced skin blistering. To summarize, our results in the NHEK model indicate the following: (a) SM stimulates multiple proteases including serine protease(s), and metalloproteases; (b) SM decreases the level of laminin-5 γ2, which is prevented by either a serine protease inhibitor or a metalloprotease inhibitor and (c) MT-MMP-1 maybe one of the proteases that is involved in skin blistering due to SM exposure.

  6. Protease-sensitive synthetic prions.

    Directory of Open Access Journals (Sweden)

    David W Colby

    2010-01-01

    Full Text Available Prions arise when the cellular prion protein (PrP(C undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc. Frequently, PrP(Sc is protease-resistant but protease-sensitive (s prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164, denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174 did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc.

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

    Science.gov (United States)

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

    2016-05-01

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

  8. Molecular Imaging of Proteases in Cancer

    Directory of Open Access Journals (Sweden)

    Yunan Yang

    2009-01-01

    Full Text Available Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence, magnetic resonance imaging (MRI, single-photon emission computed tomography (SPECT, and positron emission tomography (PET. In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction.

  9. Cysteine Protease Zymography: Brief Review.

    Science.gov (United States)

    Wilkesman, Jeff

    2017-01-01

    Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.

  10. Advances in protease engineering for laundry detergents.

    Science.gov (United States)

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Matthew J Kesic

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

  12. Mass spectrometry-assisted protease substrate screening

    DEFF Research Database (Denmark)

    Schlüter, Hartmut; Rykl, Jana; Thiemann, Joachim

    2007-01-01

    Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown....... Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed...

  13. Proteolytic crosstalk in multi-protease networks

    Science.gov (United States)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

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

  14. Current and Novel Inhibitors of HIV Protease

    Czech Academy of Sciences Publication Activity Database

    Pokorná, Jana; Machala, L.; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    Roč. 1, č. 3 (2009), s. 1209-1239 ISSN 1999-4915 R&D Projects: GA MŠk 1M0508 Grant - others:GA AV ČR(CZ) IAAX00320901 Program:IA Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV protease * protease inhibitor * HAART Subject RIV: CE - Biochemistry

  15. Tissue dissociation enzyme neutral protease assessment.

    Science.gov (United States)

    Breite, A G; Dwulet, F E; McCarthy, R C

    2010-01-01

    Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures. Copyright 2010 Elsevier Inc. All rights reserved.

  16. Production of extracellular aspartic protease in submerged ...

    African Journals Online (AJOL)

    Production of extracellular aspartic protease in submerged fermentation with Mucor mucedo DSM 809. ... The preferred method was the inoculation of the culture media with spores at a total load of 6x105 spores per flask. Key words: Milk clotting enzyme, Aspartic protease, Mucor mucedo, Sub-merged fermentation.

  17. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    user

    2013-03-20

    Mar 20, 2013 ... be absorbed and utilized by living cells. Due to their wide .... The effect of pH on protease stability was determined by pre-incubating the enzyme without substrate at different pH values (5 to 11) using different buffers. The residual ..... detergent formulations: effects of thermodynamic stabilizers and protease ...

  18. Immobilization to prevent enzyme incompatibility with proteases

    NARCIS (Netherlands)

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was

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

    African Journals Online (AJOL)

    USER

    ABSTRACT: Microbial proteases have wide industrial applications and proteases of the lactic acid bacteria (LAB) have received special attention ... Purification of the enzyme by gel filtration chromatography on Sephadex G75 following ammonium .... Gel filtration and ion exchange chromatography. The dialysate was ...

  20. Optimization of alkaline protease production from Pseudomonas ...

    African Journals Online (AJOL)

    A protease producing bacteria was isolated from meat waste contaminated soil and identified as Pseudomonas fluorescens. Optimization of the fermentation medium for maximum protease production was carried out. The culture conditions like inoculum concentration, incubation time, pH, temperature, carbon sources, ...

  1. Optimization of medium composition for thermostable protease ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... Optimization of the fermentation medium for maximization of thermostable neutral protease production by Bacillus sp. ... at 3.6 g/l and yeast extract at 3.9 g/l gived maximum protease activity of 6804 U/ml. Key words: Medium ... face method, which is used to study the effects of several factors influencing the ...

  2. Optimization of alkaline protease production from Pseudomonas ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... the metal ions tested. Key words: Alkaline protease, casein agar, meat waste contaminated soil, Pseudomonas fluorescens. INTRODUCTION. Proteases are the most important industrial enzymes that execute a wide variety of functions and have various important biotechnological applications (Mohen et al.,.

  3. 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.

  4. Generation and characterization of a collection of knock-down lines for the chloroplast Clp protease complex in tobacco.

    Science.gov (United States)

    Moreno, Juan C; Tiller, Nadine; Diez, Mercedes; Karcher, Daniel; Tillich, Michael; Schöttler, Mark A; Bock, Ralph

    2017-04-01

    Protein degradation in chloroplasts is carried out by a set of proteases that eliminate misfolded, damaged, or superfluous proteins. The ATP-dependent caseinolytic protease (Clp) is the most complex protease in plastids and has been implicated mainly in stromal protein degradation. In contrast, FtsH, a thylakoid membrane-associated metalloprotease, is believed to participate mainly in the degradation of thylakoidal proteins. To determine the role of specific Clp and FtsH subunits in plant growth and development, RNAi lines targeting at least one subunit of each Clp ring and FtsH were generated in tobacco. In addition, mutation of the translation initiation codon was employed to down-regulate expression of the plastid-encoded ClpP1 subunit. These protease lines cover a broad range of reductions at the transcript and protein levels of the targeted genes. A wide spectrum of phenotypes was obtained, including pigment deficiency, alterations in leaf development, leaf variegations, and impaired photosynthesis. When knock-down lines for the different protease subunits were compared, both common and specific phenotypes were observed, suggesting distinct functions of at least some subunits. Our work provides a well-characterized collection of knock-down lines for plastid proteases in tobacco and reveals the importance of the Clp protease in physiology and plant development. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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

    Directory of Open Access Journals (Sweden)

    Nicolas Faller

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

  6. Factor VII-activating protease

    DEFF Research Database (Denmark)

    Ramanathan, Ramshanker; Gram, Jørgen B; Sand, Niels Peter R

    2017-01-01

    : Factor VII-activating protease (FSAP) may regulate development of cardiovascular disease (CVD). We evaluated sex differences in FSAP measures and examined the association between FSAP and coronary artery calcification (CAC) in a middle-aged population. Participants were randomly selected citizens...... aged 50 or 60 without CVD, diabetes mellitus, Marburg I polymorphism, or hormone replacement therapy (HRT). FSAP protein concentration (total FSAP), FSAP urokinase-activating capacity (FSAP GP), and FSAP GP/total FSAP (specific FSAP activity) were measured. Cardiac computed tomography (CT) determined...... the Agatston score, dividing the study population in three groups: (1) Agatston score = 0 U, (2) Agatston score = 1-99 U, or (3) Agatston score more than 99 U. A total of 134 women and 116 men were included. Total FSAP, FSAP GP, and specific FSAP activity were independently higher in women (97.4%, 81.1%, 0...

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

    Directory of Open Access Journals (Sweden)

    Xun-Cheng Su

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

  8. Degradation of PsbO by the Deg protease HhoA Is thioredoxin dependent.

    Directory of Open Access Journals (Sweden)

    Irma N Roberts

    Full Text Available The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA. rHhoA cleaved reduced eukaryotic (specifically, spinach PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803, no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA was detected in the PSII-enriched membrane fraction.

  9. Gut proteases target Yersinia invasin in vivo

    Directory of Open Access Journals (Sweden)

    Freund Sandra

    2011-04-01

    Full Text Available Abstract Background Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches. Findings Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases. Conclusions We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

  10. A biotechnology perspective of fungal proteases

    Directory of Open Access Journals (Sweden)

    Paula Monteiro de Souza

    2015-06-01

    Full Text Available Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

  11. A biotechnology perspective of fungal proteases

    Science.gov (United States)

    de Souza, Paula Monteiro; Bittencourt, Mona Lisa de Assis; Caprara, Carolina Canielles; de Freitas, Marcela; de Almeida, Renata Paula Coppini; Silveira, Dâmaris; Fonseca, Yris Maria; Ferreira, Edivaldo Ximenes; Pessoa, Adalberto; Magalhães, Pérola Oliveira

    2015-01-01

    Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. PMID:26273247

  12. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

    This thesis describes the design and synthesis of peptide-based serine protease inhibitors. The targeted protease, urokinase-type plasminogen activator (uPA) activates plasminogen, which plays a major role in cancer metastasis. The peptide upain-2 (S 1 ,S 12-cyclo-AcCSWRGLENHAAC-NH2) is a highly...... of novel peptide-based protease inhibitors, efforts were made towards improved methods for peptide synthesis. The coupling of Fmoc-amino acids onto N-methylated peptidyl resins was investigated. These couplings can be low yielding and the effect of the use of microwave heating combined with the coupling...

  13. Permeability testing of biomaterial membranes

    Energy Technology Data Exchange (ETDEWEB)

    Dreesmann, L; Hajosch, R; Nuernberger, J Vaz; Schlosshauer, B [NMI Natural and Medical Sciences Institute at University Tuebingen, Markwiesenstr. 55, D-72770 Reutlingen (Germany); Ahlers, M [GELITA AG, Gammelsbacher Str. 2, D-69412 Eberbach (Germany)], E-mail: schlosshauer@nmi.de

    2008-09-01

    The permeability characteristics of biomaterials are critical parameters for a variety of implants. To analyse the permeability of membranes made from crosslinked ultrathin gelatin membranes and the transmigration of cells across the membranes, we combined three technical approaches: (1) a two-chamber-based permeability assay, (2) cell culturing with cytochemical analysis and (3) biochemical enzyme electrophoresis (zymography). Based on the diffusion of a coloured marker molecule in conjunction with photometric quantification, permeability data for a gelatin membrane were determined in the presence or absence of gelatin degrading fibroblasts. Cytochemical evaluation after cryosectioning of the membranes was used to ascertain whether fibroblasts had infiltrated the membrane inside. Zymography was used to investigate the potential release of proteases from fibroblasts, which are known to degrade collagen derivatives such as gelatin. Our data show that the diffusion equilibrium of a low molecular weight dye across the selected gelatin membrane is approached after about 6-8 h. Fibroblasts increase the permeability due to cavity formation in the membrane inside without penetrating the membrane for an extended time period (>21 days in vitro). Zymography indicates that cavity formation is most likely due to the secretion of matrix metalloproteinases. In summary, the combination of the depicted methods promises to facilitate a more rational development of biomaterials, because it provides a rapid means of determining permeability characteristics and bridges the gap between descriptive methodology and the mechanistic understanding of permeability alterations due to biological degradation.

  14. Permeability testing of biomaterial membranes

    International Nuclear Information System (INIS)

    Dreesmann, L; Hajosch, R; Nuernberger, J Vaz; Schlosshauer, B; Ahlers, M

    2008-01-01

    The permeability characteristics of biomaterials are critical parameters for a variety of implants. To analyse the permeability of membranes made from crosslinked ultrathin gelatin membranes and the transmigration of cells across the membranes, we combined three technical approaches: (1) a two-chamber-based permeability assay, (2) cell culturing with cytochemical analysis and (3) biochemical enzyme electrophoresis (zymography). Based on the diffusion of a coloured marker molecule in conjunction with photometric quantification, permeability data for a gelatin membrane were determined in the presence or absence of gelatin degrading fibroblasts. Cytochemical evaluation after cryosectioning of the membranes was used to ascertain whether fibroblasts had infiltrated the membrane inside. Zymography was used to investigate the potential release of proteases from fibroblasts, which are known to degrade collagen derivatives such as gelatin. Our data show that the diffusion equilibrium of a low molecular weight dye across the selected gelatin membrane is approached after about 6-8 h. Fibroblasts increase the permeability due to cavity formation in the membrane inside without penetrating the membrane for an extended time period (>21 days in vitro). Zymography indicates that cavity formation is most likely due to the secretion of matrix metalloproteinases. In summary, the combination of the depicted methods promises to facilitate a more rational development of biomaterials, because it provides a rapid means of determining permeability characteristics and bridges the gap between descriptive methodology and the mechanistic understanding of permeability alterations due to biological degradation

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

    Directory of Open Access Journals (Sweden)

    José Andrés Morgado-Díaz

    2005-07-01

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

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

    Science.gov (United States)

    Kernaghan, Gavin; Mayerhofer, Michael

    2017-01-01

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

  17. Secreted fungal aspartic proteases: A review.

    Science.gov (United States)

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. Copyright © 2016 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.

  18. 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: elimina......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...... for ADAM 12 involving both furin cleavage and copper binding....

  19. Tissue Dissociation Enzyme Neutral Protease Assessment

    OpenAIRE

    Breite, A.G.; Dwulet, F.E.; McCarthy, R.C.

    2010-01-01

    Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polym...

  20. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    The enzyme was active in pH range 5 to11 and temperature of 30 to 80°C. The optimum pH and the temperature for protease activity were recorded to be pH 8 and 50°C, respectively. The enzyme was stable up to 40°C and pH 9. The protease activity was inhibited by Zn2+, Ni2+ and Sn2+ and increased by Ca2+, Mg2+ ...

  1. Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

    Science.gov (United States)

    Huang, Qiwei; Li, Qingxin; Joy, Joma; Chen, Angela Shuyi; Ruiz-Carrillo, David; Hill, Jeffrey; Lescar, Julien; Kang, Congbao

    2013-12-01

    Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Acid protease production in fungal root endophytes.

    Science.gov (United States)

    Mayerhofer, Michael S; Fraser, Erica; Kernaghan, Gavin

    2015-01-01

    Fungal endophytes are ubiquitous in healthy root tissue, but little is known about their ecosystem functions, including their ability to utilize organic nutrient sources such as proteins. Root-associated fungi may secrete proteases to access the carbon and mineral nutrients within proteins in the soil or in the cells of their plant host. We compared the protein utilization patterns of multiple isolates of the root endophytes Phialocephala fortinii s.l., Meliniomyces variabilis and Umbelopsis isabellina with those of two ectomycorrhizal (ECM) fungi, Hebeloma incarnatulum and Laccaria bicolor, and the wood-decay fungus Irpex lacteus at pH values of 2-9 on liquid BSA media. We also assessed protease activity using a fluorescently labeled casein assay and gelatin zymography and characterized proteases using specific protease inhibitors. I. lacteus and U. isabellina utilized protein efficiently, while the ECM fungi exhibited poor protein utilization. ECM fungi secreted metallo-proteases and had pH optima above 4, while other fungi produced aspartic proteases with lower pH optima. The ascomycetous root endophytes M. variabilis and P. fortinii exhibited intermediate levels of protein utilization and M. variabilis exhibited a very low pH optimum. Comparing proteolytic profiles between fungal root endophytes and fungi with well defined ecological roles provides insight into the ecology of these cryptic root associates. © 2015 by The Mycological Society of America.

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

    Directory of Open Access Journals (Sweden)

    Aiman Tanveer

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

  4. Survey of immunoglobulin A protease activity among selected species of Ureaplasma and Mycoplasma: specificity for host immunoglobulin A.

    Science.gov (United States)

    Kapatais-Zoumbos, K; Chandler, D K; Barile, M F

    1985-03-01

    Because immunoglobulin A (IgA) is the predominant immunoglobulin at mucosal surfaces, IgA proteases produced by pathogenic bacteria are considered potential virulence factors for organisms that cause disease or gain entry at mucous membranes. To determine the role of IgA protease in the pathogenicity of mycoplasmal disease, a variety of human and animal mycoplasma and ureaplasma species were examined for IgA protease activity with human, murine, porcine, and canine IgA. None of the mycoplasma species examined showed detectable IgA protease activity with any of the IgAs tested. Twenty-eight strains of Ureaplasma urealyticum isolated from human urogenital tissues cleaved human IgA1, but no cleavage of human IgA2 or murine, porcine, or canine IgA was observed. Ureaplasmas isolated from nonhuman hosts (feline, canine, avian, and bovine [Ureaplasma diversum]) did not cleave human IgA1. Two strains of canine ureaplasmas were able to cleave canine IgA, but not murine IgA. Thus, ureaplasmas from other species can produce IgA protease, but the specificity of the enzyme was restricted to the IgA of the appropriate host. This finding suggests that IgA proteases could play a role in the selective host specificity of mucosal pathogens.

  5. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. [Ohio State Univ., Columbus, OH (United States)]|[Lawrence Berkeley Lab., CA (United States)

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

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

    Directory of Open Access Journals (Sweden)

    Rui Cruz

    2014-08-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Studies on the membrane-associated proteolytic activities of Escherichia coli

    International Nuclear Information System (INIS)

    Palmer, S.M.

    1986-01-01

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

  9. Evaluation of proteases and protease inhibitors in Heterodera glycines cysts obtained from laboratory and field populations

    Science.gov (United States)

    Proteases and proteases inhibitors were evaluated in a number of preparations of Heterodera glycines cysts obtained from glasshouse cultures (GH) and field (LR) populations. Using a FRET-peptide library comprising 512 peptide substrate pools that detect 4 endoprotease types (aspartic, cysteine, meta...

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  11. Purification and characterisation of a protease (tamarillin) from tamarillo fruit

    KAUST Repository

    Li, Zhao

    2018-02-16

    A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named \\'tamarillin\\'.

  12. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...

  13. Temporal Proteomics of Inducible RNAi Lines of Clp Protease Subunits Identifies Putative Protease Substrates.

    Science.gov (United States)

    Moreno, Juan C; Martínez-Jaime, Silvia; Schwartzmann, Joram; Karcher, Daniel; Tillich, Michael; Graf, Alexander; Bock, Ralph

    2018-02-01

    The Clp protease in the chloroplasts of plant cells is a large complex composed of at least 13 nucleus-encoded subunits and one plastid-encoded subunit, which are arranged in several ring-like structures. The proteolytic P-ring and the structurally similar R-ring form the core complex that contains the proteolytic chamber. Chaperones of the HSP100 family help with substrate unfolding, and additional accessory proteins are believed to assist with Clp complex assembly and/or to promote complex stability. Although the structure and function of the Clp protease have been studied in great detail in both bacteria and chloroplasts, the identification of bona fide protease substrates has been very challenging. Knockout mutants of genes for protease subunits are of limited value, due to their often pleiotropic phenotypes and the difficulties with distinguishing primary effects (i.e. overaccumulation of proteins that represent genuine protease substrates) from secondary effects (proteins overaccumulating for other reasons). Here, we have developed a new strategy for the identification of candidate substrates of plant proteases. By combining ethanol-inducible knockdown of protease subunits with time-resolved analysis of changes in the proteome, proteins that respond immediately to reduced protease activity can be identified. In this way, secondary effects are minimized and putative protease substrates can be identified. We have applied this strategy to the Clp protease complex of tobacco ( Nicotiana tabacum ) and identified a set of chloroplast proteins that are likely degraded by Clp. These include several metabolic enzymes but also a small number of proteins involved in photosynthesis. © 2018 American Society of Plant Biologists. All Rights Reserved.

  14. Temporal Proteomics of Inducible RNAi Lines of Clp Protease Subunits Identifies Putative Protease Substrates1[OPEN

    Science.gov (United States)

    Martínez-Jaime, Silvia; Karcher, Daniel; Tillich, Michael

    2018-01-01

    The Clp protease in the chloroplasts of plant cells is a large complex composed of at least 13 nucleus-encoded subunits and one plastid-encoded subunit, which are arranged in several ring-like structures. The proteolytic P-ring and the structurally similar R-ring form the core complex that contains the proteolytic chamber. Chaperones of the HSP100 family help with substrate unfolding, and additional accessory proteins are believed to assist with Clp complex assembly and/or to promote complex stability. Although the structure and function of the Clp protease have been studied in great detail in both bacteria and chloroplasts, the identification of bona fide protease substrates has been very challenging. Knockout mutants of genes for protease subunits are of limited value, due to their often pleiotropic phenotypes and the difficulties with distinguishing primary effects (i.e. overaccumulation of proteins that represent genuine protease substrates) from secondary effects (proteins overaccumulating for other reasons). Here, we have developed a new strategy for the identification of candidate substrates of plant proteases. By combining ethanol-inducible knockdown of protease subunits with time-resolved analysis of changes in the proteome, proteins that respond immediately to reduced protease activity can be identified. In this way, secondary effects are minimized and putative protease substrates can be identified. We have applied this strategy to the Clp protease complex of tobacco (Nicotiana tabacum) and identified a set of chloroplast proteins that are likely degraded by Clp. These include several metabolic enzymes but also a small number of proteins involved in photosynthesis. PMID:29229697

  15. Bacterial proteases, untapped antimicrobial drug targets.

    Science.gov (United States)

    Culp, Elizabeth; Wright, Gerard D

    2017-04-01

    Bacterial proteases are an extensive collection of enzymes that have vital roles in cell viability, stress response and pathogenicity. Although their perturbation clearly offers the potential for antimicrobial drug development, both as traditional antibiotics and anti-virulence drugs, they are not yet the target of any clinically used therapeutics. Here we describe the potential for and recent progress in the development of compounds targeting bacterial proteases with a focus on AAA+ family proteolytic complexes and signal peptidases (SPs). Caseinolytic protease (ClpP) belongs to the AAA+ family of proteases, a group of multimeric barrel-shaped complexes whose activity is tightly regulated by associated AAA+ ATPases. The opportunity for chemical perturbation of these complexes is demonstrated by compounds targeting ClpP for inhibition, activation or perturbation of its associated ATPase. Meanwhile, SPs are also a proven antibiotic target. Responsible for the cleavage of targeting peptides during protein secretion, both type I and type II SPs have been successfully targeted by chemical inhibitors. As the threat of pan-antibiotic resistance continues to grow, these and other bacterial proteases offer an arsenal of novel antibiotic targets ripe for development.

  16. Thermostable alkaline halophilic-protease production by Natronolimnobius innermongolicus WN18.

    Science.gov (United States)

    Selim, Samy; Hagagy, Nashwa; Abdel Aziz, Mohamed; El-Meleigy, El Syaed; Pessione, Enrica

    2014-01-01

    This study reports the production and biochemical characterisation of a thermostable alkaline halophilic protease from Natronolimnobius innermongolicus WN18 (HQ658997), isolated from soda Lake of Wadi An-Natrun, Egypt. The enzyme was concentrated by spinning through a centriplus, centrifugal ultrafiltration Millipore membrane with a total yield of 25%. The relative molecular mass of this protease determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ranged from 67 to 43 kDa. The extracellular protease of N. innermongolicus WN18 was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60°C in the presence of 2.5 M NaCl. This enzyme was stable in a broad pH range (6-12) with an optimum pH of 9-10 for azocasein hydrolysis. This extracellular protease, therefore, could be defined as thermostable and haloalkaliphilic with distinct properties that make the enzyme applicable for different industrial purposes.

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Willy Praira

    2010-11-01

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

  19. TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

    Science.gov (United States)

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

    2016-12-15

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

  20. Cell invasion through basement membrane

    Science.gov (United States)

    Morrissey, Meghan A; Hagedorn, Elliott J; Sherwood, David R

    2013-01-01

    Cell invasion through basement membrane is an essential part of normal development and physiology, and occurs during the pathological progression of human inflammatory diseases and cancer. F-actin-rich membrane protrusions, called invadopodia, have been hypothesized to be the “drill bits” of invasive cells, mediating invasion through the dense, highly cross-linked basement membrane matrix. Though studied in vitro for over 30 y, invadopodia function in vivo has remained elusive. We have recently discovered that invadopodia breach basement membrane during anchor cell invasion in C. elegans, a genetically and visually tractable in vivo invasion event. Further, we found that the netrin receptor DCC localizes to the initial site of basement membrane breach and directs invasion through a single gap in the matrix. In this commentary, we examine how the dynamics and structure of AC-invadopodia compare with in vitro invadopodia and how the netrin receptor guides invasion through a single basement membrane breach. We end with a discussion of our surprising result that the anchor cell pushes the basement membrane aside, instead of completely dissolving it through proteolysis, and provide some ideas for how proteases and physical displacement may work together to ensure efficient and robust invasion. PMID:24778942

  1. Cleavage entropy as quantitative measure of protease specificity.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    2013-04-01

    Full Text Available A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  2. The Clp protease system; a central component of the chloroplast protease network.

    Science.gov (United States)

    Olinares, Paul Dominic B; Kim, Jitae; van Wijk, Klaas J

    2011-08-01

    Intra-plastid proteases play crucial and diverse roles in the development and maintenance of non-photosynthetic plastids and chloroplasts. Formation and maintenance of a functional thylakoid electron transport chain requires various protease activities, operating in parallel, as well as in series. This review first provides a short, referenced overview of all experimentally identified plastid proteases in Arabidopsis thaliana. We then focus on the Clp protease system which constitutes the most abundant and complex soluble protease system in the plastid, consisting of 15 nuclear-encoded members and one plastid-encoded member in Arabidopsis. Comparisons to the simpler Clp system in photosynthetic and non-photosynthetic bacteria will be made and the role of Clp proteases in the green algae Chlamydomonas reinhardtii will be briefly reviewed. Extensive molecular genetics has shown that the Clp system plays an essential role in Arabidopsis chloroplast development in the embryo as well as in leaves. Molecular characterization of the various Clp mutants has elucidated many of the consequences of loss of Clp activities. We summarize and discuss the structural and functional aspects of the Clp machinery, including progress on substrate identification and recognition. Finally, the Clp system will be evaluated in the context of the chloroplast protease network. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts. Copyright © 2010 Elsevier B.V. All rights reserved.

  3. Effect of protease inhibitors on exflagellation in Plasmodium falciparum.

    Science.gov (United States)

    Rupp, Ingrid; Bosse, Rebecca; Schirmeister, Tanja; Pradel, Gabriele

    2008-04-01

    Enzymes involved in sexual differentiation and fertilization of the human malaria parasite Plasmodium falciparum represent potential targets for transmission blocking strategies. Parasite proteases are putatively involved in several steps during fertilization, but the types of proteases, their targets and modes of action remain hitherto unknown. We investigated the involvement of proteases in gametogenesis via exflagellation and immunofluorescence assays, using a variety of commercially available as well as newly designed protease inhibitors. The assays revealed a blockade of microgamete formation by the cysteine/serine protease inhibitors TLCK and TPCK. The serine protease inhibitor PMSF, the falcipain-targeting inhibitor RV112D, and the aspartic protease inhibitor EPNP also significantly decreased formation of microgametes. The metalloprotease inhibitor 1,10-phenanthroline, on the other hand, inhibited exflagellation by interfering with microgamete motility. Furthermore, EPNP reduced the activation of male and female gametocytes. Our data point to a major involvement of serine proteases and a non-thermolysin-like zinc metalloprotease in microgametocyte exflagellation.

  4. Synergism of Selective Tumor Vascular Thrombosis and Protease Activated Prodrug

    National Research Council Canada - National Science Library

    Liu, Cheng

    2008-01-01

    ... by administration of protease-activated prodrug. The activation of coagulation cascade and tumor vascular thrombosis as well as the following activation of the thrombolytic pathways led to explosive amplification of serine protease cascades...

  5. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    DEFF Research Database (Denmark)

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt

    2011-01-01

    En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner.......En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner....

  6. Selection of Protease Inhibitors to Prevent or Attenuate Inflammatory Processes

    Science.gov (United States)

    2007-08-01

    temperature. elevated body temperature, pH, oxygen tension): * number of chemical factors (fatty acids. lactid acid, pepsin, lysozyme, antimicrobial ...inhibit toxic serine proteases produced by the fungus Metarhizium anisopliae. The known spectrum of protease inhibitors from invertebrates includes also

  7. Ten Prominent Host Proteases in Plant-Pathogen Interactions

    Directory of Open Access Journals (Sweden)

    Emma L. Thomas

    2018-02-01

    Full Text Available Proteases are enzymes integral to the plant immune system. Multiple aspects of defence are regulated by proteases, including the hypersensitive response, pathogen recognition, priming and peptide hormone release. These processes are regulated by unrelated proteases residing at different subcellular locations. In this review, we discuss 10 prominent plant proteases contributing to the plant immune system, highlighting the diversity of roles they perform in plant defence.

  8. The yeast autophagy protease Atg4 is regulated by thioredoxin.

    Science.gov (United States)

    Pérez-Pérez, María Esther; Zaffagnini, Mirko; Marchand, Christophe H; Crespo, José L; Lemaire, Stéphane D

    2014-01-01

    Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation.

  9. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  10. Biobased Membrane

    NARCIS (Netherlands)

    Koenders, E.A.B.; Zlopasa, J.; Picken, S.J.

    2015-01-01

    The present invention is in the field of a composition for forming a bio-compatible membrane applicable to building material, such as concrete, cement, etc., to a meth od of applying said composition for forming a bio-compatible membrane, a biocompatible membrane, use of said membrane for various

  11. Production of alkaline proteases by alkalophilic Bacillus subtilis ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-11-23

    Nov 23, 2016 ... detergent industries (Moon and Parulekar, 1991). Proteases of commercial importance are produced from microbial, animal and plant sources (Patel, 1985). Almost all living organisms can produce alkaline protease at 32 to 45°C and pH 8 to 9 (Akcan and Uyar, 2011). Microbial proteases are produced ...

  12. Optimization of alkaline protease production from Bacillus subtilis ...

    African Journals Online (AJOL)

    Optimization of the strain revealed that the most suitable nitrogen source to enhance protease production was beef extract. Among various carbon sources tested, maximum production of protease was registered in medium with added glucose. The effect of metals ions indicated that maximum protease production was ...

  13. Production dynamics of extracellular protease from Bacillus species ...

    African Journals Online (AJOL)

    ... showed that Bacillus species under study are good producers of extracellular protease at high temperature. This might be an indication that proteases produced would be thermostable. Keywords Protease; proteolytic bacteria; Bacillus macerans; Bacillus licheniformis; Bacillus subtilis. African Journal of Biotechnology Vol.

  14. Purification and characterization of a protease from Thermophilic ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-10-19

    Oct 19, 2006 ... strain HS08, originally isolated from a soil sample collected from the Tulufan Crater of China, is .... Effect of temperature and pH on protease activity ... pH. To test the thermostability of the protease, the purified protease was incubated at various temperatures ranging from 40 to 80°C for. 10 to 60 min, then ...

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

    Directory of Open Access Journals (Sweden)

    Ramesh Tati

    2017-02-01

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

  16. tolerant alkaline protease from Bacillus coagulans PSB

    African Journals Online (AJOL)

    oyaide

    2013-05-22

    May 22, 2013 ... optimum activity at 60°C and pH 8.0 with casein as substrate. The enzyme was .... appropriate buffers. 50 mM of buffer solutions (sodium citrate, pH .... Table 2. Hydrolysis of protein substrates by protease from Bacillus coagulans PSB-07. Substrate. Relative activity (%). Casein. 100. Gelatin. 18. BSA. 72.

  17. HIV-1 protease-induced apoptosis

    Czech Academy of Sciences Publication Activity Database

    Rumlová, Michaela; Křížová, Ivana; Keprová, Alena; Hadravová, Romana; Doležal, Michal; Strohalmová, Karolína; Pichová, Iva; Hájek, Miroslav; Ruml, T.

    2014-01-01

    Roč. 11, May 20 (2014), 37/1-37/15 ISSN 1742-4690 R&D Projects: GA ČR GA204/09/1388 Institutional support: RVO:61388963 Keywords : HIV protease * BCA3 * AKIP-1 * apoptosis * mitochondria Subject RIV: EE - Microbiology, Virology Impact factor: 4.185, year: 2014 http://www.retrovirology.com/content/11/1/37

  18. Protease inhibitor mediated resistance to insects

    NARCIS (Netherlands)

    Outchkourov, N.S.

    2003-01-01

    Protease inhibitors (PIs) are among the defensive molecules that plants produce in order to defend themselves against herbivores. A major aim of this thesis is to develop novel insect resistance traits usingheterologous, non-plant PIs. Prerequisite for the success of the

  19. Protease inhibitor (Pi) locus, fertility and twinning

    NARCIS (Netherlands)

    Boomsma, D.I.; Frants, R.R.; Bank, R.A.; Martin, N.G.

    1992-01-01

    In a sample of 160 Dutch twin pairs and their parents, we found that mothers of dizygotic twins had frequencies of the S and Z alleles at the protease inhibitor (Pi) locus that were 3 times higher than a control sample. Mothers of identical twins also had a higher frequency of S than controls. The S

  20. Inhibition of HIV protease by monoclonal antibodies

    Czech Academy of Sciences Publication Activity Database

    Řezáčová, Pavlína; Brynda, Jiří; Fábry, Milan; Hořejší, Magdalena; Štouračová, Renata; Lescar, J.; Riottot, M. M.; Sedláček, Juraj; Bentley, G. A.

    15(5), č. 15 (2002), s. 272-276 ISSN 0952-3499 R&D Projects: GA AV ČR IAA5052502; GA ČR GV203/98/K023 Institutional research plan: CEZ:AV0Z5052915 Keywords : monoclonal antibodies * HIV protease * crystal structure Subject RIV: CE - Biochemistry Impact factor: 2.838, year: 2002

  1. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    Katharina Janek

    2016-09-01

    Full Text Available The data provide information in support of the research article, “The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors” (Janek et al., 2016 [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. Keywords: Aspartic protease, Cleavage sites, Cocoa, In-vitro proteolysis, Mass spectrometry, Peptides

  2. Proteases of Sporothrix schenckii: Cytopathological effects on a host-cell model.

    Science.gov (United States)

    Sabanero López, Myrna; Flores Villavicencio, Lérida L; Soto Arredondo, Karla; Barbosa Sabanero, Gloria; Villagómez-Castro, Julio César; Cruz Jiménez, Gustavo; Sandoval Bernal, Gerardo; Torres Guerrero, Haydee

    Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. To evaluate the proteolytic activity of S. schenckii on epithelial cells. The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors. Copyright © 2017 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

  3. Protease-resistant prions selectively decrease Shadoo protein.

    Directory of Open Access Journals (Sweden)

    Joel C Watts

    2011-11-01

    Full Text Available The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C into PrP(Sc, a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho, a protein that resembles the flexibly disordered N-terminal domain of PrP(C, were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc. Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc. Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc during prion disease.

  4. 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.

  5. Emerging principles in protease-based drug discovery.

    Science.gov (United States)

    Drag, Marcin; Salvesen, Guy S

    2010-09-01

    Proteases have an important role in many signalling pathways, and represent potential drug targets for diseases ranging from cardiovascular disorders to cancer, as well as for combating many parasites and viruses. Although inhibitors of well-established protease targets such as angiotensin-converting enzyme and HIV protease have shown substantial therapeutic success, developing drugs for new protease targets has proved challenging in recent years. This in part could be due to issues such as the difficulty of achieving selectivity when targeting protease active sites. This Perspective discusses the general principles in protease-based drug discovery, highlighting the lessons learned and the emerging strategies, such as targeting allosteric sites, which could help harness the therapeutic potential of new protease targets.

  6. Pathophysiological significance and therapeutic applications of snake venom protease inhibitors.

    Science.gov (United States)

    Thakur, Rupamoni; Mukherjee, Ashis K

    2017-06-01

    Protease inhibitors are important constituents of snake venom and play important roles in the pathophysiology of snakebite. Recently, research on snake venom protease inhibitors has provided valuable information to decipher the molecular details of various biological processes and offer insight for the development of some therapeutically important molecules from snake venom. The process of blood coagulation and fibrinolysis, in addition to affecting platelet function, are well known as the major targets of several snake venom protease inhibitors. This review summarizes the structure-functional aspects of snake venom protease inhibitors that have been described to date. Because diverse biological functions have been demonstrated by protease inhibitors, a comparative overview of their pharmacological and pathophysiological properties is also highlighted. In addition, since most snake venom protease inhibitors are non-toxic on their own, this review evaluates the different roles of individual protease inhibitors that could lead to the identification of drug candidates and diagnostic molecules. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. A Multifunctional Protease Inhibitor To Regulate Endolysosomal Function

    Science.gov (United States)

    2011-01-01

    Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin–pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses. PMID:21910425

  8. Microbial alkaline proteases: Optimization of production parameters and their properties

    Directory of Open Access Journals (Sweden)

    Kanupriya Miglani Sharma

    2017-06-01

    Full Text Available Proteases are hydrolytic enzymes capable of degrading proteins into small peptides and amino acids. They account for nearly 60% of the total industrial enzyme market. Proteases are extensively exploited commercially, in food, pharmaceutical, leather and detergent industry. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. This review summarizes a fraction of the enormous reports available on various aspects of alkaline proteases. Diverse sources for isolation of alkaline protease producing microorganisms are reported. The various nutritional and environmental parameters affecting the production of alkaline proteases in submerged and solid state fermentation are described. The enzymatic and physicochemical properties of alkaline proteases from several microorganisms are discussed which can help to identify enzymes with high activity and stability over extreme pH and temperature, so that they can be developed for industrial applications.

  9. Site-SpecificCu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N), Using Copper Free Click Chemistry

    DEFF Research Database (Denmark)

    Jeppesen, Troels E; Kristensen, Lotte K; Nielsen, Carsten H

    2018-01-01

    A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migrati...

  10. Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: evidence of biogenic membrane flippase in plants.

    Science.gov (United States)

    Sahu, Santosh Kumar; Gummadi, Sathyanarayana N

    2008-09-30

    Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.

  11. Membranous nephropathy

    Science.gov (United States)

    ... check for hepatitis B, hepatitis C, and syphilis Complement levels Cryoglobulin test Treatment The goal of treatment ... not as helpful for people with membranous nephropathy. Medicines used treat membranous nephropathy include: Angiotensin-converting enzyme ( ...

  12. Membrane compartmentalization of melanosomal gp75.

    Science.gov (United States)

    Giacomini, P; Fraioli, R; Cuomo, M; Natali, P G

    1992-03-01

    A melanosomal integral membrane glycoprotein of 75 kD (gp75) has been previously identified as the human homologue of the product specified by the murine brown locus. We presently report that this molecule may be susceptible to limited proteolysis and extrinsic radioiodination in intact, live cells. Consequently, it is suggested that its cellular location might include the plasma membrane and/or a cellular compartment easily accessible to proteases and to chemically catalyzed vectorial iodination. This is of interest in view of the potential applicative value of gp75 as a target for the radioimmunoscintography of melanoma lesions.

  13. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

    This thesis describes the design and synthesis of peptide-based serine protease inhibitors. The targeted protease, urokinase-type plasminogen activator (uPA) activates plasminogen, which plays a major role in cancer metastasis. The peptide upain-2 (S 1 ,S 12-cyclo-AcCSWRGLENHAAC-NH2) is a highly...... specific inhibitor of uPA. With the aim of creating better inhibitors based on the upain-2 scaffold, the following three strategies were explored: First, it was attempted to predefine the structure of upain-2 in solution by incorporating turn-inducing sequences and peptidomimetics. Additionally...... bond across the ring. The second bridge was made by a disulfide bridge, amide bond formation or via ring-closing metathesis. A, with upain-2 equipotent, bicyclic inhibitor was obtained and its binding to uPA was studied by ITC, NMR and X-ray. The knowledge of how selective inhibitors bind uPA has been...

  14. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

    Directory of Open Access Journals (Sweden)

    Madhusudhan Budatha

    Full Text Available Mice deficient for the fibulin-5 gene (Fbln5(-/- develop pelvic organ prolapse (POP due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/- mice, herein named V1 (25 kDa. V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/- mice. PRSS3 was (a localized in epithelial secretions, (b detected in media of vaginal organ culture from both Fbln5(-/- and wild type mice, and (c cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin and Elafin] was dysregulated in Fbln5(-/- epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

  15. 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.

  16. 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. PMID:24658532

  17. Role of Proteases in Chronic Obstructive Pulmonary Disease

    Directory of Open Access Journals (Sweden)

    Kailash C. Pandey

    2017-08-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is generally associated with progressive destruction of airways and lung parenchyma. Various factors play an important role in the development and progression of COPD, like imbalance of proteases, environmental and genetic factors and oxidative stress. This review is specifically focused on the role of proteases and their imbalance in COPD. There are three classes (serine, mettalo, and cysteine of proteases involved in COPD. In serine proteases, neutrophil elastase, cathepsin G, and proteinase-3 are involved in destruction of alveolar tissue. Matrix-mettaloproteinase-9, 12, 13, plays an influential role in severity of COPD. Among cysteine proteases, caspase-3, caspases-8 and caspase-9 play an important role in controlling apoptosis. These proteases activities can be regulated by inhibitors like α-1-antitrypsin, neutrophil elastase inhibitor, and leukocyte protease inhibitor. Studies suggest that neutrophil elastase may be a therapeutic target for COPD, and specific inhibitor against this enzyme has potential role to control the disease. Current study suggests that Dipeptidyl Peptidase IV is a potential marker for COPD. Since the expression of proteases and its inhibitors play an important role in COPD pathogenesis, therefore, it is worth investigating the role of proteases and their regulation. Understanding the biochemical basis of COPD pathogenesis using advanced tools in protease biochemistry and aiming toward translational research from bench-to-bedside will have great impact to deal with this health problem.

  18. PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

    Directory of Open Access Journals (Sweden)

    R. Satheeskumar

    2013-10-01

    Full Text Available Partial purification and characterization of alkalophilic protease production from Pseudomonas aeruginosa was isolated from the gut of marine and coastal waters shrimp Penaeus monodon. The protease production was assayed in submerged fermentation to produce maximum protease activity (423 ± 0.09 U/ml. The enzyme was precipitated with ammonium sulphate and partially purified by ion exchange chromatography through DEAE Sephadex A-50 column. In 10th fraction showed maximum protease activity (734 ± 0.18 U/ml with increase in purification fold. The molecular weight of protease from Pseudomonas aeruginosa was recorded as 60 kDa. The stability of protease was tested at various pH and temperature; it showed maximum protease activity at pH-9 and temperature 50ºC. Among the various surfactants tested for enzyme stability, maximum activity was retained in poly ethylene glycol. The compatibility of protease enzyme with various commercial detergents; the enzyme retained maximum protease activity in tide. The results are indicated that all these properties make the bacterial proteases are most suitable for wide industrial applications.

  19. Corruption of innate immunity by bacterial proteases.

    Science.gov (United States)

    Potempa, Jan; Pike, Robert N

    2009-01-01

    The innate immune system of the human body has developed numerous mechanisms to control endogenous and exogenous bacteria and thus prevent infections by these microorganisms. These mechanisms range from physical barriers such as the skin or mucosal epithelium to a sophisticated array of molecules and cells that function to suppress or prevent bacterial infection. Many bacteria express a variety of proteases, ranging from non-specific and powerful enzymes that degrade many proteins involved in innate immunity to proteases that are extremely precise and specific in their mode of action. Here we have assembled a comprehensive picture of how bacterial proteases affect the host's innate immune system to gain advantage and cause infection. This picture is far from being complete since the numbers of mechanisms utilized are as astonishing as they are diverse, ranging from degradation of molecules vital to innate immune mechanisms to subversion of the mechanisms to allow the bacterium to hide from the system or take advantage of it. It is vital that such mechanisms are elucidated to allow strategies to be developed to aid the innate immune system in controlling bacterial infections.

  20. Corruption of Innate Immunity by Bacterial Proteases

    Science.gov (United States)

    Potempa, Jan; Pike, Robert N.

    2009-01-01

    The innate immune system of the human body has developed numerous mechanisms to control endogenous and exogenous bacteria and thus prevent infections by these microorganisms. These mechanisms range from physical barriers such as the skin or mucosal epithelium to a sophisticated array of molecules and cells that function to suppress or prevent bacterial infection. Many bacteria express a variety of proteases, ranging from non-specific and powerful enzymes that degrade many proteins involved in innate immunity to proteases that are extremely precise and specific in their mode of action. Here we have assembled a comprehensive picture of how bacterial proteases affect the host’s innate immune system to gain advantage and cause infection. This picture is far from being complete since the numbers of mechanisms utilized are as astonishing as they are diverse, ranging from degradation of molecules vital to innate immune mechanisms to subversion of the mechanisms to allow the bacterium to hide from the system or take advantage of it. It is vital that such mechanisms are elucidated to allow strategies to be developed to aid the innate immune system in controlling bacterial infections. PMID:19756242

  1. Membrane Biophysics

    CERN Document Server

    Ashrafuzzaman, Mohammad

    2013-01-01

    Physics, mathematics and chemistry all play a vital role in understanding the true nature and functioning of biological membranes, key elements of living processes. Besides simple spectroscopic observations and electrical measurements of membranes we address in this book the phenomena of coexistence and independent existence of different membrane components using various theoretical approaches. This treatment will be helpful for readers who want to understand biological processes by applying both simple observations and fundamental scientific analysis. It provides a deep understanding of the causes and effects of processes inside membranes, and will thus eventually open new doors for high-level pharmaceutical approaches towards fighting membrane- and cell-related diseases.

  2. The Clp protease system is required for copper ion-dependent turnover of the PAA2/HMA8 copper transporter in chloroplasts.

    Science.gov (United States)

    Tapken, Wiebke; Kim, Jitae; Nishimura, Kenji; van Wijk, Klaas J; Pilon, Marinus

    2015-01-01

    The distribution of essential metal ions over subcellular compartments for use as cofactors requires control of membrane transporters. PAA2/HMA8 is a copper-transporting P1B -type ATPase in the thylakoid membrane, required for the maturation of plastocyanin. When copper is highly available to the plant this transporter is degraded, which implies the action of a protease. In order to identify the proteolytic machinery responsible for PAA2/HMA8 turnover in Arabidopsis, mutant lines defective in five different chloroplast protease systems were analyzed. Plants defective in the chloroplast caseinolytic protease (Clp) system were specifically impaired in PAA2/HMA8 protein turnover on media containing elevated copper concentrations. However, the abundance of a core Clp component was not directly affected by copper. Furthermore, the expression and activity of both cytosolic and chloroplast-localized superoxide dismutases (SODs), which are known to be dependent on copper, were not altered in the clp mutants, indicating that the loss of PAA2/HMA8 turnover in these lines was not caused by a lack of stromal copper. The results suggest that copper excess in the stroma triggers selection of the thylakoid-localized PAA2 transporter for degradation by the Clp protease, but not several other chloroplast proteases, and support a novel role for this proteolytic system in cellular copper homeostasis. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  4. Bio-Inspired Polymer Membrane Surface Cleaning

    Directory of Open Access Journals (Sweden)

    Agnes Schulze

    2017-03-01

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

  5. Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

    Directory of Open Access Journals (Sweden)

    Thomas A Prohaska

    Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

  6. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

    Directory of Open Access Journals (Sweden)

    Olivier Barré

    Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

  7. Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

    International Nuclear Information System (INIS)

    Watanabe, Yoshihisa; Okui, Akira; Mitsui, Shinichi; Kawarabuki, Kentaro; Yamaguchi, Tatsuyuki; Uemura, Hidetoshi; Yamaguchi, Nozomi

    2004-01-01

    We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression

  8. A look inside HIV resistance through retroviral protease interaction maps.

    Directory of Open Access Journals (Sweden)

    Aleksejs Kontijevskis

    2007-03-01

    Full Text Available Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular-chemical mechanisms involved in substrate cleavage by retroviral proteases.

  9. Magnesium Enhanced Fibrinolytic Activity of Protease from Schizophyllum commune

    Directory of Open Access Journals (Sweden)

    Chung-Lun Lu

    2010-06-01

    Full Text Available Prevention and therapy of thrombotic diseases have attracted much attention in developed countries during recent years. Investigators have been looking for cheaper and safer thrombolytic agents for therapy of thrombotic diseases. Recently, we have discovered a fibrinolytic protease from Schizophyllum commune. In this study, the protease was proven to degrade blood clot effectively. Seven divalent metal ions were used to test the selectiveness on enhancing protease activity. The treated rat blood was traced by thromboelastography to assess the viscoelastic properties of whole blood. As the result, fibrinolytic activity of the protease was enhanced remarkably by Mg2+ in reducing the strength of blood clot and showed the innovative anti-thrombotic effects. This is the first study of anti-thrombotic effects from fungal-derived fibrinolytic protease using thromboelastography and delineates the efficacy of magnesium supplementation in enhancement of thrombolytic activity from S. commune fibrinolytic protease.

  10. Erwinia carotovora extracellular proteases : characterization and role in soft rot

    OpenAIRE

    Kyöstiö, Sirkka R. M.

    1990-01-01

    Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene enc...

  11. Microbial alkaline proteases: Optimization of production parameters and their properties

    OpenAIRE

    Kanupriya Miglani Sharma; Rajesh Kumar; Surbhi Panwar; Ashwani Kumar

    2017-01-01

    Proteases are hydrolytic enzymes capable of degrading proteins into small peptides and amino acids. They account for nearly 60% of the total industrial enzyme market. Proteases are extensively exploited commercially, in food, pharmaceutical, leather and detergent industry. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. This review summarizes a fraction of the enormous repor...

  12. Extracellular protease from the antarctic yeast Candida humicola.

    OpenAIRE

    Ray, M K; Devi, K U; Kumar, G S; Shivaji, S

    1992-01-01

    The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulf...

  13. Recombinant E. coli expressing Vitreoscilla haemoglobin prefers ...

    Indian Academy of Sciences (India)

    2012-08-13

    Aug 13, 2012 ... 5.0 32.2 4.95. −1.9. −2.11. Proteins involved in nucleic acid metabolism. 19 Chain A, Purine nucleoside phosphorylase. deoD. 45. 103. 6/25. 27. 5.6 25.6 5.54. -. −1.57. Proteins involved in cellular transport mechanisms. 20 Outer membrane protein. 3b (a), protease VII. ompT 27. 73. 5/24. 30.5 5.5 35.5 5.55.

  14. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    International Nuclear Information System (INIS)

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-01-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  15. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    Directory of Open Access Journals (Sweden)

    Suk Ho Choi

    2011-09-01

    Full Text Available Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods: The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1 The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2 The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3 Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II chloride inhibited it. 4 The fibrinolytic protease cleaved preferentially A-chain and slowly B-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions: The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

  16. Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

    Energy Technology Data Exchange (ETDEWEB)

    Kocab, S.; Erdem, B. [Middle East Technical University, Ankara (Turkey). Dept. of Biological Sciences

    2002-08-01

    In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70{sup o}C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes. (author)

  17. Comparative analysis of serine protease-related genes in the honey bee genome: possible involvement in embryonic development and innate immunity

    OpenAIRE

    Zou, Z; Lopez, Dawn L; Kanost, Michael R; Evans, Jay D; Jiang, Haobo

    2006-01-01

    We have identified 44 serine protease (SP) and 13 serine protease homolog (SPH) genes in the genome of Apis mellifera. Most of these genes encode putative secreted proteins, but four SPs and three SPHs may associate with the plasma membrane via a transmembrane region. Clip domains represent the most abundant non-catalytic structural units in these SP-like proteins −12 SPs and six SPHs contain at least one clip domain. Some of the family members contain other modules for protein–protein intera...

  18. Complexity of cancer protease biology: Cathepsin K expression and function in cancer progression

    NARCIS (Netherlands)

    Verbovšek, Urška; van Noorden, Cornelis J. F.; Lah, Tamara T.

    2015-01-01

    Proteases, including lysosomal cathepsins, are functionally involved in many processes in cancer progression from its initiation to invasion and metastatic spread. Only recently, cathepsin K (CatK), the cysteine protease originally reported as a collagenolytic protease produced by osteoclasts,

  19. Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

    Directory of Open Access Journals (Sweden)

    Nicole Hofmann

    Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

  20. Host Plants Indirectly Influence Plant Virus Transmission by Altering Gut Cysteine Protease Activity of Aphid Vectors*

    Science.gov (United States)

    Pinheiro, Patricia V.; Ghanim, Murad; Rebelo, Ana Rita; Santos, Rogerio S.; Orsburn, Benjamin C.; Gray, Stewart

    2017-01-01

    The green peach aphid, Myzus persicae, is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops. PMID:27932519

  1. Processing by rhomboid protease is required for Providencia stuartii TatA to interact with TatC and to form functional homo-oligomeric complexes

    OpenAIRE

    Fritsch, Maximilian J; Krehenbrink, Martin; Tarry, Michael J; Berks, Ben C; Palmer, Tracy

    2012-01-01

    The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. In Escherichia coli three membrane proteins, TatA, TatB and TatC, are essential components of the machinery. TatA from Providencia stuartii is homologous to E. coli TatA but is synthesized as an inactive pre-protein with an N-terminal extension of eight amino acids. Removal of this extension by the rhomboid protease AarA is required to activate P. s...

  2. 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

    ) 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......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...

  3. Membrane paradigm

    International Nuclear Information System (INIS)

    Price, R.H.; Thorne, K.S.

    1986-01-01

    The membrane paradigm is a modified frozen star approach to modeling black holes, with particles and fields assuming a complex, static, boundary-layer type structure (membrane) near the event horizon. The membrane has no effects on the present or future evolution of particles and fields above itself. The mathematical representation is a combination of a formalism containing terms for the shear and bulk viscosity, surface pressure, momentum, temperature, entropy, etc., of the horizon and the 3+1 formalism. The latter model considers a family of three-dimensional spacelike hypersurfaces in one-dimensional time. The membrane model considers a magnetic field threading the hole and undergoing torque from the hole rotation. The field is cleaned by the horizon and distributed over the horizon so that ohmic dissipation is minimized. The membrane paradigm is invalid inside the horizon, but is useful for theoretically probing the properties of slowly evolving black holes

  4. Membrane processes

    Science.gov (United States)

    Staszak, Katarzyna

    2017-11-01

    The membrane processes have played important role in the industrial separation process. These technologies can be found in all industrial areas such as food, beverages, metallurgy, pulp and paper, textile, pharmaceutical, automotive, biotechnology and chemical industry, as well as in water treatment for domestic and industrial application. Although these processes are known since twentieth century, there are still many studies that focus on the testing of new membranes' materials and determining of conditions for optimal selectivity, i. e. the optimum transmembrane pressure (TMP) or permeate flux to minimize fouling. Moreover the researchers proposed some calculation methods to predict the membrane processes properties. In this article, the laboratory scale experiments of membrane separation techniques, as well their validation by calculation methods are presented. Because membrane is the "heart" of the process, experimental and computational methods for its characterization are also described.

  5. Karakterisasi protease Bacillus sp. UGM5

    Directory of Open Access Journals (Sweden)

    Titik Purwati Widowati

    1999-07-01

    Full Text Available The objective of this experiment is to indentify the characters of proetease produced by Bacillus sp.UGM5.the protease secreted by Bacillus sp.UGM5 was first isolated,purified and then charactirezed.The crude enzyme has spesific actifity of 1.14 U/mg,however,the spesific activity of purified enzyme was increased by 23.8 times fold and recovery was 33.69%.The Page of nondenatured crude enzymes showes two type of proreases,however ,the SDS-Page of denatured purified enzyme showed four protein-bends with molecular weights of 55.5 kDa,18kDa respecetively.The optimum pH and temperature for the enzyme acrivity are 8.5 and 420C and belongs to serin protease type,with Km 3 X 10-3mM and Vmax 0.0890mM/30 minutes.The activity is not inhibited by Ca+2,Fe+2 and EDTA.

  6. Lopinavir-Ritonavir: a new protease inhibitor.

    Science.gov (United States)

    Mangum, E M; Graham, K K

    2001-11-01

    Lopinavir is a new protease inhibitor that is structurally related to ritonavir. It recently was approved by the Food and Drug Administration as a coformulation with ritonavir under the brand name Kaletra. Ritonavir substantially increases lopinavir drug exposure by inhibiting cytochrome P450 isoenzyme 3A4. Based on limited data, lopinavir-ritonavir demonstrates safety and efficacy in both antiretroviral-naive and protease inhibitor-experienced patients. It has the ability to durably suppress human immunodeficiency virus (HIV) RNA for up to 2 years in antiretroviral-naïve patients. Compared with nelfinavir, it had superior virologic control at 48 weeks in antiretroviral-naïve patients. Its side effects include diarrhea, abnormal stools, abdominal pain, nausea, vomiting, and asthenia. A number of patients experienced grade 3-4 laboratory abnormalities in liver function tests, cholesterol, and triglycerides while receiving this drug combination. The exact resistance patterns of lopinavir-ritonavir are unknown, but the Department of Health and Human Services strongly recommends it for the initial treatment of HIV-infected adults and adolescents.

  7. Peptide synthesis in neat organic solvents with novel thermostable proteases

    NARCIS (Netherlands)

    Toplak, Ana; Nuijens, Timo; Quaedflieg, Peter J L M; Wu, Bian; Janssen, Dick B

    Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the

  8. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Science.gov (United States)

    TECHNICAL ABSTRACT A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated the enzyme had...

  9. Focal Cerebral Ischemia Induces Active Proteases That Degrade Microvascular Matrix

    Science.gov (United States)

    Fukuda, Shunichi; Fini, Catherine A.; Mabuchi, Takuma; Koziol, James A.; Eggleston, Leonard L.; del Zoppo, Gregory J.

    2010-01-01

    Background and Purpose Focal cerebral ischemia causes microvessel matrix degradation and generates proteases known to degrade this matrix. However, proof that the proteases generated do indeed degrade vascular matrix is lacking. Here we demonstrate that active proteases derived from ischemic tissue after middle cerebral artery occlusion (MCAO) and transferred to normal tissue can degrade vascular matrix. Methods In an ex vivo bioassay, the effects of supernatants from ischemic and normal basal ganglia of nonhuman primates, proteases, and control buffer on the immunoreactivity of vascular matrix constituents in normal brain tissue sections were quantified. Protease families were identified with specific inhibitors. Results Plasmin, active matrix metalloproteinase (MMP)-2, and active MMP-9 significantly reduced microvessel-associated collagen, laminin, and heparan sulfate proteoglycans (HSPG). The vascular HSPG perlecan was more sensitive than collagen or laminin in the bioassay and in the ischemic core 2 hours after MCAO. Two-hour and 7-day ischemic tissue samples significantly degraded matrix perlecan and collagen. Inhibitor studies confirmed that while active MMPs were generated, active cysteine proteases significantly degraded microvessel perlecan. The cysteine proteases cathepsins B and L were generated in the microvasculature and adjacent neurons or glial cells 2 hours after MCAO and decreased perlecan in the bioassay. Conclusions This is the first direct evidence that active proteases are generated in ischemic cerebral tissues that are acutely responsible for vascular matrix degradation. Degradation of vascular perlecan, the most sensitive matrix component thus far identified, may be due to cathepsins B and L, generated very rapidly after MCAO. PMID:15001799

  10. Localisation of a multicatalytic protease complex in Trypanosoma ...

    African Journals Online (AJOL)

    Recently, a multicatalytic protease complex, the proteasome, has also become of interest because of its potential involvement in cell-cycle development. The proteasome is the central protease of the non lysosomal ubiquitin dependent pathway of protein degradation. Here we report the subcellular localization of 20S ...

  11. Acidic extracellular proteases from microrganisms of fairly acidic niche.

    Science.gov (United States)

    Bossi, Alessandra; Bonizzato, Luca; Zapparoli, Giacomo

    2006-01-01

    The protein population secreted from three wine-contaminant microorganisms was studied by two-dimensional electrophoresis and screened for proteases. Proteolytic enzymes were electrophoretically purified and their activity, optimum pH and temperature determined. The protease of Acetobacter aceti maintained its activity over the range of pHs 3.0-5.0, thus being of potential biotechnological interest.

  12. Partial purification and characterization of alkaline proteases from ...

    African Journals Online (AJOL)

    Alkaline proteases from the digestive tract of anchovy were partially purified by ammonium sulfate fractionation, dialysis and Sephadex G-75 gel filtration. The purification fold and yield were 6.23 and 4.49%, respectively. The optimum activities of partially purified alkaline proteases were observed at 60°C and at pH 11.0.

  13. Cold denaturation of the HIV-1 protease monomer

    DEFF Research Database (Denmark)

    Rösner, Heike Ilona; Caldarini, Martina; Prestel, Andreas

    2017-01-01

    The HIV-1-protease is a complex protein which in its active form adopts a homodimer dominated by -sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1-protease which is populated above 0ºC and therefore directly accessible to various spectroscopic...

  14. Extracellular protease produced by Bacillus subtilis isolated from ...

    African Journals Online (AJOL)

    In a study to evaluate the microbiological safety of some paracetamol oral solutions sold in some Nigerian drug stores, 40.0% of the samples examined was contaminated with protease-producing Bacillus subtilis. The production of extracellular protease was induced by casein in the minimal medium and was found to be the ...

  15. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    During germination of barley seeds, the mobilization of protein is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction of the...

  16. A Protease Isolated from the Latex of Plumeria rubra Linn ...

    African Journals Online (AJOL)

    Purpose: To isolate, purify and characterize protease from the latex of the plant. Methods: Protease was isolated from the latex of Plumeria rubra Linn using acetone precipitation method and purified by a sequence of DEAE cellulose column chromatography, followed by two successive column purification in Sephadex G-50 ...

  17. Protease-induced solubilisation of carbohydrates from brewers' spent grain

    NARCIS (Netherlands)

    Faulds, C.B.; Collins, S.; Robertson, J.A.; Treimo, J.; Eijsink, V.G.H.; Hinz, S.W.A.; Schols, H.A.; Buchert, J.; Waldron, K.W.

    2009-01-01

    The impact of microbial proteases on the release of carbohydrates from BSG was studied. The proteases were able to release the non-cellulosic glucose, a portion of feruloylated arabinoxylan and over 50% of the protein from brewers' spent grain (BSG) after 24 h hydrolysis. The non-cellulosic glucose

  18. Cloning and characterization of a novel cysteine protease gene ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Latex has been reported to contain a number of proteins such as protease-like enzymes (Arima et al 2000;. Arribere et al 1998), chitinase (Haque and Bradbury 1999;. Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis. SHI-QING PENG. 1,#,*, JIA-HONG ZHU. 1,#, HUI-LIANG LI.

  19. Reaction mechanism of O-acylhydroxamate with cysteine proteases

    Indian Academy of Sciences (India)

    WINTEC

    such as, cardiovascular, oncology, osteoporosis, and arthritis. Till now, twenty one families of cysteine proteases have been discovered5, almost half of them are found in viruses and others are found in bacteria, fungi, pro- tozoa and plants. In mammals, two main groups of cysteine proteases are present: cytosolic calpains.

  20. Improvement of acid protease production by a mixed culture of ...

    African Journals Online (AJOL)

    The synthesis of acid protease by Aspergillus oryzae AS3042 was enhanced significantly with the mixed culture of Aspergillus niger SL-09 using solid-state fermentation technique. The influence of carbon sources, nitrogen sources and the addition of phytic acid on acid protease production were investigated. The enzyme ...

  1. Comparison of protease production from newly isolated bacterial ...

    African Journals Online (AJOL)

    Fermentation medium (by using sub-merged fermentation technique) was incubated for 48 h at 37°C temperature and agitation speed of 200 rpm. The protease was partially purified with 70% ammonium sulphate. Four different supports were used for the immobilization of the bacterial protease by physical adsorption ...

  2. Purification of an Intracellular Fibrinolytic Protease from Ganoderma ...

    African Journals Online (AJOL)

    Erah

    Method: The intracellular fibrinolytic protease produced by Ganoderma lucidum VK12 was isolated from the mycelia grown in MCDBF broth ... The inhibitory effect of different metal ions and commercial protease inhibitors on enzyme activity was studied. ... sodium hydroxide and 2.9 %w/v sodium carbonate in glass-distilled ...

  3. Some physicochemical properties of acid protease produced during ...

    African Journals Online (AJOL)

    Acid protease synthesis by Aspergillus niger increased in direct proportion to growth up to the early hours of the death phase. Maximum enzyme synthesis was obtained at the 96th hour of fermentation, which was 12 hours into the death phase. The acid protease synthesized had optimum pH of 4.0, while the enzyme was ...

  4. High-level expression of alkaline protease using recombinant ...

    African Journals Online (AJOL)

    AJL

    2012-02-16

    Feb 16, 2012 ... compared with that of wild-type B. licheniformis CICIM B5102. Key word: Alkaline protease, Bacillus amyloliquefaciens, Bacillus licheniformis. INTRODUCTION. Proteases are one of the most important industrial enzyme groups, accounting for approximately 60% of the total enzyme sales (Beg et al., 2003).

  5. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned...

  6. Isolation of protease producing novel Bacillus cereus and detection ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... The highest protease activity was determined at 30°C temperature and 6.4 pH conditions and after the 18th hour, it decreased evidently. Key words: Protease, production, optimization, Bacillus sp. INTRODUCTION. Enzymes have been produced in large industrial scale for several decades (Falch, 1991).

  7. Production of alkaline proteases by alkalophilic Bacillus subtilis ...

    African Journals Online (AJOL)

    Among various nitrogen sources, yeast extract was found to be the best inducer of alkaline protease. Among metal salts, KNO3 and NH4Cl were found to increase protease production. The maximum enzyme production (3600 U/ml) was observed with pomegranate peels of fermentation medium in the presence of yeast ...

  8. Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores.

    Science.gov (United States)

    Hagerman, A E; Blau, D M; McClure, A L

    1985-12-01

    A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.

  9. Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease.

    Science.gov (United States)

    Hizukuri, Y; Akiyama, K; Akiyama, Y

    2017-01-01

    Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metalloprotease family of I-CLiPs, plays an essential role in activation of a cell envelope stress response through cleavage of anti-σ E protein RseA, a single-span transmembrane protein. A recent study showed that it also cleaves remnant signal peptides generated upon membrane translocation of secretory proteins. Here, we describe several methods for characterization of the proteolytic functions and structure of RseP mainly in vivo, including a proteolytic activity assay using model substrates, an in vitro analysis of cleavage of signal peptides in a detergent solution and in the membrane vesicles, structural analysis of membrane-embedded RseP based on the thiol modifiability of introduced cysteine residues, and the protein interaction analysis by in vivo cross-linking protocols. © 2017 Elsevier Inc. All rights reserved.

  10. Alkaline protease production by a strain of marine yeasts

    Science.gov (United States)

    Ping, Wang; Zhenming, Chi; Chunling, Ma

    2006-07-01

    Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45°C. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5g soluble starch and 2.0g NaNO3 in 100mL seawater with initial pH 6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5°C, aeration rate 8.0L min-1 and agitation speed 150r min-1 Under the optimal conditions, 623.1 U mg-1 protein of alkaline protease was reached in the culture within 30h of fermentation.

  11. Cross genome comparisons of serine proteases in Arabidopsis and rice

    Directory of Open Access Journals (Sweden)

    Sowdhamini R

    2006-08-01

    Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

  12. Positive selection of digestive Cys proteases in herbivorous Coleoptera.

    Science.gov (United States)

    Vorster, Juan; Rasoolizadeh, Asieh; Goulet, Marie-Claire; Cloutier, Conrad; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Primordial membranes

    DEFF Research Database (Denmark)

    Hanczyc, Martin M; Monnard, Pierre-Alain

    2017-01-01

    Cellular membranes, which are self-assembled bilayer structures mainly composed of lipids, proteins and conjugated polysaccharides, are the defining feature of cell physiology. It is likely that the complexity of contemporary cells was preceded by simpler chemical systems or protocells during...... the various evolutionary stages that led from inanimate to living matter. It is also likely that primitive membranes played a similar role in protocell 'physiology'. The composition of such ancestral membranes has been proposed as mixtures of single hydrocarbon chain amphiphiles, which are simpler versions...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-12-02

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

  15. From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

    Czech Academy of Sciences Publication Activity Database

    Cígler, Petr; Kožíšek, Milan; Řezáčová, Pavlína; Brynda, Jiří; Otwinowski, Z.; Pokorná, Jana; Plešek, Jaromír; Grüner, Bohumír; Marešová, Lucie; Máša, Martin; Sedláček, Juraj; Bodem, J.; Kräusslich, H. G.; Král, V.; Konvalinka, Jan

    2005-01-01

    Roč. 102, č. 43 (2005), s. 15394-15399 ISSN 0027-8424 R&D Projects: GA MŠk(CZ) 1M0508; GA MŠk(CZ) LC523 Grant - others:5th Framework(XE) QLK2-CT-2001-02360 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z40320502 Keywords : carboranes * HIV protease * X-ray structure Subject RIV: CE - Biochemistry Impact factor: 10.231, year: 2005

  16. Characterization of a secreted Chlamydia protease

    DEFF Research Database (Denmark)

    Shaw, A.C.; Vandahl, B.B.; Larsen, M.R.

    2002-01-01

    Chlamydiae are obligate intracellular bacteria that are important human pathogens. The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified. Most likely, effector proteins are secreted in order...... to promote infection. Effector proteins cannot be identified by motif or similarity searches. As a new strategy for identification of secreted proteins we have compared 2D-PAGE profiles of [35S]-labelled Chlamydia proteins from whole lysates of infected cells to 2D-PAGE profiles of proteins from purified...... Chlamydia. Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method. Two protein spots were identified among the candidates. These represent fragments of the 'chlamydial protease- or proteasome-like activity factor' (CPAF) and were clearly present in 2D...

  17. Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation Process

    Directory of Open Access Journals (Sweden)

    Catherine S. Adamson

    2012-01-01

    Full Text Available Protease-mediated maturation of HIV-1 virus particles is essential for virus infectivity. Maturation occurs concomitant with immature virus particle release and is mediated by the viral protease (PR, which sequentially cleaves the Gag and Gag-Pol polyproteins into mature protein domains. Maturation triggers a second assembly event that generates a condensed conical capsid core. The capsid core organizes the viral RNA genome and viral proteins to facilitate viral replication in the next round of infection. The fundamental role of proteolytic maturation in the generation of mature infectious particles has made it an attractive target for therapeutic intervention. Development of small molecules that target the PR active site has been highly successful and nine protease inhibitors (PIs have been approved for clinical use. This paper provides an overview of their development and clinical use together with a discussion of problems associated with drug resistance. The second-half of the paper discusses a novel class of antiretroviral drug termed maturation inhibitors, which target cleavage sites in Gag not PR itself. The paper focuses on bevirimat (BVM the first-in-class maturation inhibitor: its mechanism of action and the implications of naturally occurring polymorphisms that confer reduced susceptibility to BVM in phase II clinical trials.

  18. Identification of covalent active site inhibitors of dengue virus protease

    Directory of Open Access Journals (Sweden)

    Koh-Stenta X

    2015-12-01

    Full Text Available Xiaoying Koh-Stenta,1 Joma Joy,1 Si Fang Wang,1 Perlyn Zekui Kwek,1 John Liang Kuan Wee,1 Kah Fei Wan,2 Shovanlal Gayen,1 Angela Shuyi Chen,1 CongBao Kang,1 May Ann Lee,1 Anders Poulsen,1 Subhash G Vasudevan,3 Jeffrey Hill,1 Kassoum Nacro11Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR, Singapore; 2Novartis Institute for Tropical Diseases, Singapore; 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, SingaporeAbstract: Dengue virus (DENV protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.Keywords: flavivirus protease, small molecule optimization, covalent inhibitor, active site binding, pyrazole ester derivatives

  19. Bacillus amyloliquefaciens SUBSP. plantarum PROBIOTIC STRAINS AS PROTEASE PRODUCERS

    Directory of Open Access Journals (Sweden)

    E. V. Маtseliukh

    2015-04-01

    Full Text Available Proteases from probiotic strains of the genus Bacillus, just like the antibiotics, bacteriocins and other hydrolytic enzymes, are one of the main factors that determine their biological activity. The aim of this work was to study the synthesis and biochemical properties of proteases from two strains Bacillus amyloliquefaciens subsp. plantarum UCM B-5139 and UCM B-5140 that included in the probiotic Endosporin. The cultivation of strains was carried out in flasks under rotating for two days. The influence of physico-chemical parameters of the reaction medium on proteolytic activity was studied on partially purified protease preparations. Lytic activity was determined by turbidimetric method. On the second day of cultivation B. amyloliquefaciens subsp. plantarum UCM В-5139 and UCM В-5140 synthesized the metal-dependent peptidase and serine protease, respectively. The optimum conditions of their action were the following: temperature 37–40 °C and pH 6.5–7.0. Isolated proteases are able to lyse the living cells of Staphylococcus aureus and Candida albicans. Thus we demonstrated that B. amyloliquefaciens subsp. plantarum UCM B-5140 and UCM B-5139, included in the probiotic veterinary preparation Endosporin, produced proteolytic enzymes that hydrolyze the native insoluble proteins (elastin, fibrin and collagen. These enzymes belong to the group of neutral metal-dependent and serine proteases. They are active under physiological conditions against gram-positive bacteria and yeasts. The application of these proteases in biotechnology is considered.

  20. Structure of HIV-1 protease determined by neutron crystallography

    International Nuclear Information System (INIS)

    Adachi, Motoyasu; Kuroki, Ryota

    2009-01-01

    HIV-1 protease is an aspartic protease, and plays an essential role in replication of HIV. To develop HIV-1 protease inhibitors through structure-based drug design, it is necessary to understand the catalytic mechanism and inhibitor recognition of HIV-1 protease. We have determined the crystal structure of HIV-1 protease in complex with KNI-272 to 1.9 A resolution by neutron crystallography in combination with 1.4 A resolution X-ray diffraction data. The results show that the carbonyl group of hydroxymethylcarbonyl (HMC) in KNI-272 forms a hydrogen bonding interaction with protonated Asp 25 and the hydrogen atom from the hydroxyl group of HMC forms a hydrogen bonding interaction with the deprotonated Asp125. This is the first neutron report for HIV-1/inhibitor complex and shows directly the locations of key hydrogen atoms in catalysis and in the binding of a transition-state analog. The results confirm key aspect of the presumed catalytic mechanism of HIV-1 protease and will aid in the further development of protease inhibitors. (author)

  1. Optimizing PHB and Protease Production by Box Behnken Design

    Directory of Open Access Journals (Sweden)

    Amro Abd al fattah Amara

    2013-04-01

    Full Text Available Mixed culture is more suitable to adapt more flexible fermentation process and produce different product simultaneously. In this study a mixed Bacillus culture was investigated for their ability to produce the bioplastic "Polyhydroxybutyrate" and both of the mesophilic and the thermophilic proteases in one flask. Box-Behnken experimental design was used. The produced amount of PHB has been increased significantly. Meanwhile there is a competition between PHB and proteases. The maximum produced amount of PHB using Box-Behnken design was 2.82 g/l/48 h with protease activity equal to 41.9 Units/ml/48 h for thermophilic proteases and 99.65 Units/ml/48 h for mesophilic proteases. Excel solver was used for extra-optimization for the optimum conditions obtained from Box-Behnken experiments and its model. The maximum PHB obtained after using Excel solver was 2.88 g/l/48 h. The maximum mesophilic and thermophilic activities obtained at the same PHB production conditions were 175.68 and 243.38 Units/ml respectively. The model accuracy as obtained from Excel solver was 118.8%, which prove the power of the experimental design in optimizing such complicated process. The strategies used in this study are recommended for the production of PHB and different proteases simultaneously using Bacillus mixed culture. ABSTRAK: Kultur campuran adalah lebih sesuai bagi proses penapaian yang fleksibel dan ia boleh menghasilkan produk yang berbeza secara serentak. Dalam kajian ini keupayaan  menghasilkan "Polyhydroxybutyrate" bioplastik serta mesofilik dan termofilik protease dalam satu flask oleh  kultur Bacillus campuran telah disiasat. Eksperimen rekabentuk Box-Behnken telah digunakan. Jumlah PHB yang dikeluarkan meningkat dengan ketara dan terdapat persaingan antara PHB dan protease. Jumlah keluaran PHB maksima menggunakan rekabentuk Box-Behnken adalah 2.82 g/l/48 jam dengan aktiviti protease sama dengan 41.9 Unit/ml/48 jam untuk protease termofilik dan 99.65 Unit

  2. 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.

  3. Two-Dimensional Zymography of Proteases from Steatotic Duck Liver.

    Science.gov (United States)

    Wilkesman, Jeff; Padrón, María Fernanda; Kurz, Liliana; Rémignon, Hervé

    2017-01-01

    Protease activity present in liver cells with steatosis can be electrophoretically characterized. Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metalloprotease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the determination of both M r. and pI of the metalloprotease and cathepsin activity present in the homogenates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind steatosis development.

  4. Protease-associated cellular networks in malaria parasite Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Lilburn Timothy G

    2011-12-01

    Full Text Available Abstract Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome in the malaria parasite Plasmodium falciparum and its sibling species 123, providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database 4, and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H system 5, blood stage microarray experiments 678, proteomics 9101112, literature text mining, and sequence homology analysis. Seventy-seven (77 out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs. These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins, range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide

  5. In vivo expression of proteases and protease inhibitor, a serpin, by periodontal pathogens at teeth and implants.

    Science.gov (United States)

    Eckert, Martin; Mizgalska, Danuta; Sculean, Anton; Potempa, Jan; Stavropoulos, Andreas; Eick, Sigrun

    2018-03-02

    Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK-proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri-implant health and disease conditions. Biofilm and gingival/peri-implant crevicular fluid (GCF or PISF respectively) samples from healthy tooth and implant sites (n=10), gingivitis and mucositis sites (n=12), and periodontitis and peri-implantitis sites (n=10). Concentration of interleukins (IL)-8, IL-1β and IL-10 in GCF was determined by ELISA. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by qPCR, while genes expression was estimated by qRT-PCR. Presence of P. gingivalis and T. forsythia, as well as the level of IL-8 and IL-1β, were associated with disease severity in the periodontal and peri-implant tissues. In biofilm samples harboring T. forsythia genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK-proteases genes and miropin. At the same time, detectable mRNA expression of individual genes was in the range from 20.7% to 58.6% samples (for forsylisin and miropsin-1, respectively). In comparison to the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK-proteases and especially miropin might play a role in pathogenesis of both periodontal and peri-implant diseases. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

    Science.gov (United States)

    Sharma, Ranu; Suresh, C G

    2015-01-01

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

  7. Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli andIn Vitro.

    Science.gov (United States)

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

    2017-10-01

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

  8. Zebra chip disease decreases tuber (Solanum tuberosum L.) protein content by attenuating protease inhibitor levels and increasing protease activities.

    Science.gov (United States)

    Kumar, G N Mohan; Knowles, Lisa O; Knowles, N Richard

    2015-11-01

    Zebra chip disease of potato decreases protease inhibitor levels resulting in enhanced serine-type protease activity, decreased protein content and altered protein profiles of fully mature tubers. Zebra-chip (ZC), caused by Candidatus Liberibacter solanacearum (CLso), is a relatively new disease of potato that negatively affects growth, yield, propagation potential, and fresh and process qualities of tubers. Diseased plants produce tubers with characteristic brown discoloration of vascular tissue accompanied by elevated levels of free amino acids and reducing sugars. Here we demonstrate that ZC disease induces selective protein catabolism in tubers through modulating protease inhibitor levels. Soluble protein content of tubers from CLso-infected plants was 33% lower than from non-infected plants and electrophoretic analyses revealed substantial reductions in major tuber proteins. Patatin (~40 kDa) and ser-, asp- (22 kDa) and cys-type (85 kDa) protease inhibitors were either absent or greatly reduced in ZC-afflicted tubers. In contrast to healthy (non-infected) tubers, the proteolytic activity in CLso infected tubers was high and the ability of extracts from infected tubers to inhibit trypsin (ser-type) and papain (cys-type) proteases greatly attenuated. Moreover, extracts from CLso-infected tubers rapidly catabolized proteins purified from healthy tubers (40 kDa patatin, 22 kDa protease inhibitors, 85 kDa potato multicystatin) when subjected to proteolysis individually. In contrast, crude extracts from non-infected tubers effectively inhibited the proteolytic activity from ZC-afflicted tubers. These results suggest that the altered protein profile of ZC afflicted tubers is largely due to loss of ser- and cys-type protease inhibitors. Further analysis revealed a novel PMSF-sensitive (ser) protease (ca. 80-120 kDa) in CLso infected tubers. PMSF abolished the proteolytic activities responsible for degrading patatin, the 22 kDa protease inhibitor(s) and potato

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

    Directory of Open Access Journals (Sweden)

    Garabaya Cecilia

    2006-03-01

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

  10. Isolation of protease producing novel Bacillus cereus and detection ...

    African Journals Online (AJOL)

    user

    2011-02-14

    1991). Industrial enzymes-developments in production and application, Biotechnol. Adv. 9: 643-658. Ferrero MA, Castro GR, Abate CM, Baigori MD, Sineriz F (1996). Thermostable alkaline proteases of Bacillus licheniformis MIR ...

  11. Immune pressure analysis of protease and reverse transcriptase ...

    African Journals Online (AJOL)

    /dn) were analyzed for 33 HIV-1 subtype C protease (PR) and reverse transcriptase (RT) nucleotide sequences each from antiretroviral naïve South African chronically infected individuals. The ds/dn ratios were calculated using the ...

  12. Analysis and protease-catalysed synthesis of sucrose alkanoate regioisomers

    DEFF Research Database (Denmark)

    Lie, Aleksander

    2014-01-01

    laurate in DMF using serine proteases and a metalloprotease. A broad range of elution strategies for the chromatographic analysis of sucrose alkanoate regioisomers was systematically investigated using design of experiments strategies and statistical and multivariate analysis and modelling. Efficiency...

  13. Effect of Gastrointestinal Protease Digestion on Bioactivity of Marine Peptides

    DEFF Research Database (Denmark)

    Jensen, Ida-Johanne; Andersen, Lisa Lystbæk; Ossum, Carlo Gunnar

    2014-01-01

    executed without concerning subsequent digestion after intake and the aim of this work was hence to investigate how the in vitro antioxidative, antihypertensive and caspase activating activities of peptides are affected by digestion with gastrointestinal (GI) proteases. Five different fish protein...... hydrolysates were chosen to study the effect of in vitro digestion on bioactivity. The protein concentration decreased in all samples during digestion and the molecular weight distribution of the peptides shifted towards lower values. Thus, in vitro digestion with GI proteases resulted in a further degradation...... of the peptides obtained by hydrolysis. The antihypertensive effect increased in all samples after digestion with GI proteases whereas the antioxidative capacity decreased. The effect on the caspase activity depended on the proteases used in the preparation of hydrolysates. In conclusion, the caspase activity...

  14. The Place of protease inhibitors in antiretroviral treatment

    Directory of Open Access Journals (Sweden)

    S.B. Tenore

    Full Text Available With the introduction of highly active antiretroviral therapy, a number of drugs have been developed. The best choice concerning which antiretroviral analogs to start is always under discussion, especially in the choice between non-nucleoside reverse transcriptase inhibitors-based therapies and ritonavir-boosted protease inhibitors. Both are proven to control viral replication and lead to immunological gain. The choice between a non-nucleoside analog reverse transcriptase inhibitor and a protease inhibitor as a third antiretroviral drug in the therapy should consider factors related to the individual, as well as the inclusion of the best therapy in the patient's daily activities and potential adherence. The protease inhibitor-based therapies showed similar efficacy among the various inhibitors with characteristics concerning the adverse events from each medicine. For the treatment of protease-resistant patients, darunavir and tipranavir showed good efficacy with higher genetic barrier to resistance.

  15. Sequential Detection of Thermophilic Lipase and Protease by Zymography.

    Science.gov (United States)

    Kurz, Liliana; Hernández, Zully; Contreras, Lellys M; Wilkesman, Jeff

    2017-01-01

    Lipase and protease present in cell-free fractions of thermophilic Bacillus sp. cultures were analyzed by polyacrylamide gel (PAG) electrophoresis. After run, the gel is electrotransferred to another PAG copolymerized with glycerol tributyrate, olive oil, and gelatin. This multi-substrate gel was incubated first for lipase detection, until bands appeared, and then stained with Coomassie for protease detection. Advantages of this sequential procedure are the detection of two different enzyme activities on a single PAG, beside time and resource saving.

  16. Functional protease profiling for diagnosis of malignant disease.

    Science.gov (United States)

    Findeisen, Peter; Neumaier, Michael

    2012-01-01

    Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2010-04-01

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

  18. Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

    International Nuclear Information System (INIS)

    Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian

    2007-01-01

    The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection

  19. 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.

  20. 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.

  1. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

    Directory of Open Access Journals (Sweden)

    Huan Zhang

    2017-01-01

    Full Text Available Serine protease inhibitors (serpins are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

  2. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum.

    Science.gov (United States)

    Zhang, Huan; Fei, Rui; Xue, Baigong; Yu, Shanshan; Zhang, Zuoming; Zhong, Sheng; Gao, Yuanqi; Zhou, Xiaoli

    2017-01-07

    Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum , was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI) of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

  3. Characterizing Protease Specificity: How Many Substrates Do We Need?

    Directory of Open Access Journals (Sweden)

    Michael Schauperl

    Full Text Available Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points. Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4' with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

  4. Regulation of intestinal permeability: The role of proteases.

    Science.gov (United States)

    Van Spaendonk, Hanne; Ceuleers, Hannah; Witters, Leonie; Patteet, Eveline; Joossens, Jurgen; Augustyns, Koen; Lambeir, Anne-Marie; De Meester, Ingrid; De Man, Joris G; De Winter, Benedicte Y

    2017-03-28

    The gastrointestinal barrier is - with approximately 400 m 2 - the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases.

  5. Insights into the Cyanobacterial Deg/HtrA Proteases

    Directory of Open Access Journals (Sweden)

    Otilia eCheregi

    2016-05-01

    Full Text Available Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g. caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serine-type ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologues. Homology modeling was used to find specific features of the Deg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior.

  6. Robotic membranes

    DEFF Research Database (Denmark)

    Ramsgaard Thomsen, Mette

    2008-01-01

    , Vivisection and Strange Metabolisms, were developed at the Centre for Information Technology and Architecture (CITA) at the Royal Danish Academy of Fine Arts in Copenhagen as a means of engaging intangible digital data with tactile physical material. As robotic membranes, they are a dual examination...

  7. Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

    OpenAIRE

    Saitoh, Eiichi; Yamamoto, Shinya; Okamoto, Eishiro; Hayakawa, Yoshimi; Hoshino, Takashi; Sato, Ritsuko; Isemura, Satoko; Ohtsubo, Sadami; Taniguchi, Masayuki

    2007-01-01

    We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the fi rst-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic...

  8. Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: Function of the Asn-His interaction in the catalytic triad

    NARCIS (Netherlands)

    Snijder, H.J.; van Eerde, J.H.; Kalk, K.H.; Dekker, N.; Egmond, M.R.; Dijkstra, B.W.

    2010-01-01

    Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral-membrane enzyme with a unique His-Ser-Asn catalytic triad. In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged

  9. Antibody proteases: induction of catalytic response.

    Science.gov (United States)

    Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D

    2002-10-01

    Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

  10. Modelling of potentially promising SARS protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Plewczynski, Dariusz [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland); Hoffmann, Marcin [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Grotthuss, Marcin von [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Knizewski, Lukasz [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland); Rychewski, Leszek [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Eitner, Krystian [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Ginalski, Krzysztof [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland)

    2007-07-18

    In many cases, at the beginning of a high throughput screening experiment some information about active molecules is already available. Active compounds (such as substrate analogues, natural products and inhibitors of related proteins) are often identified in low throughput validation studies on a biochemical target. Sometimes the additional structural information is also available from crystallographic studies on protein and ligand complexes. In addition, the structural or sequence similarity of various protein targets yields a novel possibility for drug discovery. Co-crystallized compounds from homologous proteins can be used to design leads for a new target without co-crystallized ligands. In this paper we evaluate how far such an approach can be used in a real drug campaign, with severe acute respiratory syndrome (SARS) coronavirus providing an example. Our method is able to construct small molecules as plausible inhibitors solely on the basis of the set of ligands from crystallized complexes of a protein target, and other proteins from its structurally homologous family. The accuracy and sensitivity of the method are estimated here by the subsequent use of an electronic high throughput screening flexible docking algorithm. The best performing ligands are then used for a very restrictive similarity search for potential inhibitors of the SARS protease within the million compounds from the Ligand.Info small molecule meta-database. The selected molecules can be passed on for further experimental validation.

  11. Protease Production by Different Thermophilic Fungi

    Science.gov (United States)

    Macchione, Mariana M.; Merheb, Carolina W.; Gomes, Eleni; da Silva, Roberto

    A comparative study was carried out to evaluate protease production in solid-state fermentation (SSF) and submerged fermentation (SmF) by nine different thermophilic fungi — Thermoascus aurantiacus Miehe, Thermomyces lanuginosus, T. lanuginosus TO.03, Aspergillus flavus 1.2, Aspergillus sp. 13.33, Aspergillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36 and Rhizomucor sp. 13.37 — using substrates containing proteins to induce enzyme secretion. Soybean extract (soybean milk), soybean flour, milk powder, rice, and wheat bran were tested. The most satisfactory results were obtained when using wheat bran in SSF. The fungi that stood out in SSF were T. lanuginosus, T. lanuginosus TO.03, Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37, and those in SmF were T. aurantiacus, T. lanuginosus TO.03, and 13.37. In both fermentation systems, A. flavus 1.2 and R. pusillus 13.36 presented the lowest levels of proteolytic activity.

  12. Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta

    OpenAIRE

    Cao, Xiaolong; He, Yan; Hu, Yingxia; Zhang, Xiufeng; Wang, Yang; Zou, Zhen; Chen, Yunru; Blissard, Gary W.; Kanost, Michael R.; Jiang, Haobo

    2014-01-01

    Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., 2015), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sush...

  13. Plant-derived protease inhibitors LC-pi (Lavatera cashmeriana) inhibit human lung cancer cell proliferation in vitro.

    Science.gov (United States)

    Rakashanda, Syed; Qazi, Asif Khurshid; Majeed, Rabiya; Andrabi, Syed Mubashir; Hamid, Abid; Sharma, P R; Amin, Shajrul

    2015-01-01

    The objective of this study was to check the anticancer activity of purified protease inhibitors of Lavatera cashmeriana viz LC-pi I, II, III, and IV (Lavatera cashmeriana protease inhibitors) on A549 (lung) cell. It was found that LC-pi I and II significantly inhibited the proliferation of A549 cells with IC₅₀ value of 54 μg/ml and 38 μg/ml, respectively, whereas inhibition by LC-pi III and IV was negligible. LC-pi I and II were further found to inhibit formation of colonies in a dose-dependent manner. Also, both inhibitors were found to induce apoptosis causing chromatin condensation and DNA fragmentation, without loss of mitochondrial membrane potential. Cell cycle revealed a significant increase of subG₀/G₁ phase cells that are apoptotic cells. We also demonstrated a dose-dependent decrease in migration of A549 cells on cell migration assay by both inhibitors. Taken together, we demonstrate that LC-pi I and II inhibited proliferation through arresting cells before apoptosis, inducing apoptosis and inhibiting cell migration in human lung cancer cells, but the study warrants further investigation. Our results support the notion that plant protease inhibitors may have the potential to advance as chemopreventive agents.

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

    Directory of Open Access Journals (Sweden)

    Magdalena Opalińska

    2017-11-01

    Full Text Available Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4’s physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4 and Pam18-2 and known (Tim17-2 substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

  15. Host Plants Indirectly Influence Plant Virus Transmission by Altering Gut Cysteine Protease Activity of Aphid Vectors.

    Science.gov (United States)

    Pinheiro, Patricia V; Ghanim, Murad; Alexander, Mariko; Rebelo, Ana Rita; Santos, Rogerio S; Orsburn, Benjamin C; Gray, Stewart; Cilia, Michelle

    2017-04-01

    The green peach aphid, Myzus persicae , is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Evaluation of Immunoglobulin A1 (IgA1) Protease and IgA1 Protease-Inhibitory Activity in Human Female Genital Infection with Neisseria gonorrhoeae

    OpenAIRE

    Hedges, Spencer R.; Mayo, Matthew S.; Kallman, Lisa; Mestecky, Jiri; Hook, Edward W.; Russell, Michael W.

    1998-01-01

    Immunoglobulin A1 (IgA1) protease, an enzyme that selectively cleaves human IgA1, may be a virulence factor for pathogenic organisms such as Neisseria gonorrhoeae. Host protection from the effects of IgA1 protease includes antibody-mediated inhibition of IgA1 protease activity, and it is believed that the relative balance between IgA1 protease and inhibitory antibodies contributes to the pathogenesis of disease caused by IgA1 protease-producing organisms. We have examined the levels of these ...

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

    Directory of Open Access Journals (Sweden)

    Ming-Yang Zhou

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

  18. Cysteine Protease (Capparin from Capsules of Caper (Capparis spinosa

    Directory of Open Access Journals (Sweden)

    Yasar Demir

    2008-01-01

    Full Text Available Proteases are enzymes that perform very important functions in organisms and are used for a variety of objectives in vitro. In recent years, proteases have been used for clinical, pharmaceutical (alimentary digestion, anti-inflammatory, etc. and industrial applications (cheese production, meat tenderizing, leather tanning. In this research, a protease has been purified from capsules of caper (Capparis spinosa and characterized. Caper plants have been used for food and medicine since ancient times. The plant grows abundantly in certain regions of Turkey. Ammonium sulphate fractionation and a CM Sephadex column were used for purification of the enzyme. The purification enzyme has an optimum pH=5.0 and its optimum temperature was 60 °C. The vmax and Km values determined by Lineweaver-Burk graphics were 1.38 μg/(L·min and 0.88 μg/L, respectively. The purification degree and the molecular mass of the enzyme (46 kDa were determined by SDS-PAGE and gel filtration chromatography. It was investigated whether the purified and characterized protease could cause milk to congeal or digest chicken and cow meat. The results show that protease can be used for industrial production.

  19. Penaeus vannamei protease stabilizing process of ZnS nanoparticles.

    Science.gov (United States)

    Razzaghi, Mozhgan; Homaei, Ahmad; Mosaddegh, Elaheh

    2018-01-31

    The protease enzyme purified from the Penaeus vannamei shrimp has unique properties, so improving the stability of this enzyme can improve their practical applications. In this study, ZnS nanoparticles, which have special properties for enzyme immobilization, were synthesized using a chemical precipitation method, and Penaeus vannamei protease was successfully immobilized on them. The size, structure, and morphology of the ZnS nanoparticles, and the immobilization of the protease were studied, using Transmission Electron Microscopy (TEM), Fourier Transform Infrared (FT-IR) spectroscopy, UV-Vis spectroscopy and Dynamic Light Scattering (DLS) analysis. We show that the immobilized enzyme has improved functionality at high temperatures, extreme pH conditions (pH3 and 12), and during storage. Immobilization increased the optimum temperature range of the enzyme, but did not change the pH optimum, which remained at pH7. Immobilization of P. vannamei protease enzyme increased the K m and decreased k cat /K m . These results indicate that P. vannamei protease immobilized on ZnS nanoparticles, has improved properties due to its high stability and unique properties, can be used for biotechnology applications. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Extracellular protease from the antarctic yeast Candida humicola.

    Science.gov (United States)

    Ray, M K; Devi, K U; Kumar, G S; Shivaji, S

    1992-06-01

    The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.

  1. Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.

    Science.gov (United States)

    Verma, Amit; Ansari, Mohammad W; Anwar, Mohmmad S; Agrawal, Ruchi; Agrawal, Sanjeev

    2014-05-01

    Proteases have found a wide application in the several industrial processes, such as laundry detergents, protein recovery or solubilization, prion degradation, meat tenderizations, and in bating of hides and skins in leather industries. But the main hurdle in industrial application of proteases is their economical production on a large scale. The present investigation aimed to exploit the locally available inexpensive agricultural and household wastes for alkaline protease production using Thermoactinomyces sp. RS1 via solid-state fermentation (SSF) technique. The alkaline enzyme is potentially useful as an additive in commercial detergents to mitigate pollution load due to extensive use of caustic soda-based detergents. Thermoactinomyces sp. RS1 showed good protease production under SSF conditions of 55 °C, pH 9, and 50 % moisture content with potato peels as solid substrate. The presented findings revealed that crude alkaline protease produced by Thermoactinomyces sp. RS1 via SSF is of potential application in silver recovery from used X-ray films.

  2. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    Science.gov (United States)

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  3. Identification of a new subtilisin-like protease NbSLP2 interacting with cytoskeletal protein septin in Microsporidia Nosema bombycis.

    Science.gov (United States)

    Liu, Fangyan; Ma, Qiang; Dang, Xiaoqun; Wang, Ying; Song, Yue; Meng, Xianzhi; Bao, Jialing; Chen, Jie; Pan, Guoqing; Zhou, Zeyang

    2017-09-01

    Nosema bombycis is the pathogen of pébrine which brings heavy losses to sericulture every year. As a member of serine proteases, subtilisin-like protease (SLP) is related to the pathogenicity in fungi. In this study, we characterized a novel 63.8kDa subtilisin-like protease NbSLP2 with a predicted transmembrane domain from Microsporidia, N. bombycis. RT-PCR showed that the transcript of NbSLP2 was detected from third day post infection. Immunofluorescence assay (IFA) indicated that NbSLP2 mainly scattered around the spore wall of N. bombycis. Co-immunoprecipitation data and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed that NbSLP2 directly interacts with septin2 of N. bombycis, which is a cytoskeletal protein. IFA showed that NbSLP2 and Nbseptin2 co-localized beneath the spore wall. NbSLP2 can be pulled down by Nbseptin2, further confirming the interaction between NbSLP2 and Nbseptin2. As an important serine protease with a transmembrane domain, NbSLP2 interacting with Nbseptin2, a scaffold protein adjacent to the membrane may provide advantages to stabilize the NbSLP2 for its hydrolysis function. Copyright © 2017. Published by Elsevier Inc.

  4. Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

    NARCIS (Netherlands)

    Kaman, W.E.; Voskamp-Visser, I.; de Jongh, D.M.C.; Endtz, H.P.; van Belkum, A.; Hays, J.P.; Bikker, F.J.

    2013-01-01

    Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these

  5. Enzymatic cleaning of biofouled thin-film composite reverse osmosis (RO) membrane operated in a biofilm membrane reactor.

    Science.gov (United States)

    Khan, Mohiuddin; Danielsen, Steffen; Johansen, Katja; Lorenz, Lindsey; Nelson, Sara; Camper, Anne

    2014-02-01

    Application of environmentally friendly enzymes to remove thin-film composite (TFC) reverse osmosis (RO) membrane biofoulants without changing the physico-chemical properties of the RO surface is a challenging and new concept. Eight enzymes from Novozyme A/S were tested using a commercially available biofouling-resistant TFC polyamide RO membrane (BW30, FilmTech Corporation, Dow Chemical Co.) without filtration in a rotating disk reactor system operated for 58 days. At the end of the operation, the accumulated biofoulants on the TFC RO surfaces were treated with the three best enzymes, Subtilisin protease and lipase; dextranase; and polygalacturonase (PG) based enzymes, at neutral pH (~7) and doses of 50, 100, and 150 ppm. Contact times were 18 and 36 h. Live/dead staining, epifluorescence microscopy measurements, and 5 μm thick cryo-sections of enzyme and physically treated biofouled membranes revealed that Subtilisin protease- and lipase-based enzymes at 100 ppm and 18 h contact time were optimal for removing most of the cells and proteins from the RO surface. Culturable cells inside the biofilm declined by more than five logs even at the lower dose (50 ppm) and shorter incubation period (18 h). Subtilisin protease- and lipase-based enzyme cleaning at 100 ppm and for 18 h contact time restored the hydrophobicity of the TFC RO surface to its virgin condition while physical cleaning alone resulted in a 50° increase in hydrophobicity. Moreover, at this optimum working condition, the Subtilisin protease- and lipase-based enzyme treatment of biofouled RO surface also restored the surface roughness measured with atomic force microscopy and the mass percentage of the chemical compositions on the TFC surface estimated with X-ray photoelectron spectroscopy to its virgin condition. This novel study will encourage the further development and application of enzymes to remove biofoulants on the RO surface without changing its surface properties.

  6. The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

    Directory of Open Access Journals (Sweden)

    Marian Dorcas Quain

    2013-08-01

    Full Text Available Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

  7. Screening of Alkaline Protease-Producing Streptomyces diastaticus and Optimization of Enzyme Production

    Directory of Open Access Journals (Sweden)

    Elham Dawoodi

    2014-12-01

    Full Text Available Background and Aim: Alkaline proteases are used in pharmaceutical, film and photography, silk production and food, leather and detergent industries. Actinomycetes are gram positive bacteria that produce different enzymes such as proteases. The aims of this research were isolation of native alkaline protease-producing Actinomycete spp. from different soil samples as well as optimizing the conditions for enzyme production. Materials and Methods: The different soil samples were collected from different locations of the provinces of Khouzestan, Chahar Mahalo Bakhtiari and Isfahan, Iran. After determining of the best alkaline protease producing species using Lowry method, the optimization of alkaline protease was performed. Results: The alkaline protease producing Actinomycete spp. was isolated from soil. The most enzyme activity was measured in S.diastaticus. The best concentration of sucrose as the carbon source for the highest production of alkaline protease was 10 g/l. The optimum pH and temperature for the alkaline protease production by S. diastaticus were 10 and 30°C respectively. The maximum activity of alkaline protease was measured at 200 rpm as the best aeration speed. Conclusions: This is the first report of alkaline protease production by Streptomyces diastaticus in Iran. The accomplished examinations in this research confirmed the previous theories of alkaline protease production by Actinomycetes relatively. Regarding the immense applications of alkaline proteases in several industries and isolation of a native alkaline protease producing Actinomycete, The production potential of this enzyme in our country could be accessible in the near future.

  8. Diversity of cultivable protease-producing bacteria in sediments of Jiaozhou Bay, China

    Directory of Open Access Journals (Sweden)

    Xi-Ying eZhang

    2015-09-01

    Full Text Available Although protease-producing bacteria are key players in the degradation of organic nitrogen and essential for the nitrogen recycling in marine sediments, diversity of both these bacteria and their extracellular proteases is still largely unknown. This study investigated the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the eutrophied Jiaozhou Bay, China through phylogenetic analysis and protease inhibitor tests. The abundance of the cultivable protease-producing bacteria was up to 104 cells/g in all six sediment samples. The cultivated protease-producing bacteria mostly belonged to the phyla Proteobacteria and Firmicutes with the predominant genera being Photobacterium (39.4%, Bacillus (25.8% and Vibrio (19.7%. Protease inhibitor tests revealed that extracellular proteases secreted by the bacteria were mainly serine proteases and/or metalloproteases with relatively low proportions of cysteine proteases. This study represents the first comprehensive analysis on the diversity of protease-producing bacteria and their extracellular proteases in sediments of a eutrophic bay.

  9. Severe dystrophic cardiomyopathy caused by the enteroviral protease 2A-mediated C-terminal dystrophin cleavage fragment.

    Science.gov (United States)

    Barnabei, Matthew S; Sjaastad, Frances V; Townsend, DeWayne; Bedada, Fikru B; Metzger, Joseph M

    2015-07-01

    Enterovirus infection can cause severe cardiomyopathy in humans. The virus-encoded 2A protease is known to cleave the cytoskeletal protein dystrophin. It is unclear, however, whether cardiomyopathy results from the loss of dystrophin or is due to the emergence of a dominant-negative dystrophin cleavage product. We show for the first time that the 2A protease-mediated carboxyl-terminal dystrophin cleavage fragment (CtermDys) is sufficient to cause marked dystrophic cardiomyopathy. The sarcolemma-localized CtermDys fragment caused myocardial fibrosis, heightened susceptibility to myocardial ischemic injury, and increased mortality during cardiac stress testing in vivo. CtermDys cardiomyopathy was more severe than in hearts completely lacking dystrophin. In vivo titration of CtermDys peptide content revealed an inverse relationship between the decay of membrane-bound CtermDys and the restoration of full-length dystrophin at the sarcolemma, in support of a physiologically relevant loss of dystrophin function in this model. CtermDys gene titration and dystrophin replacement studies further established a target threshold of 50% membrane-bound intact dystrophin necessary to prevent mice from CtermDys cardiomyopathy. Conversely, the NtermDys fragment did not compete with dystrophin and had no pathological effect. Thus, CtermDys must be localized to the sarcolemma, with intact dystrophin dystrophin and compensatory utrophin from binding at the membrane. Therefore, membrane-bound CtermDys is a new potential translational target for virus-mediated cardiomyopathy. Copyright © 2015, American Association for the Advancement of Science.

  10. Membrane topology of hedgehog acyltransferase.

    Science.gov (United States)

    Matevossian, Armine; Resh, Marilyn D

    2015-01-23

    Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. Knowledge of the topological organization of Hhat transmembrane helices would enhance our understanding of Hhat-mediated Shh palmitoylation. Bioinformatics analysis of transmembrane domains within human Hhat using 10 different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains 10 transmembrane domains and 2 re-entrant loops. The invariant His and highly conserved Asp residues within the membrane-bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His-379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C-terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O-acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  12. Cleaning protocols for crystallization robots: preventing protease contamination

    Science.gov (United States)

    Naschberger, Andreas; Fürnrohr, Barbara G.; Dunzendorfer-Matt, Theresia; Bonagura, Christopher A.; Wright, David; Scheffzek, Klaus; Rupp, Bernhard

    2015-01-01

    The protease in the commonly used commercial low-foam enzyme cleaner Zymit cannot be completely blocked by EDTA, a widely used inhibitor of metalloproteases, at concentrations of up to 5 mM. Severe protein degradation was observed in crystallization drops after EDTA-containing wash steps unless residual Zymit protease was removed with NaOH at a concentration of at least 0.1 M. Wash steps with 0.1% SDS were also ineffective in completely removing the remaining Zymit activity. Protocols including wash steps with at least 0.1 M NaOH, as for example specified in the original ZENM protocol, are recommended to completely deactivate Zymit protease activity. PMID:25615978

  13. Protease activity in the larval stage of the parasitoid wasp, Eulophus pennicornis (Nees) (Hymenoptera: Eulophidae); effects of protease inhibitors.

    Science.gov (United States)

    Down, R E; Ford, L; Mosson, H J; Fitches, E; Gatehouse, J A; Gatehouse, A M

    1999-08-01

    Hymenopteran, parasitoid wasps have good potential for use in integrated pest management (IPM); for example, the gregarious ectoparasitoid, Eulophus pennicornis, has been suggested as a biological control agent for larvae of the tomato moth (Lacanobia oleracea L.). However, the processes by which such parasitic larvae are able to utilize the nutritional resource provided by the host have been little studied. Protease activity was present in E. pennicornis larvae, and characterization of the enzymes responsible for proteolysis was performed using a range of synthetic substrates and specific inhibitors. Serine protease enzymes was both trypsin- and chymotrypsin-like activities were present. A range of plant-derived serine protease inhibitors was tested for activity against these enzymes. Certain inhibitors, notably soybean Kunitz inhibitor (SKTI), inhibited enzyme activity by > 80% at < 10(-5) M. When SKTI was fed to L. oleracea larvae in an artificial diet, the inhibitor was subsequently detected within the larval haemolymph, showing that protease inhibitors in the host diet can be delivered to a parasitoid via the host haemolymph. If transgenic plants expressing foreign protease inhibitors for protection against insect pests are to form a component of IPM systems, possible adverse effects, whether direct or indirect, of transgene expression on parasitoids like E. pennicornis should be considered.

  14. Purification and characterization of alkaline proteases from aspergillus terreus

    International Nuclear Information System (INIS)

    Hussain, A.; Mannan, A.; Zubair, H.; Mirza, B.

    2010-01-01

    Proteases belong to an important class of enzymes known as hydrolases and catalyze hydrolysis of proteins. They act primarily to degrade proteins that are used for energy production and as biosynthetic precursors. In the following study, protease produced from Aspergillus terreus was found to be thermo stable and included in the category of alkaline serine and metallo protease. During partial purification, presence of enzyme in 60% (NH/sub 4/)/sub 2/SO/sub 4/ indicated small molecular weight polypeptide; later purification with Sephadex G-75 fractionation yielded a single proteolytic active molecule. At final purification step, the increase in specific activity of the enzyme was 7.5 fold with 23% yield. SDS-PAGE analysis revealed that alkaline protease of Aspergillus terreus is a monomer with approximate molecular weight of 35 kDa. Optimum pH for protease activity was found in the range of 7.5-11.0 (maximum at pH 8.5), thus apparently classified as an alkaline protease. The enzyme was thermo stable towards high temperature (60 deg. C), however it denatured irreversibly at 70 deg. C showing 80% loss of activity. The maximum proteolytic activity was found at 40 deg. C. The enzyme was effectively inhibited by PMSF, EDTA and urea whereas iodoacetamide and thiourea did not result in any loss in activity while cysteine was found to be activator molecule. The study with metal ions Mg/sup +2/, Mn/sup +2/ and Fe/sup +3/ (1 mM each) showed minute stimulatory effects on enzyme activity. Co/sup +2/ and Ca/sup +2/ (1 mM) had neither excitatory nor inhibitory effect while Hg/sup +2/ and Cu/sup +2/ (1 mM) slightly reduced the enzyme activity. (author)

  15. Design of HIV protease inhibitors based on inorganic polyhedral metallacarboranes

    Czech Academy of Sciences Publication Activity Database

    Řezáčová, Pavlína; Pokorná, Jana; Brynda, Jiří; Kožíšek, Milan; Cígler, Petr; Lepšík, Martin; Fanfrlík, Jindřich; Řezáč, Jan; Grantz Šašková, Klára; Sieglová, Irena; Plešek, Jaromír; Šícha, Václav; Grüner, Bohumír; Oberwinkler, H.; Sedláček, Juraj; Kräusslich, H. G.; Hobza, Pavel; Král, V.; Konvalinka, Jan

    2009-01-01

    Roč. 52, č. 22 (2009), s. 7132-7141 ISSN 0022-2623 R&D Projects: GA AV ČR IAAX00320901; GA MŠk LC512; GA MŠk LC523 EU Projects: European Commission(XE) 37693 - HIV PI RESISTANCE Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514; CEZ:AV0Z40320502 Keywords : HIV protease inhibitors * aspartic proteases * viral resistance * cobalt bis(dicarbollide) * crystal structure Subject RIV: CC - Organic Chemistry Impact factor: 4.802, year: 2009

  16. Chemistry and biology of natural product derived protease inhibitors

    OpenAIRE

    Stolze, Sara Christina

    2012-01-01

    Im Rahmen dieser Dissertation sollten Naturstoffe und davon abgeleitete Derivate synthetisiert und im Hinblick auf ihre biologische Aktivität als Protease-Inhibitoren untersucht werden. Für die Naturstoffklasse der 3-Amino-6-Hydroxy-2-piperidon(Ahp)-Cyclodepsipeptide, die als nicht-kovalente Serin-Protease-Inhibitoren bekannt sind, konnte eine Festphasensynthese basierend auf einem allgemeinen Ahp-Vorläufermolekül entwickelt werden. Für den Ahp-Vorläufer wurde eine Lösungssynthese entwicke...

  17. Dissecting Degradomes: Analysis of Protease-Coding Genes.

    Science.gov (United States)

    Álvarez-Eguiluz, Ángel; Díaz-Navarro, Ander; Puente, Xose S

    2018-01-01

    Proteases constitute up to 3% of all protein-coding genes in a vertebrate genome and participate in numerous physiological and pathological processes. The characterization of the degradome of one organism, the set of all genes encoding proteolytic enzymes, and the comparison to the degradome of other species have proved useful to identify genetic differences that are helpful to elucidate the molecular basis of diverse biological processes, the different susceptibility to disease, and the evolution of the structure and function of proteases. Here we describe the main procedures involved in the characterization of the degradome of an organism for which its genome sequence is available.

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

    Science.gov (United States)

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

    2013-12-05

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

  19. Boosted protease inhibitors and the electrocardiographic measures of QT and PR durations

    DEFF Research Database (Denmark)

    Soliman, Elsayed Z; Lundgren, Jens D; Roediger, Mollie P

    2011-01-01

    There are contradictory reports regarding the effects of protease inhibitors on the ECG measures of QT and PR interval durations. The effect of interrupting use of protease inhibitors on QT and PR progression is also unknown.......There are contradictory reports regarding the effects of protease inhibitors on the ECG measures of QT and PR interval durations. The effect of interrupting use of protease inhibitors on QT and PR progression is also unknown....

  20. Evaluation of Immunoglobulin A1 (IgA1) Protease and IgA1 Protease-Inhibitory Activity in Human Female Genital Infection with Neisseria gonorrhoeae

    Science.gov (United States)

    Hedges, Spencer R.; Mayo, Matthew S.; Kallman, Lisa; Mestecky, Jiri; Hook, Edward W.; Russell, Michael W.

    1998-01-01

    Immunoglobulin A1 (IgA1) protease, an enzyme that selectively cleaves human IgA1, may be a virulence factor for pathogenic organisms such as Neisseria gonorrhoeae. Host protection from the effects of IgA1 protease includes antibody-mediated inhibition of IgA1 protease activity, and it is believed that the relative balance between IgA1 protease and inhibitory antibodies contributes to the pathogenesis of disease caused by IgA1 protease-producing organisms. We have examined the levels of these two opposing factors in genital tract secretions and sera from women with uncomplicated infection with N. gonorrhoeae. When IgA1 in cervical mucus was examined by Western blotting, no evidence of cleavage fragments characteristic of IgA1 protease activity was seen in gonococcus-infected or control patients. Cleavage fragments typical of IgA1 protease were detected, however, after the addition of exogenous IgA1 protease to cervical mucus. Degraded IgA1 was detected in some vaginal wash samples, but the fragment pattern was not typical of IgA1 protease activity. All N. gonorrhoeae isolates from the infected patients produced IgA1 protease in vitro. All but two serum samples and 16 of 65 cervical mucus samples displayed inhibitory activity against gonococcal IgA1 protease, but there was no significant difference in the level of inhibitory activity between gonococcus-infected and noninfected patients in either cervical mucus or serum. There was no difference in the levels of IgA1 protease-inhibitory activity in serum or cervical mucus collected from patients at recruitment and 2 weeks later. These results suggest that cleavage of IgA1 by gonococcal IgA1 protease within the lumen of the female lower genital tract is unlikely to be a significant factor in the pathogenesis of infections by N. gonorrhoeae. PMID:9826361

  1. Higher Desolvation Energy Reduces Molecular Recognition in Multi-Drug Resistant HIV-1 Protease

    Directory of Open Access Journals (Sweden)

    Ladislau C. Kovari

    2012-05-01

    Full Text Available Designing HIV-1 protease inhibitors that overcome drug-resistance is still a challenging task. In this study, four clinical isolates of multi-drug resistant HIV-1 proteases that exhibit resistance to all the US FDA-approved HIV-1 protease inhibitors and also reduce the substrate recognition ability were examined. A multi-drug resistant HIV-1 protease isolate, MDR 769, was co-crystallized with the p2/NC substrate and the mutated CA/p2 substrate, CA/p2 P1’F. Both substrates display different levels of molecular recognition by the wild-type and multi-drug resistant HIV-1 protease. From the crystal structures, only limited differences can be identified between the wild-type and multi-drug resistant protease. Therefore, a wild-type HIV-1 protease and four multi-drug resistant HIV-1 proteases in complex with the two peptides were modeled based on the crystal structures and examined during a 10 ns-molecular dynamics simulation. The simulation results reveal that the multi-drug resistant HIV-1 proteases require higher desolvation energy to form complexes with the peptides. This result suggests that the desolvation of the HIV-1 protease active site is an important step of protease-ligand complex formation as well as drug resistance. Therefore, desolvation energy could be considered as a parameter in the evaluation of future HIV-1 protease inhibitor candidates.

  2. A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis

    DEFF Research Database (Denmark)

    Halls, C.E.; Rogers, S. W.; Ouffattole, M.

    2006-01-01

    A Kunitz-type protease inhibitor co-purified from cauliflower florets with a granulin domain cysteine protease that cleaved barley proaleurain to yield a molecular form the same size as that for mature aleurain. The purified cauliflower protease required treatment with SDS detergent to become...

  3. Teaching Foundational Topics and Scientific Skills in Biochemistry within the Conceptual Framework of HIV Protease

    Science.gov (United States)

    Johnson, R. Jeremy

    2014-01-01

    HIV protease has served as a model protein for understanding protein structure, enzyme kinetics, structure-based drug design, and protein evolution. Inhibitors of HIV protease are also an essential part of effective HIV/AIDS treatment and have provided great societal benefits. The broad applications for HIV protease and its inhibitors make it a…

  4. A preliminary study of the protease activities in germinating brown rice (Oryza sativa L.).

    Science.gov (United States)

    Li, Cuijuan; Cao, Xiaohong; Gu, Zhenxin; Wen, Huanbin

    2011-03-30

    Proteases hydrolyse storage proteins to provide precursors for perpetuating species. The aim of this study was to investigate and characterise different proteases in germinating brown rice. The protease activity of brown rice increased sevenfold during 7 days of germination. It was highest on day 6 when determined at pH 3.5. With casein as substrate the proteases showed two catalytic groups: acidic proteases with an optimal pH of 3.5 and alkaline proteases with an optimal pH of 8.0. The acidic protease activity was inhibited by Ba(2+) and Pb(2+) but stimulated by Zn(2+) , while the alkaline protease activity was inhibited by Ca(2+) and Pb(2+) but stimulated by Mg(2+) and Zn(2+) . SDS-gelatin-PAGE assay showed two protease activity bands at pH 3.5, while two different bands with higher molecular weights were observed at pH 8.0. Inhibition assay revealed that pepstatin A and E-64 inhibited 67.63 and 38.26% respectively of the protease activity at pH 3.5, indicating the presence of aspartic and cysteine proteases. Metalloproteases played a major role under alkaline conditions (88.37% inhibition with EDTA). Germinated brown rice proteases fall into different classes with different properties. This study is helpful for their further purification. Copyright © 2010 Society of Chemical Industry.

  5. A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications.

    Science.gov (United States)

    Juntunen, Kari; Mäkinen, Susanna; Isoniemi, Sari; Valtakari, Leena; Pelzer, Alexander; Jänis, Janne; Paloheimo, Marja

    2015-09-01

    A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.

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

    African Journals Online (AJOL)

    MBI

    2017-03-07

    Mar 7, 2017 ... *1Murtala, Y., 1Babandi, A., 1Babagana, K., 1Rajah, M. R., 3Yakasai, H. M., 1Ibrahim, ... many traditional spices and food additives in ..... by fungi. J. Gen. Microbiol. 84, 327–331. Adulyatham, P. and Owusu-Apenten, R. (2005). Stabilization and partial purification of a protease from ginger rhizome (Zingiber.

  7. Purification and characterization of protease from Bacillus cereus ...

    African Journals Online (AJOL)

    chitti

    2013-09-16

    Sep 16, 2013 ... Purification and characterization of protease from. Bacillus cereus SU12 isolated from oyster. Saccostrea cucullata. S. Umayaparvathi*, S. Meenakshi, M. Arumugam and T. Balasubramanian. Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608.

  8. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)

    ashok

    2012-08-28

    Aug 28, 2012 ... This research study was mainly focused on phenotypic, biochemical characterization, 16s rRNA sequence based species level identification of isolate and determination of the higher production of alkaline protease through optimization study (carbon, nitrogen, incubation period, temperature, pH and.

  9. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.; Purslow, P.P.

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin...

  10. Retroviral proteases and their roles in virion maturation

    Czech Academy of Sciences Publication Activity Database

    Konvalinka, Jan; Kräusslich, H. G.; Müller, B.

    2015-01-01

    Roč. 479, SI (2015), s. 403-417 ISSN 0042-6822 R&D Projects: GA ČR GBP208/12/G016; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : retrovirus * aspartic protease * maturation * human immunodeficiency virus * Gag Subject RIV: CE - Biochemistry Impact factor: 3.200, year: 2015

  11. 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...

  12. Optimization of mycelial biomass and protease production by ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-04-20

    Apr 20, 2009 ... 164: 81. De Azeredo LAI, De Lima MB, Coelho RRR, Freire DMG (2006). A low- cost fermentation medium for thermophilic protease production by. Streptomyces sp. 594 using feather meal and corn steep liquor. Curr. Microbiol. 53: 335-339. Dedman V (2000). “Native bread” Polyporus mylittae. Fungimap.

  13. Alkaline protease from senesced leaves of invasive weed Lantana ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-17

    Dec 17, 2008 ... SDS-PAGE was performed on a slab gel containing 10% ( w/v) polyacrylamide by the method of Laemmli (Laemmli, 1971). Native. PAGE was performed on 7% (w/v) polyacrylamide gel. Compatibility with detergents. The compatibility of protease with local laundry detergents was studied in the presence of ...

  14. Alkaline protease production by alkaliphilic marine bacteria isolated ...

    African Journals Online (AJOL)

    The molecular mass determined using SDS-PAGE, was nearly 31.0 39 kDa. Some fundamental properties like effects of different temperatures, pH, metal ions (Ca2+, Mg2+, Cu2+, Pb3+, Mn2+ and Cd2+) and ethylene diamine tetraacetic acid (EDTA) on protease activity were also studied. Maximum activities were obtained ...

  15. Physical and chemical properties of the acid protease from ...

    African Journals Online (AJOL)

    samsung

    2016-03-02

    Mar 2, 2016 ... using casein as substrate. Activities were compared with the enzyme activity in absence of any inhibitor (100%). Determination of the molecular mass. The molecular mass of the purified protease was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-. PAGE) using 15% (w/v) ...

  16. Influence of radiation and photolysis on intracellular proteases

    International Nuclear Information System (INIS)

    Jamadar, V.K.; Jamdar, S.N.; Harikumar, P.; Hari Mohan; Dandekar, S.P.

    2002-01-01

    In contrast to gamma radiation, photoinactivation of aminopeptidase was more profound in air than in N 2 , indicating that oxidative reactions are predominantly involved in photoinduced inactivation. The results are discussed in view of possible differences in the mechanisms underlying radiation and photoinduced inactivation of proteases

  17. Isolation and screening of alkaline protease producing bacteria and ...

    African Journals Online (AJOL)

    Soil samples from different habitats including tanneries, soap industries, garden soil and soil compost were screened for the presence of alkalophilic Bacillus isolates capable of producing alkaline protease in large quantities. One hundred and eighteen (118) isolates were found having proteolytic activity on skim milk agar ...

  18. Isolation of alkaline protease from Bacillus subtilis AKRS3 ...

    African Journals Online (AJOL)

    This research study was mainly focused on phenotypic, biochemical characterization, 16s rRNA sequence based species level identification of isolate and determination of the higher production of alkaline protease through optimization study (carbon, nitrogen, incubation period, temperature, pH and sodium chloride ...

  19. Production of Thermostable Alkaline Protease from Streptomyces sp ...

    African Journals Online (AJOL)

    Bacterial extracellular alkaline proteases have been found to have broad spectrum industrial applications because of their stability characteristics among the bacteria. The Actinomyces are of enormous importance as they can be recovered easier than other bacteria after fermentation. Thus, the study was aimed at sourcing ...

  20. Alkaline protease production on date waste by an alkalophilic ...

    African Journals Online (AJOL)

    This research focused on isolation and characterization of a new strain of Bacillus sp. from alkaline soil, which was able to producing extracellular alkaline protease and amylase from date waste at pH ranging from 8 to 11 and temperatures of 20 to 50°C. Purification was conducted by fractionation, concentration, and cation ...

  1. Alkaline Protease from Bacillus firmus 7728 | Rao | African Journal ...

    African Journals Online (AJOL)

    Extracellular alkaline protease producing Bacillus firmus MTCC 7728 was isolated from the soil samples taken from the leather factories in Nacharam industrial area, Hyderabad. Maximum activity was found after 48 h of fermentation. Optimum pH and temperature for maximum enzyme activity were 9 and 40°C, respectively.

  2. Optimization of protease production by an actinomycete Strain, PS ...

    African Journals Online (AJOL)

    Actinomycetes were isolated from the sediment samples of an estuarine shrimp pond located along the south east coast of India. During the investigation, a total of 28 strains of actinomycetes were isolated and examined for their protease activity. Among them, one strain PS-18A which was tentatively identified as ...

  3. Breakdown of the innate immune system by bacterial proteases

    NARCIS (Netherlands)

    Laarman, A.J.

    2011-01-01

    Bacteria have developed many strategies to circumvent our immune system to survive and colonize human tissues. One of these strategies is by secreting proteases that specifically target the innate immune system. Aureolysin is a metalloprotease from Staphylococcus aureus which target the main

  4. Manipulating the autolytic pathway of a Bacillus protease

    NARCIS (Netherlands)

    VandenBurg, B; Eijsink, VGH; Vriend, G; Veltman, OR; Venema, G; HopsuHavu, VK; Jarvinen, M; Kirschke, H

    1997-01-01

    Autolytic degradation of Bacillus subtilis thermolysin-like proteinase (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously, we reported five autolysis sites in B. subtilis neutral protease (Van den Burg et al., 1990, Biochem. J. 272:93-97).

  5. Determining and overcoming resistance to HIV protease inhibitors

    Czech Academy of Sciences Publication Activity Database

    Prejdová, Jana; Souček, Milan; Konvalinka, Jan

    2004-01-01

    Roč. 4, - (2004), s. 137-152 ISSN 1568-0053 Grant - others:5th Framework(XE) QLK2-CT-2001-02360 Institutional research plan: CEZ:AV0Z4055905 Keywords : HIV protease inhibitors Subject RIV: CE - Biochemistry

  6. Targeted degradomics in protein terminomics and protease substrate discovery

    DEFF Research Database (Denmark)

    Savickas, Simonas; auf dem Keller, Ulrich

    2017-01-01

    extensive degradomics target lists that now can be tested with help of selected and parallel reaction monitoring (S/PRM) in complex biological systems, where proteases act in physiological environments. In this minireview, we describe the general principles of targeted degradomics, outline the generic...

  7. Delay of Iris flower senescence by protease inhibitors

    NARCIS (Netherlands)

    Pak, C.; Doorn, van W.G.

    2005-01-01

    asterisk inside a circle sign Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than

  8. Ionic liquids and proteases: A clean alliance for semisynthesis

    Czech Academy of Sciences Publication Activity Database

    Wehofsky, N.; Wespe, Ch.; Čeřovský, Václav; Pech, A.; Hoess, E.; Rudolph, R.; Bordusa, F.

    2008-01-01

    Roč. 9, č. 9 (2008), s. 1493-1499 ISSN 1439-4227 Grant - others:DFG(DE) SPP1191; DFG(DE) SFB610 Institutional research plan: CEZ:AV0Z40550506 Keywords : chemoenzymatic synthesis * ionic liquids * peptides * proteases * substrate mimetics Subject RIV: CC - Organic Chemistry Impact factor: 3.322, year: 2008

  9. Serine protease from midgut of Bombus terrestris males

    Czech Academy of Sciences Publication Activity Database

    Brabcová, Jana; Kindl, Jiří; Valterová, Irena; Pichová, Iva; Zarevúcka, Marie; Brabcová, J.; Jágr, Michal; Mikšík, Ivan

    2013-01-01

    Roč. 82, č. 3 (2013), s. 117-128 ISSN 0739-4462 R&D Projects: GA ČR GA203/09/1446; GA TA ČR TA01020969 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Bombus terrestris * midgut * serine protease * bumblebee Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C) Impact factor: 1.160, year: 2013

  10. Oxidant and solvent stable alkaline protease from Aspergillus flavus ...

    African Journals Online (AJOL)

    The enzyme retained 80% of its original activity in the presence of non ionic and ionic surfactants and 100% with 10% H2O2 after 1 h of incubation at 30°C. In addition, the enzyme showed excellent compatibility with some commercial powder detergents. The compatibility of our protease with several detergents, oxidants ...

  11. ElaD, a Deubiquitinating protease expressed by E. coli.

    Directory of Open Access Journals (Sweden)

    André Catic

    Full Text Available BACKGROUND: Ubiquitin and ubiquitin-like proteins (Ubl are designed to modify polypeptides in eukaryotes. Covalent binding of ubiquitin or Ubls to substrate proteins can be reversed by specific hydrolases. One particular set of cysteine proteases, the CE clan, which targets ubiquitin and Ubls, has homologs in eukaryotes, prokaryotes, and viruses. FINDINGS: We have cloned and analyzed the E. coli protein elaD, which is distantly related to eukaryotic CE clan members of the ULP/SENP protease family that are specific for SUMO and Nedd8. Previously misannotated as a putative sulfatase/phosphatase, elaD is an efficient and specific deubiquitinating enzyme in vitro. Interestingly, elaD is present in all intestinal pathogenic E. coli strains, but conspicuously absent from extraintestinal pathogenic strains (ExPECs. Further homologs of this protease can be found in Acanthamoeba Polyphaga Mimivirus, and in Alpha-, Beta-and Gammaproteobacteria. CONCLUSION: The expression of ULP/SENP-related hydrolases in bacteria therefore extends to plant pathogens and medically relevant strains of Escherichia coli, Legionella pneumophila, Rickettsiae, Chlamydiae, and Salmonellae, in which the elaD ortholog sseL has recently been identified as a virulence factor with deubiquitinating activity. As a counterpoint, our phylogenetic and functional examination reveals that ancient eukaryotic ULP/SENP proteases also have the potential of ubiquitin-specific hydrolysis, suggesting an early common origin of this peptidase clan.

  12. Production of Microbial Protease from Selected Soil Fungal Isolates ...

    African Journals Online (AJOL)

    Production of Microbial Protease from Selected Soil Fungal Isolates. ... Nigerian Journal of Biotechnology ... and 500C. The optimal pH on the enzyme production was observed to be between pH 3.5 and 5.5 for the organisms. Keywords: Soil microorganism, fungal isolate, incubation period, microbial enzyme. Nig J. Biotech.

  13. Production of Microbial Protease from Selected Soil Fungal Isolates

    African Journals Online (AJOL)

    Dr Oseni

    Nig J. Biotech. Vol. 23 (2011) 28 - 34. ISSN: 0189 17131. Available online at www.biotechsocietynigeria.org. Production of Microbial Protease from Selected Soil Fungal. Isolates. Oseni, O.A.. Department of Medical Biochemistry, Ekiti State University, Ado-Ekiti, Nigeria. (Received 20.07.11, Accepted 09.11.11). Abstract.

  14. Accelerated ripening of Kashar cheese with encapsulated protease ...

    African Journals Online (AJOL)

    In this study, protease enzymes were encapsulated in Κ-carragenan, gellan and sodium alginate using emulsion and extrusion techniques and were then added in cheese milk together with rennet. The effects of the encapsulating material and ripening period on the chemical, textural and sensory characteristics of Kashar ...

  15. Purification and characterization of a protease from Thermophilic ...

    African Journals Online (AJOL)

    The purification and characterization of a thermophilic neutral protease from Thermophilic bacillus strain HS08, originally isolated from a soil sample collected from the Tulufan Crater of China, is presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of DEAE-Sepharose anion ...

  16. High-level expression of alkaline protease using recombinant ...

    African Journals Online (AJOL)

    The apr gene was cloned into plasmid pUB110, resulting in the recombinant plasmid pUB-apr, which was then transformed into Bacillus amyloliquefaciens CICIM B4803. The protease productivity was significantly improved in the transformants of B. amyloliquefaciens CICIM B4803. A transformant with high alkaline ...

  17. Staphylococcal Proteases Aid in Evasion of the Human Complement System

    DEFF Research Database (Denmark)

    Jusko, Monika; Potempa, Jan; Kantyka, Tomasz

    2014-01-01

    lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component...

  18. Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

    Czech Academy of Sciences Publication Activity Database

    Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

    2015-01-01

    Roč. 16, č. 11 (2015), s. 1616-1621 ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity-based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.850, year: 2015

  19. An oxidant, detergent and salt stable alkaline protease from Bacillus ...

    African Journals Online (AJOL)

    A novel soil bacterium, Bacillus cereus SIU1 was earlier isolated from non-saline, slightly alkaline soil of Eastern Uttar Pradesh, India. The isolate B. cereus SIU1 was grown in modified glucose yeast extract (modified GYE) medium at pH 9.0 and 45°C. It produced maximum protease at 20 h incubation. The enzyme was ...

  20. Purification and characterization of a milk-clotting protease from ...

    African Journals Online (AJOL)

    The protein homogeneity (a single electrophoretic band) of the monomeric protease was confirmed by both methods after precipitation with 80% saturated ammonium sulphate. Moreover, the fractional precipitation technique with this salt (40 and 80%) was useless in the experimental conditions employed and an important ...

  1. Molecular Basis for Drug Resistance in HIV-1 Protease

    Directory of Open Access Journals (Sweden)

    Celia A. Schiffer

    2010-11-01

    Full Text Available HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.

  2. A Protease Isolated from the Latex of Plumeria rubra Linn ...

    African Journals Online (AJOL)

    Erah

    [3]. The extensive use of proteolytic enzymes in the treatment of inflammation and wound healing process is a well established fact. Bromelain from Ananas comosu has been shown to possess both anti-inflammatory and wound healing properties [4] while curcain, a protease from Jatropha curcas, was reported to have ...

  3. molecular biology approach to the search for novel hiv proteases ...

    African Journals Online (AJOL)

    ... which could be tested in the animal models of HIV infection before subjection to clinical trials. Optimistically, the magic HIV therapeutics may be hidden in such insects and may require the application of molecular biology techniques to unravel. KEY WORDS: Antiretroviral drugs, malaria, proteases, restriction enzymes, ...

  4. A Protease Isolated from the Latex of Plumeria rubra Linn ...

    African Journals Online (AJOL)

    Erah

    Spinacia oleracea and Petroselinum crispum leaves [5]. Proteases are important enzymes of plant metabolism and are instrumental in regulating senescence [6]. They are responsible for the degradation of proteins. Proteolytic enzymes are used extensively in industrial and medical applications [7]. Plumeria rubra Linn.

  5. Production of alkaline protease by Teredinobacter turnirae cells ...

    African Journals Online (AJOL)

    The conditions for immobilizing the new alkaline protease-producing bacteria strain Teredinobacter turnirae by entrapment in calcium alginate gel were investigated. The influence of alginate concentration (20, 25 and 30 g/l) and initial cell loading (ICL) on enzyme production were studied. The production of alkaline ...

  6. From biological membranes to biomimetic model membranes

    Directory of Open Access Journals (Sweden)

    Eeman, M.

    2010-01-01

    Full Text Available Biological membranes play an essential role in the cellular protection as well as in the control and the transport of nutrients. Many mechanisms such as molecular recognition, enzymatic catalysis, cellular adhesion and membrane fusion take place into the biological membranes. In 1972, Singer et al. provided a membrane model, called fluid mosaic model, in which each leaflet of the bilayer is formed by a homogeneous environment of lipids in a fluid state including globular assembling of proteins and glycoproteins. Since its conception in 1972, many developments were brought to this model in terms of composition and molecular organization. The main development of the fluid mosaic model was made by Simons et al. (1997 and Brown et al. (1997 who suggested that membrane lipids are organized into lateral microdomains (or lipid rafts with a specific composition and a molecular dynamic that are different to the composition and the dynamic of the surrounding liquid crystalline phase. The discovery of a phase separation in the plane of the membrane has induced an explosion in the research efforts related to the biology of cell membranes but also in the development of new technologies for the study of these biological systems. Due to the high complexity of biological membranes and in order to investigate the biological processes that occur on the membrane surface or within the membrane lipid bilayer, a large number of studies are performed using biomimicking model membranes. This paper aims at revisiting the fundamental properties of biological membranes in terms of membrane composition, membrane dynamic and molecular organization, as well as at describing the most common biomimicking models that are frequently used for investigating biological processes such as membrane fusion, membrane trafficking, pore formation as well as membrane interactions at a molecular level.

  7. The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

    Science.gov (United States)

    Beers, Eric P; Jones, Alan M; Dickerman, Allan W

    2004-01-01

    The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

  8. An Epithelial-Derived, Integral Membrane, Kunitz-Type serine Protease Inhibitor in Breast Cancer

    National Research Council Canada - National Science Library

    Lin, Chen-Yong

    2004-01-01

    .... During matriptase activation induced either by S1P or suramin, HAI-1 along with matriptase is translocated and accumulated at cell-cell junctions or in vesicle-like structures, which were named...

  9. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.; (Monash); (Queensland Inst. of Med. Rsrch.)

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  10. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    International Nuclear Information System (INIS)

    Samarel, A.M.; Ferguson, A.G.; Decker, R.S.; Lesch, M.

    1989-01-01

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation

  11. Molecular model of the specificity pocket of the hepatitis C virus protease: implications for substrate recognition.

    OpenAIRE

    Pizzi, E; Tramontano, A; Tomei, L; La Monica, N; Failla, C; Sardana, M; Wood, T; De Francesco, R

    1994-01-01

    We have built a model of the specificity pocket of the protease of hepatitis C virus on the basis of the known structures of trypsin-like serine proteases and of the conservation pattern of the protease sequences among various hepatitis C strains. The model allowed us to predict that the substrate of this protease should have a cysteine residue in position P1. This hypothesis was subsequently proved by N-terminal sequencing of two products of the protease. The success of this "blind" test inc...

  12. 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.

  13. A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14.

    Directory of Open Access Journals (Sweden)

    Takayuki Shindo

    Full Text Available Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa. In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.

  14. 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…

  15. Identification of cysteine proteases and screening of cysteine protease inhibitors in biological samples by a two-dimensional gel system of zymography and reverse zymography.

    Science.gov (United States)

    Saitoh, Eiichi; Yamamoto, Shinya; Okamoto, Eishiro; Hayakawa, Yoshimi; Hoshino, Takashi; Sato, Ritsuko; Isemura, Satoko; Ohtsubo, Sadami; Taniguchi, Masayuki

    2007-11-18

    We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37 degrees C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin beta chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.

  16. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus

    Directory of Open Access Journals (Sweden)

    Fabíola Dorneles Inácio

    2015-01-01

    Full Text Available Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine.

  17. Production and partial characterization of alkaline protease from bacillus subtilis mutant induced by gamma radiation

    International Nuclear Information System (INIS)

    Ibrahim, H.M.M.; Bashandy, A.S.

    2010-01-01

    Fourteen bacterial isolates belonging to B.subtilis were locally isolated from soil and screened for alkaline protease production. Only one strain, the highly potent one, was selected as alkaline protease producer and subjected to further studies to optimize its production. Alkaline protease production was maximum at 35 degree C after 72 h of incubation and at ph 10.0. molasses as a carbon source and combination of peptone and yeast extract as a nitrogen source enhanced greatly alkaline protease production. The mutant strain induced by gamma radiation showed higher alkaline protease production by 1.97 fold as compared with the parent strain. The alkaline protease enzyme was active at 40 degree C and ph 10. It was compatible with many commercial detergents and showed high stability (84 %) of its original activity with Ariel detergent. Moreover, alkaline protease enhanced the washing performance, and retained 95 % of its activity in the formulated dry powder.

  18. Molecular model of the specificity pocket of the hepatitis C virus protease: implications for substrate recognition.

    Science.gov (United States)

    Pizzi, E; Tramontano, A; Tomei, L; La Monica, N; Failla, C; Sardana, M; Wood, T; De Francesco, R

    1994-02-01

    We have built a model of the specificity pocket of the protease of hepatitis C virus on the basis of the known structures of trypsin-like serine proteases and of the conservation pattern of the protease sequences among various hepatitis C strains. The model allowed us to predict that the substrate of this protease should have a cysteine residue in position P1. This hypothesis was subsequently proved by N-terminal sequencing of two products of the protease. The success of this "blind" test increases our confidence in the overall correctness of our proposed alignment of the enzyme sequence with those of other proteases of known structure and constitutes a first step in the construction of a complete model of the viral protease domain.

  19. 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.

  20. Molecular Interactions at Membranes

    DEFF Research Database (Denmark)

    Jagalski, Vivien

    Biological membranes are essential and complex structures in every living cell consisting of a fluid lipid bilayer sheet and membrane proteins. Its significance makes biological membranes not only interesting for medical research, but also has made it a target for toxins in the course of evolution....... Today, we know more than ever before about the properties of biological membranes. Advanced biophysical techniques and sophisticated membrane models allow us to answer specific questions about the structure of the components within membranes and their interactions. However, many detailed structural...... mechanisms of membrane compounds, including compounds associated with membranes, are still unknown due to the challenges that arise when probing the hydrophobic nature of the membrane's interior. For integral membrane proteins that span through the entire membrane, the amphiphilic environment is essential...

  1. Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

    Science.gov (United States)

    Maddur, Ashoka A; Swanson, Richard; Izaguirre, Gonzalo; Gettins, Peter G W; Olson, Steven T

    2013-11-01

    Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to (i) serpin reactive center loop insertion into sheet A with full protease translocation but incomplete protease distortion followed by, (ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism.

  2. Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease.

    Science.gov (United States)

    de Almeida Nogueira, Natália Pereira; Morgado-Díaz, José Andrés; Menna-Barreto, Rubem Figueiredo Sadok; Paes, Marcia Cristina; da Silva-López, Raquel Elisa

    2013-10-01

    It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10(-5)M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10(-6)M ShPI-I, in the same periods of time, reduced parasite viability about 91-95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. The possible role of pancreatic proteases in the turnover of intestinal brush border proteins.

    Science.gov (United States)

    Alpers, D H; Tedesco, F J

    1975-08-05

    sucrase, maltase and lactase, but not alkaline phosphatase and trehalase. The enzyme most potent in this respect was the pancreatic protease, elastase. Non-proteolytic enzymes (amylase, lipase, phospholipase A) were inactive in removing enzyme from the brush border. The addition of elastase to pancreatectomized animals in vivo restored the rapid turnover rate of large brush border proteins. 6. A model is thus proposed for the normal catabolism of some large intestinal brush border proteins. It is suggested that the surface of intestinal absorptive cells is being constantly remodelled, and that certain surface enzymes are in part removed from the membrane by the action of pancreatic proteases. A possible special role for elastase is suggested.

  4. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

    International Nuclear Information System (INIS)

    Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

    1990-01-01

    The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

  5. Consequences of membrane protein overexpression in Escherichia coli.

    Science.gov (United States)

    Wagner, Samuel; Baars, Louise; Ytterberg, A Jimmy; Klussmeier, Anja; Wagner, Claudia S; Nord, Olof; Nygren, Per-Ake; van Wijk, Klaas J; de Gier, Jan-Willem

    2007-09-01

    Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a down-regulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.

  6. Magnetically controlled permeability membranes

    KAUST Repository

    Kosel, Jurgen

    2013-10-31

    A bioactive material delivery system can include a thermoresponsive polymer membrane and nanowires distributed within the thermoresponsive polymer membrane. Magnetic activation of a thermoresponsive polymer membrane can take place via altering the magnetization or dimensions of nanowires dispersed or ordered within the membrane matrix.

  7. Advances in zymography techniques and patents regarding protease analysis.

    Science.gov (United States)

    Wilkesman, Jeff; Kurz, Liliana

    2012-08-01

    Detection of enzymatic activity on gel electrophoresis, namely zymography, is a technique that has received increasing attention in the last 10 years, according to the number of articles published. A growing amount of enzymes, mainly proteases, are now routinely detected by zymography. Detailed analytical studies are beginning to be published, as well as new patents have been developed. This new article updates the information covered in our last review, condensing the recent publications dealing with the identification of proteolytic enzymes in electrophoretic gel supports and its variations. The new advances of this method are basically focused towards two dimensional zymography and transfer zymography. Though comparatively fewer patents have been published, they basically coincide in the study of matrix metalloproteases. The tendency is foreseen to be very productive in the area of zymoproteomics, combining electrophoresis and mass spectrometry for the analysis of proteases.

  8. Keratinolytic protease: a green biocatalyst for leather industry.

    Science.gov (United States)

    Fang, Zhen; Yong, Yang-Chun; Zhang, Juan; Du, Guocheng; Chen, Jian

    2017-11-01

    Depilation/unhairing is the crucial but heavy pollution process in leather industry. Traditional inorganic sulfide treatment was the most widely used depilation technique in the past decades, which was usually detrimental to leather quality and resulted in serious environmental pollution. Using biocatalysts to substitute inorganic sulfide showed great advantages in environment protection and unhairing efficiency. Keratinolytic protease is one of the excellent biocatalysts to hydrolyze disulfide bond-rich proteins of hair and has little damage to leather. Biological treatment with keratinolytic proteases could largely reduce the quantity and toxicity of wastewater effluent from the leather industry. But low thermostability and substrate specificity or specific activity of these enzymes limited their practical application. Therefore, recent progresses on protein engineering strategies (site-directed mutagenesis, protein fusion, N/C-terminus truncation, and domain swapping) used to enhance the keratinolytic enzyme performance were presented.

  9. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

    Science.gov (United States)

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-09-01

    Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process. Copyright © 2011 Elsevier GmbH. All rights reserved.

  10. Experience of islet isolation without neutral protease supplementation

    OpenAIRE

    Kin, Tatsuya; O'Gorman, Doug; Senior, Peter; Shapiro, AM James

    2010-01-01

    We have reported improved islet isolation outcomes using a new digestion protocol where the pancreas is perfused only with collagenase, and neutral protease (NP) is administered during the digestion phase. Since the inception of this protocol, we have had some cases where administration of NP was not required. Our new protocol was utilized in 94 islet isolations. The timing of adding NP was dependent on the progression of digestion but in 10 cases the progression was rapid and most islets in ...

  11. Schistosome serine protease inhibitors: parasite defense or homeostasis?

    Directory of Open Access Journals (Sweden)

    Landys A. Lopez Quezada

    2011-06-01

    Full Text Available Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL. Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases.Serpinas são uma família de inibidores macromoleculares estruturalmente conservados encontrados em inúmeros sistemas biológicos. O término e a anotação dos genomas de Schistosoma mansoni e de Schistosoma japonicum permitiram a identificação por análise filogenética de dois principais clados de serpinas. S. mansoni mostra uma multiplicidade maior de genes de serpinas, talvez refletindo uma adaptação à infecção de um hospedeiro humano. Alvos putativos das serpinas de esquistossomos podem ser preditos a partir da sequência do "loop" do centro reativo. Serpinas de esquistossomos podem ter importantes papeis tanto na regulação pós-traducional de proteases derivadas do esquistossoma, quanto nos mecanismos de defesa contra a ação de proteases do hospedeiro.

  12. Characterization and Preparation of Broken Rice Proteins Modified by Proteases

    Directory of Open Access Journals (Sweden)

    Lixia Hou

    2010-01-01

    Full Text Available Broken rice is an underutilized by-product of milling. Proteins prepared from broken rice by treatments with alkaline protease and papain have been characterized with regard to nutritional and functional properties. The protein content and the protein recovery were 56.45 and 75.45 % for alkaline protease treatment, and 65.45 and 46.32 % for papain treatment, respectively. Protease treatment increased the lysine and valine content, leading to a more balanced amino acid profile. Broken rice proteins had high emulsifying capacity, 58.3–71.6 % at neutral pH, and adequate water holding capacity, ranging from 1.96 to 2.93 g/g of proteins. At pH=7.0, the broken rice protein had the highest water holding capacity and the best interfacial activities (emulsifying capacity, emulsifying stability, foaming capacity and foaming stability, which may be the result of the higher solubility at pH=7.0. The interfacial activities increased with the increase in the mass fraction of broken rice proteins. The proteins prepared by the papain treatment had higher water holding capacity (p>0.05, emulsifying capacity (p0.05 than alkaline protease treatment at the same pH or mass fraction. To test the fortification of food products with broken rice proteins, pork sausages containing the proteins were prepared. Higher yield of the sausages was obtained with the increased content of broken rice proteins, in the range of 2.0–9.0 %. The results indicate that broken rice proteins have potential to be used as the protein fortification ingredient for food products.

  13. Engineering Environmentally-Stable Proteases to Specifically Neutralize Protein Toxins

    Science.gov (United States)

    2013-10-01

    synthesized as a fusion protein on the surface of M13 phage . The random library of subtilisin phage is mixed with the GA- PLFRAL-S-GB substrate...factor (LF), representing four functionally distinct families of toxins. The centerpiece of our design effort is a phage -display selection method for...hour assay. 15. SUBJECT TERMS Enterotoxin, protease, directed evolution, subtilisin, protein engineering, phage -display, enzymology 16. SECURITY

  14. Protease-catalysed Direct Asymmetric Mannich Reaction in Organic Solvent

    Science.gov (United States)

    Xue, Yang; Li, Ling-Po; He, Yan-Hong; Guan, Zhi

    2012-10-01

    We reported the first enzyme-catalysed, direct, three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus (SGP) in acetonitrile. Yields of up to 92% with enantioselectivities of up to 88% e.e. and diastereoselectivities of up to 92:8 (syn:anti) were achieved under the optimised conditions. This enzyme's catalytic promiscuity expands the application of this biocatalyst and provides a potential alternative method for asymmetric Mannich reactions.

  15. Rhomboid protease inhibitors: Emerging tools and future therapeutics

    Czech Academy of Sciences Publication Activity Database

    Stříšovský, Kvido

    2016-01-01

    Roč. 60, Dec (2016), s. 52-62 ISSN 1084-9521 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 EU Projects: European Commission(XE) 304154 - Rhomboid substrates Institutional support: RVO:61388963 Keywords : rhomboid protease * inhibitor * disease * mechanism * substrate specificity Subject RIV: CE - Biochemistry Impact factor: 6.614, year: 2016 http://www.sciencedirect.com/science/article/pii/S1084952116302592

  16. ISOLASI DAN KARAKTERISASI PROTEASE ALKALIN DARI ISOLAT BAKTERI LIMBAH TERNAK DI EXFARM FAKULTAS PETERNAKAN UNSOED

    Directory of Open Access Journals (Sweden)

    Zusfahair

    2011-05-01

    Full Text Available Protease is one of the widely used enzymes for the industry. The potential resource of microorganism that produced protease is milk cow waste. In this research, isolation and characterization has been done toward isolated protease from milk cow waste of the Exfarm’s Animal Husbandry Faculty at University of Jenderal Soedirman, Purwokerto. The research used experiment method and the parameters observed were the genus of bacteria which produce protease and the activity of protease. The characterizations of protease were determination of optimum pH and temperature, the influence of metal ions, EDTA, surfactant, and commercial detergent toward enzyme activity, and also the study of enzyme stability. The results from the research showed that the isolated bacteria from the Exfarm’s of Animal Husbandry Faculty of UNSOED, which produced protease was Salmonella sp. Characterization of isolated Salmonella sp. from 45% ammonium sulphate fraction indicated that the optimum temperature was 50 ºC, optimum pH was 8, the enzyme was activated by Ca2+ dan Mg2+ ion, whereas it was inhibited by Zn2+, Cu2+ ions and EDTA. The addition of Tween-80 with the concentration of 0.2% and 0.4% increased protease activity, however the addition of Tween-80 with concentration higher than 0.6% decreased the protease activity. Enzyme protease from isolated Salmonella sp. was relatively stable with the addition of commercial detergent such as Attack, Surf, and Bukrim.

  17. Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b

    Science.gov (United States)

    Li, Jing; Peng, Ying; Wang, Xianghong; Chi, Zhenming

    2010-12-01

    The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin-1. Under the optimal conditions, 72.5 UmL-1 of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.

  18. Detergent-compatible proteases: microbial production, properties, and stain removal analysis.

    Science.gov (United States)

    Niyonzima, Francois Niyongabo; More, Sunil

    2015-01-01

    Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods.

  19. Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2010-07-01

    Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Polymeric Membrane Reactors

    OpenAIRE

    José M. Sousa; Luís M. Madeira; João C. Santos; Adélio Mendes

    2008-01-01

    The aim of this chapter is the study of membrane reactors with polymeric membranes, particularly catalytic polymeric membranes. After an introduction where the main advantages and disadvantages of the use of polymeric membranes are summarised, a review of the main areas where they have been applied, integrated in chemical reactors, is presented. This excludes the field of bio-membranes processes, which is analysed in a specific chapter of this book. Particular attention is then given to model...

  3. Studies on detection and analysis of proteases in leaf extract of medicinally important plants.

    Science.gov (United States)

    Chinnadurai, Gandhi Shree; Krishnan, Sivakumar; Perumal, Palani

    2018-02-01

    The whole plant or the extracts obtained from them have long been used as medicine to treat various human diseases and disorders. Notably, those plants endowed with protease activity have been traditionally used as the agents for treating tumors, digestion disorders, swelling, blood coagulation, fibrinolysis and also for immune-modulation. Proteases occupy a pivotal position in enzyme based industries. Plant proteases have been increasingly exploited for pharmaceutical, food, leather and textile processing industries. Earlier investigations have focused on the occurrence of proteases in medicinally unimportant plants. Therefore it has been aimed to study the occurrence of proteolytic enzymes from medicinally important plants establish any correlation exists between protease activity and medicinal use of individual plants. Crude extract were obtained from the leaves of 80 different medicinal plants. Tris-HCl buffer was used as the extraction buffer and the supernatants obtained were used for determination of total protein and protease activity using spectrophotometric methods. Qualitative screening for the presence of protease was carried out with agar diffusion method by incorporating the substrate. SDS-PAGE was used to analyse the isoforms of protease and for determination of relative molecular mass. Relatively higher protease activities were observed in the extracts of leaves of Pongamia pinnata (Fabaceae), Wrightia tinctoria (Apocyanaceae) Acalypha indica (Euphorbiaceae), Adhatoda vasica (Acanthaceae) and Curcuma longa (Zingiberaceae). No correlation was found between the total protein content and protease activity in individual plant species. SDS-PAGE analysis indicated the presence of multiple forms of protease of higher molecular weight range in several plant species. We found a strong correlation between the protease activity and medicinal application of the plant CONCLUSION: The present study has unequivocally revealed that the leaves of medicinal plants

  4. Protease-activated receptors in kidney disease progression.

    Science.gov (United States)

    Palygin, Oleg; Ilatovskaya, Daria V; Staruschenko, Alexander

    2016-12-01

    Protease-activated receptors (PARs) are members of a well-known family of transmembrane G protein-coupled receptors (GPCRs). Four PARs have been identified to date, of which PAR1 and PAR2 are the most abundant receptors, and have been shown to be expressed in the kidney vascular and tubular cells. PAR signaling is mediated by an N-terminus tethered ligand that can be unmasked by serine protease cleavage. The receptors are activated by endogenous serine proteases, such as thrombin (acts on PARs 1, 3, and 4) and trypsin (PAR2). PARs can be involved in glomerular, microvascular, and inflammatory regulation of renal function in both normal and pathological conditions. As an example, it was shown that human glomerular epithelial and mesangial cells express PARs, and these receptors are involved in the pathogenesis of crescentic glomerulonephritis, glomerular fibrin deposition, and macrophage infiltration. Activation of these receptors in the kidney also modulates renal hemodynamics and glomerular filtration rate. Clinical studies further demonstrated that the concentration of urinary thrombin is associated with glomerulonephritis and type 2 diabetic nephropathy; thus, molecular and functional mechanisms of PARs activation can be directly involved in renal disease progression. We briefly discuss here the recent literature related to activation of PAR signaling in glomeruli and the kidney in general and provide some examples of PAR1 signaling in glomeruli podocytes. Copyright © 2016 the American Physiological Society.

  5. Semi purifikasi dan karakterisasi enzim protease Bacillus sp.

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    Elidar Naiola

    2012-02-01

    Full Text Available The aim of the research was to find the partial purified of enzyme protease from Bacillus sp. The crude enzyme of protease was produce in rice brand medium (100 gram of rice brand in a liter tofu liquid waste. The enzyme was semi-purified by the procedure of precipitation using ethanol in different percentages of saturation, gel filtration using Sephadex G 100 and Ion Exchanged Chromatography using DEAE Sephadex A50. Specific activities of the enzyme during purification were 5.71 U/mg (crude enzyme; 6.75 U/mg (ethanol precipitations; 37.16 U/mg (gel filtration and 43.02 U/mg (Ion Exchanged Chromatography. The optimum temperature for enzyme reaction was 45 €“50 °C, while the optimum pH was 7.0 €“8.0. Protease was relatively stable after heating until 37 €“50 °C for 60 minutes. Metal ions had different effects to the enzyme. CaCl2, FeCl3, MnCl2, ZnCl2 and MgCl2 increased enzyme activity, CdCl2 and HgCl2 gave an inhibitory effect, and another of metal ions had no effects to the enzyme.

  6. Protease activated receptors (PARS) mediation in gyroxin biological activity

    International Nuclear Information System (INIS)

    Silva, Jose Alberto Alves da

    2009-01-01

    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 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)

  7. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives

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    Nilesh P. Nirmal

    2014-01-01

    Full Text Available A fungal strain (Conidiobolus brefeldianus MTCC 5184 isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found to be very effective in increasing the stability of FAP, which was found to be concentration dependent. Fivefold increase in residual activity of FAP was observed in the presence of trehalose (50% and sorbitol (50% at 50°C for 4 h, compared to FAP without additive. Other additives like calcium at 20 mM and 10–15% ammonium sulphate showed lower stability improvement than trehalose and sorbitol. NaCl, MgCl2, K2HPO4, and glycine were found to be poor stabilizers and showed only a marginal improvement. PEG 6000 did not show any increase in stability but was found to be slightly inhibitory.

  8. Pathogen-secreted proteases activate a novel plant immune pathway.

    Science.gov (United States)

    Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J; Sheen, Jen; Ausubel, Frederick M

    2015-05-14

    Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

  9. Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

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    Surasak Jittavisutthikul

    2015-04-01

    Full Text Available There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN. Human hepatic (Huh7 cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β, indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.

  10. 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.

  11. PURIFIKASI DAN KARAKTERISASI PARSIAL ENZIM PROTEASE DARI GETAH TANAMAN BIDURI (Calotropis gigantea [Purification and Partial Characterization of Protease from Biduri (Calotropis gigantea Latex

    Directory of Open Access Journals (Sweden)

    Yuli Witono1

    2007-06-01

    Full Text Available The main objectives of this research we to purify protease from biduri (Calotropis gigantean latex and its partial characterization in relation with this application in the food processing. Protease was extracted from biduri latex by using ammonium sulphate 35-80%, dialyzed and then purified subsequently through sephadex G-25 gel and CM sephadex C-50 caution exchanger. Biduri protease has specific activity of 59 unit/g in casein substrate. Optimum pH was 7 and temperature 550C. Apparent Km was 21.63 g/ml and reaction maximum velocity (Vmax being 18.9 mg/ml/min. SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis analysis showed the apparent molecular weight of the protease was 25.2 kD. Moreover, the protease can be inactivated at 900C for 10 min, or 600C for 30 min.

  12. Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression.

    Science.gov (United States)

    Sepúlveda-González, M Eugenia; Parra-Ortega, Berenice; Betancourt-Cervantes, Yuliana; Hernández-Rodríguez, César; Xicohtencatl-Cortes, Juan; Villa-Tanaca, Lourdes

    2016-01-01

    The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen. Copyright © 2014 Asociación Española de Micología. Published by Elsevier Espana. All rights reserved.

  13. Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients.

    Science.gov (United States)

    Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard; Schemann, Michael

    2018-01-01

    The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin-the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to

  14. Mucus Detachment by Host Metalloprotease Meprin β Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB

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    Rielana Wichert

    2017-11-01

    Full Text Available The host metalloprotease meprin β is required for mucin 2 (MUC2 cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB. This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease.

  15. Thermodynamic and structural analysis of HIV protease resistance to darunavir - analysis of heavily mutated patient- derived HIV-1 proteases

    Czech Academy of Sciences Publication Activity Database

    Kožíšek, Milan; Lepšík, Martin; Grantz Šašková, Klára; Brynda, Jiří; Konvalinka, Jan; Řezáčová, Pavlína

    2014-01-01

    Roč. 281, č. 7 (2014), s. 1834-1847 ISSN 1742-464X R&D Projects: GA ČR GAP207/11/1798 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : enthropic contribution * HIV protease inhibitors * isothermal titration calorimetry * resistance mutation * X-ray crystallography Subject RIV: CE - Biochemistry Impact factor: 4.001, year: 2014

  16. The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

    Czech Academy of Sciences Publication Activity Database

    Dostál, Jiří; Brynda, Jiří; Hrušková-Heidingsfeldová, Olga; Pachl, Petr; Pichová, Iva; Řezáčová, Pavlína

    2012-01-01

    Roč. 27, č. 1 (2012), s. 160-165 ISSN 1475-6366 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945; GA ČR GA203/09/0820 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : secreted aspartic protease * virulence factor * X-ray structure * candidiasis Subject RIV: CE - Biochemistry Impact factor: 1.495, year: 2012

  17. GS-8374, a Prototype Phosphonate-Containing Inhibitor of HIV-1 Protease, Effectively Inhibits Protease Mutants with Amino Acid Insertions

    Czech Academy of Sciences Publication Activity Database

    Grantz Šašková, Klára; Kožíšek, Milan; Stray, K.; Jong de, D.; Řezáčová, Pavlína; Brynda, Jiří; Maarseveen van, N. M.; Nijhuis, M.; Cihlář, T.; Konvalinka, Jan

    2014-01-01

    Roč. 88, č. 6 (2014), s. 3586-3590 ISSN 0022-538X R&D Projects: GA ČR GAP207/11/1798 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : virus type-1 protease * antiviral activity * drug resistance Subject RIV: EE - Microbiology, Virology Impact factor: 4.439, year: 2014

  18. New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina

    Science.gov (United States)

    2010-01-01

    Background Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi. Results Phylogenetic analysis of 189 subtilisin-like serine protease genes from Pezizomycotina suggests five strongly-supported monophyletic clades. The subtilisin-like serine protease genes previously identified or presumed as endocellular proteases were clustered into one clade and diverged the earliest in the phylogeny. In addition, the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi were clustered together, indicating that they might have overlapping pathogenic mechanisms against insects and nematodes. Our experimental bioassays supported this conclusion. Interestingly, although they both function as cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together in the phylogenetic tree. Our evolutionary analysis revealed evidence for positive selection on the subtilisin-like serine protease genes of the nematode-trapping fungi. Conclusions Our study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. Pezizomycotina subtilisins most likely evolved

  19. Sheet Membrane Spacesuit Water Membrane Evaporator

    Science.gov (United States)

    Bue, Grant; Trevino, Luis; Zapata, Felipe; Dillion, Paul; Castillo, Juan; Vonau, Walter; Wilkes, Robert; Vogel, Matthew; Frodge, Curtis

    2013-01-01

    A document describes a sheet membrane spacesuit water membrane evaporator (SWME), which allows for the use of one common water tank that can supply cooling water to the astronaut and to the evaporator. Test data showed that heat rejection performance dropped only 6 percent after being subjected to highly contaminated water. It also exhibited robustness with respect to freezing and Martian atmospheric simulation testing. Water was allowed to freeze in the water channels during testing that simulated a water loop failure and vapor backpressure valve failure. Upon closing the backpressure valve and energizing the pump, the ice eventually thawed and water began to flow with no apparent damage to the sheet membrane. The membrane evaporator also serves to de-gas the water loop from entrained gases, thereby eliminating the need for special degassing equipment such as is needed by the current spacesuit system. As water flows through the three annular water channels, water evaporates with the vapor flowing across the hydrophobic, porous sheet membrane to the vacuum side of the membrane. The rate at which water evaporates, and therefore, the rate at which the flowing water is cooled, is a function of the difference between the water saturation pressure on the water side of the membrane, and the pressure on the vacuum side of the membrane. The primary theory is that the hydrophobic sheet membrane retains water, but permits vapor pass-through when the vapor side pressure is less than the water saturation pressure. This results in evaporative cooling of the remaining water.

  20. A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

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    Isadora Maria Villas Boas

    Full Text Available The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system.The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere

  1. Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

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    Luciana Bonome Zeminian

    2013-02-01

    Full Text Available The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3 have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

  2. Two SUMO Proteases SUMO PROTEASE RELATED TO FERTILITY1 and 2 Are Required for Fertility in Arabidopsis.

    Science.gov (United States)

    Liu, Linpo; Jiang, Ying; Zhang, Xiaomei; Wang, Xu; Wang, Yanbing; Han, Yuzhen; Coupland, George; Jin, Jing Bo; Searle, Iain; Fu, Yong-Fu; Chen, Fulu

    2017-12-01

    In plants, the posttranslational modification small ubiquitin-like modifier (SUMO) is involved in regulating several important developmental and cellular processes, including flowering time control and responses to biotic and abiotic stresses. Here, we report two proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2, that regulate male and female gamete and embryo development and remove SUMO from proteins in vitro and in vivo. spf1 mutants exhibit abnormal floral structures and embryo development, while spf2 mutants exhibit largely a wild-type phenotype. However, spf1 spf2 double mutants exhibit severe abnormalities in microgametogenesis, megagametogenesis, and embryo development, suggesting that the two genes are functionally redundant. Mutation of SPF1 and SPF2 genes also results in misexpression of generative- and embryo-specific genes. In vitro, SPF1 and SPF2 process SUMO1 precursors into a mature form, and as expected in vivo, spf1 and spf2 mutants accumulate SUMO conjugates. Using a yeast two-hybrid screen, we identified EMBRYO SAC DEVELOPMENT ARREST9 (EDA9) as an SPF1-interacting protein. In vivo, we demonstrate that EDA9 is sumolyated and that, in spf1 mutants, EDA9-SUMO conjugates increase in abundance, demonstrating that EDA9 is a substrate of SPF1. Together, our results demonstrate that SPF1 and SPF2 are two SUMO proteases important for plant development in Arabidopsis ( Arabidopsis thaliana ). © 2017 American Society of Plant Biologists. All Rights Reserved.

  3. Proteases and antiproteases in chronic neutrophilic lung disease - relevance to drug discovery.

    LENUS (Irish Health Repository)

    Greene, Catherine M

    2009-10-01

    Chronic inflammatory lung diseases such as cystic fibrosis and emphysema are characterized by higher-than-normal levels of pulmonary proteases. While these enzymes play important roles such as bacterial killing, their dysregulated expression or activity can adversely impact on the inflammatory process. The existence of efficient endogenous control mechanisms that can dampen or halt this overexuberant protease activity in vivo is essential for the effective resolution of inflammatory lung disease. The function of pulmonary antiproteases is to fulfil this role. Interestingly, in addition to their antiprotease activity, protease inhibitors in the lung also often possess other intrinsic properties that contribute to microbial killing or termination of the inflammatory process. This review will outline important features of chronic inflammation that are regulated by pulmonary proteases and will describe the various mechanisms by which antiproteases attempt to counterbalance exaggerated protease-mediated inflammatory events. These proteases, antiproteases and their modifiers represent interesting targets for therapeutic intervention.

  4. Tailored Ahp-cyclodepsipeptides as Potent Non-covalent Serine Protease Inhibitors.

    Science.gov (United States)

    Köcher, Steffen; Rey, Juliana; Bongard, Jens; Tiaden, André N; Meltzer, Michael; Richards, Peter J; Ehrmann, Michael; Kaiser, Markus

    2017-07-10

    The S1 serine protease family is one of the largest and most biologically important protease families. Despite their biomedical significance, generic approaches to generate potent, class-specific, bioactive non-covalent inhibitors for these enzymes are still limited. In this work, we demonstrate that Ahp-cyclodepsipeptides represent a suitable scaffold for generating target-tailored inhibitors of serine proteases. For efficient synthetic access, we developed a practical mixed solid- and solution-phase synthesis that we validated through performing the first chemical synthesis of the two natural products Tasipeptin A and B. The suitability of the Ahp-cyclodepsipeptide scaffold for tailored inhibitor synthesis is showcased by the generation of the most potent human HTRA protease inhibitors to date. We anticipate that our approach may also be applied to other serine proteases, thus opening new avenues for a systematic discovery of serine protease inhibitors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Cell Entry of Porcine Epidemic Diarrhea Coronavirus Is Activated by Lysosomal Proteases*

    Science.gov (United States)

    Liu, Chang; Ma, Yuanmei; Yang, Yang; Zheng, Yuan; Shang, Jian; Zhou, Yusen; Jiang, Shibo; Du, Lanying; Li, Jianrong; Li, Fang

    2016-01-01

    Porcine epidemic diarrhea coronavirus (PEDV) is currently devastating the United States pork industry by causing an 80–100% fatality rate in infected piglets. Coronavirus spike proteins mediate virus entry into cells, a process that requires the spike proteins to be proteolytically activated. It has been a conundrum which proteases activate PEDV entry. Here we systematically investigated the roles of different proteases in PEDV entry using pseudovirus entry, biochemical, and live virus infection assays. We found that the PEDV spike is activated by lysosomal cysteine proteases but not proprotein convertases or cell surface serine proteases. Extracellular trypsin activates PEDV entry when lysosomal cysteine proteases are inhibited. We further pinpointed cathepsin L and cathepsin B as the lysosomal cysteine proteases that activate the PEDV spike. These results advance our understanding of the molecular mechanism for PEDV entry and identify potential antiviral targets for curbing the spread of PEDV. PMID:27729455

  6. Effect of amino acids on the repression of alkaline protease synthesis in haloalkaliphilic Nocardiopsis dassonvillei

    Directory of Open Access Journals (Sweden)

    Amit K. Sharma

    2016-12-01

    Full Text Available A newly isolated salt-tolerant alkaliphilic actinomycete, Nocardiopsis dassonvillei strain OK-18 grows on mineral salts medium with glucose as carbon source. It also grows and produces protease with amino acids as sole carbon source. The synthesis of extracellular alkaline protease parallel to growth was repressible by substrate concentrations. The absolute production of the protease was delinked with growth under nutritional stress, as protease production was high, despite poor growth. When amino acids served as the sole source of carbon and nitrogen, the enzyme production was significantly controlled by the number of amino acids. Maximal protease production was achieved with proline, asparagine, tyrosine, alanine, methionine and valine as sole source of carbon and nitrogen in minimal medium. With the increasing number of different amino acids in the presence and absence of glucose, the protease production was synergistically lower as compared to complex medium.

  7. Impaired folding of the mitochondrial small TIM chaperones induces clearance by the i-AAA protease.

    Science.gov (United States)

    Baker, Michael J; Mooga, Ved P; Guiard, Bernard; Langer, Thomas; Ryan, Michael T; Stojanovski, Diana

    2012-12-14

    The intermembrane space of mitochondria contains a dedicated chaperone network-the small translocase of the inner membrane (TIM) family-for the sorting of hydrophobic precursors. All small TIMs are defined by the presence of a twin CX(3)C motif and the monomeric proteins are stabilized by two intramolecular disulfide bonds formed between the cysteines of these motifs. The conserved cysteine residues within small TIM members have also been shown to participate in early biogenesis events, with the most N-terminal cysteine residue important for import and retention within the intermembrane space via the receptor and disulfide oxidase, Mia40. In this study, we have analyzed the in vivo consequences of improper folding of small TIM chaperones by generating site-specific cysteine mutants and assessed the fate of the incompletely oxidized proteins within mitochondria. We show that no individual cysteine residue is required for the function of Tim9 or Tim10 in yeast and that defective assembly of the small TIMs induces their proteolytic clearance from mitochondria. We delineate a clearance mechanism for the mutant proteins and their unassembled wild-type partner protein by the mitochondrial ATP-dependent protease, Yme1 (yeast mitochondrial escape 1). Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. 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.

  9. Production and Preliminary Characterization of Alkaline Protease from Aspergillus flavus and Aspergillus terreus

    OpenAIRE

    Chellapandi, P.

    2010-01-01

    Proteases are being an industrial candidate, which are widely used in food, bakery, and beverage and detergent industry. In leather industry, alkaline proteases are exhibiting a prominent role in unhairing and bating processes. An extensive use of filamentous fungi, especially Aspergillus species has been studied elaborately. Although, the significant application of alkaline protease produced from these strains in leather industry is being limited. Aspergillus flavus and Aspergillus terreus f...

  10. Screening of Alkaline Protease-Producing Streptomyces diastaticus and Optimization of Enzyme Production

    OpenAIRE

    Elham Dawoodi; Keivan Beheshtimaal; Hashem Nayeri

    2014-01-01

    Background and Aim: Alkaline proteases are used in pharmaceutical, film and photography, silk production and food, leather and detergent industries. Actinomycetes are gram positive bacteria that produce different enzymes such as proteases. The aims of this research were isolation of native alkaline protease-producing Actinomycete spp. from different soil samples as well as optimizing the conditions for enzyme production. Materials and Methods: The different soil samples were collected fro...

  11. Use of MALDI-TOF Mass Spectrometry for Specificity Studies of Biomedically Important Proteases

    OpenAIRE

    Siigur, Jüri; Trummal, Katrin; TÕnismägi, Külli; Samel, Mari; Siigur, Ene; Vija, Heikki; Tammiste, Indrek; Subbi, Juhan

    2002-01-01

    Proteases play crucial role starting from fertilization until to cell death. Our studies of the two Viperidae venoms (Levantine viper Vipera lebetina, Common viper Vipera berus) have demonstrated the existence of biomedically important proteases, both coagulants and anticoagulants that may be useful as diagnostic tools or potential therapeutics. We showed that venoms of both snakes contain: (i) metalloproteases and serine proteases that degrade fibrinogen, but not fibrin; (ii) factor X activa...

  12. Cytokeratin 8 Is an Epithelial Cell Receptor for Pet, a Cytotoxic Serine Protease Autotransporter of Enterobacteriaceae

    OpenAIRE

    Nava-Acosta, Raul; Navarro-Garcia, Fernando

    2013-01-01

    ABSTRACT The group of proteins known as serine protease autotransporters of Enterobacteriaceae (SPATE) is a growing family of serine proteases secreted to the external milieu by the type V secretion system. Pet toxin and some other SPATE belong to the class 1 cytotoxic SPATE, which have comparable protease strength on fodrin. Pet is internalized and is directed to its intracellular substrate by retrograde transport. However, the epithelial cell receptor for Pet has yet to be identified. We sh...

  13. Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding*

    Science.gov (United States)

    Stewart, Sarah E.; Bird, Catherina H.; Tabor, Rico F.; D'Angelo, Michael E.; Piantavigna, Stefania; Whisstock, James C.; Trapani, Joseph A.; Martin, Lisandra L.; Bird, Phillip I.

    2015-01-01

    Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding. PMID:26542805

  14. The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts.

    Directory of Open Access Journals (Sweden)

    Steffen Frey

    Full Text Available During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

  15. Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein.

    Science.gov (United States)

    Azarnezhad, Asaad; Sharifi, Zohreh; Seyedabadi, Rahmatollah; Hosseini, Arshad; Johari, Behrooz; Sobhani Fard, Mahsa

    2016-01-01

    As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity. Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli ( E. coli) BL21 . Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA. Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100 μg/ml . Immunogenicity of rPR was confirmed by Western blotting and ELISA. Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86% specificity and 90% sensitivity in immunoassay tests.

  16. Purification and characterization of an alkaline protease from Micrococcus sp. isolated from the South China Sea

    Science.gov (United States)

    Hou, Enling; Xia, Tao; Zhang, Zhaohui; Mao, Xiangzhao

    2017-04-01

    Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0-11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn2+. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.

  17. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    International Nuclear Information System (INIS)

    Jin Xin; Li Jufang; Huang Pingying; Dong Xuyan; Guo Lulu; Yang Liang; Cao Yuancheng; Wei Fang; Zhao Yuandi

    2010-01-01

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  18. Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

    DEFF Research Database (Denmark)

    Studdert, C A; Herrera Seitz, M K; Plasencia, I

    2001-01-01

    other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests......A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5...... that Natronococcus occultus extracellular protease may be a novel enzyme. Udgivelsesdato: 2001-null...

  19. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    Energy Technology Data Exchange (ETDEWEB)

    Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  20. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    International Nuclear Information System (INIS)

    Spannaus, Ralf; Bodem, Jochen

    2014-01-01

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity

  1. Synthetic Biological Membrane (SBM)

    Data.gov (United States)

    National Aeronautics and Space Administration — The ultimate goal of the Synthetic Biological Membrane project is to develop a new type of membrane that will enable the wastewater treatment system required on...

  2. Oxygen transport membrane

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to a novel composite oxygen transport membrane as well as its preparation and uses thereof.......The present invention relates to a novel composite oxygen transport membrane as well as its preparation and uses thereof....

  3. Hybrid adsorptive membrane reactor

    Science.gov (United States)

    Tsotsis, Theodore T [Huntington Beach, CA; Sahimi, Muhammad [Altadena, CA; Fayyaz-Najafi, Babak [Richmond, CA; Harale, Aadesh [Los Angeles, CA; Park, Byoung-Gi [Yeosu, KR; Liu, Paul K. T. [Lafayette Hill, PA

    2011-03-01

    A hybrid adsorbent-membrane reactor in which the chemical reaction, membrane separation, and product adsorption are coupled. Also disclosed are a dual-reactor apparatus and a process using the reactor or the apparatus.

  4. Premature rupture of membranes

    Science.gov (United States)

    ... gov/ency/patientinstructions/000512.htm Premature rupture of membranes To use the sharing features on this page, ... water that surrounds your baby in the womb. Membranes or layers of tissue hold in this fluid. ...

  5. Transmembrane Signalling: Membrane messengers

    Science.gov (United States)

    Cockroft, Scott L.

    2017-05-01

    Life has evolved elaborate means of communicating essential chemical information across cell membranes. Inspired by biology, two new artificial mechanisms have now been developed that use synthetic messenger molecules to relay chemical signals into or across lipid membranes.

  6. Antiacanthain A: New proteases isolated from Bromelia antiacantha Bertol. (Bromeliaceae).

    Science.gov (United States)

    Vallés, Diego; Cantera, Ana M B

    2018-03-06

    Crude extract (CE) from pulp of Bromelia antiacantha Bertol. mature fruit, contains at least 3 cysteine proteases with proteolytic activity. By single step cation exchange chromatography (Hi-trap SP-HP) of partially purified CE, the protease with the lowest pI, Antiacanthain A (AntA), was isolated. It showed maximum activity at pH9, and 75% of remaining activity was maintained over a wide pH range (pH6-10). The AntA activity exhibits a constant increase up to 70°C. Maintains almost 100% of its activity at 45 at pH6 and 9. A 60% of AntA was active by titration with specific inhibitor, E64. Amidasic activity was studied with pyroglutamyl-phenyl-leucyl-paranitroaniline (PFLNA) substrate having higher AntA catalytic efficiency of (k cat /K m =470s -1 M -1 ) relative to stem bromelain (k cat /K m =305s -1 M -1 ). Esterase activity using p-nitrophenyl esters of N-α-CBZ-l-Lysine (z-L-LysONp) showed a 10-fold higher catalytic efficiency for AntA (k cat /K m =6376s -1 M -1 ) relative to stem bromelain (k cat /K m =688s -1 M -1 ). Incubation with 8M Urea did not affect AntA activity and remained unchanged for 18h, with 6M GndHCl resulted in a 41% decrease in activity after 30min incubation, maintained this activity 18h. AntA exhibits high sequence identity with proteases of the Bromeliaceae family. Copyright © 2018. Published by Elsevier B.V.

  7. Idiopathic epiretinal membrane

    NARCIS (Netherlands)

    Bu, Shao-Chong; Kuijer, Roelof; Li, Xiao-Rong; Hooymans, Johanna M M; Los, Leonoor I

    2014-01-01

    Background: Idiopathic epiretinal membrane (iERM) is a fibrocellular membrane that proliferates on the inner surface of the retina at the macular area. Membrane contraction is an important sight-threatening event and is due to fibrotic remodeling. Methods: Analysis of the current literature

  8. Model cell membranes

    DEFF Research Database (Denmark)

    Günther-Pomorski, Thomas; Nylander, Tommy; Cardenas Gomez, Marite

    2014-01-01

    The high complexity of biological membranes has motivated the development and application of a wide range of model membrane systems to study biochemical and biophysical aspects of membranes in situ under well defined conditions. The aim is to provide fundamental understanding of processes control...

  9. Membrane contactor applications

    NARCIS (Netherlands)

    Klaassen, R.; Feron, P.H.M.; Jansen, A.

    2008-01-01

    In a membrane contactor the membrane separation is completely integrated with an extraction or absorption operation in order to exploit the benefits of both technologies fully. Membrane contactor applications that have been developed can be found in both water and gas treatment. Several recently

  10. On "spinning" membrane models

    NARCIS (Netherlands)

    Bergshoeff, E.; Sezgin, E.; Townsend, P.K.

    1988-01-01

    Several alternative actions for a bosonic membrane have recently been proposed. We show that a linearly realized locally world-volume-supersymmetric (spinning membrane) extension of any of these actions implies an analogous extension of the standard Dirac membrane action. We further show that a

  11. Meniscus Membranes For Separation

    Science.gov (United States)

    Dye, Robert C.; Jorgensen, Betty; Pesiri, David R.

    2005-09-20

    Gas separation membranes, especially meniscus-shaped membranes for gas separations are disclosed together with the use of such meniscus-shaped membranes for applications such as thermal gas valves, pre-concentration of a gas stream, and selective pre-screening of a gas stream. In addition, a rapid screening system for simultaneously screening polymer materials for effectiveness in gas separation is provided.

  12. Meniscus membranes for separations

    Science.gov (United States)

    Dye, Robert C [Irvine, CA; Jorgensen, Betty [Jemez Springs, NM; Pesiri, David R [Aliso Viejo, CA

    2004-01-27

    Gas separation membranes, especially meniscus-shaped membranes for gas separations are disclosed together with the use of such meniscus-shaped membranes for applications such as thermal gas valves, pre-concentration of a gas stream, and selective pre-screening of a gas stream. In addition, a rapid screening system for simultaneously screening polymer materials for effectiveness in gas separation is provided.

  13. Plasma membrane ATPases

    DEFF Research Database (Denmark)

    Palmgren, Michael Broberg; Bækgaard, Lone; Lopez Marques, Rosa Laura

    2011-01-01

    membrane include ABC transporters, vacuolar (V-type) H+ pumps, and P-type pumps. These pumps all utilize ATP as a fuel for energizing pumping. This review focuses on the physiological roles of plasma membrane P-type pumps, as they represent the major ATP hydrolytic activity in this membrane....

  14. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    Science.gov (United States)

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

  15. Characterization of a novel aspartyl protease inhibitor from Haemonchus contortus

    OpenAIRE

    Li, Baojie; Gadahi, Javaid Ali; Gao, Wenxiang; Zhang, Zhenchao; Ehsan, Muhammad; Xu, Lixin; Song, Xiaokai; Li, Xiangrui; Yan, Ruofeng

    2017-01-01

    Background Aspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. Studies on Ostertagia ostertagi, Ascaris suum and Brugia malayi indicated that aspins might play roles in nematode infection. In a recent study, proteins differentially expressed between free-living third-stage larvae (L3) and activated L3 (xL3) of Haemonchus contortus were identified by 2D-DIGE. API was found downregulated in xL3 when compared with L3...

  16. Expression and activation of proteases in co-cultures.

    Science.gov (United States)

    Paduch, Roman; Kandefer-Szerszeń, Martyna

    2011-01-01

    The present study concerned the expression and activation of metalloproteinase-2 (MMP-2), metalloproteinase-9 (MMP-9) and the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system in co-cultures of human colon carcinoma cell spheroids (HT29, LS180, SW948) with human normal colon epithelium (CCD 841 CoTr), myofibroblasts (CCD-18Co) and endothelial cells (HUVEC). Additionally, the influence of monensin on the production and function of the proteases was tested. Tumor cells expressed small amounts of MMP-2, MMP-9 and uPA. Normal cells generally produced proportionally higher concentrations of these proteases (especially MMP-2, compared with significantly smaller yields of MMP-9 and significantly lower amounts of uPAR than tumors. In co-cultures of tumor spheroids with normal cell monolayers, the concentration of the proteases was equal to the sum of the enzymes produced in monocultures of both types of cells. The highest activity of uPA, measured as the reduction of the chromogenic substrate (S-2444), was detected in supernatants and lysates of endothelial cells. Interestingly, in normal cells, the higher expression of proteases, mainly uPA, measured as the level of protein concentration, was closely linked with their lower activity and inversely, in tumor cells, the low level of the expression of the enzymes correlated with their high enzymatic activity. In zymography analysis, mainly pro-MMPs were detected both in culture supernatants and cell lysates. The highest amounts of active forms of the MMPs were detected in tumor spheroids co-cultured with endothelial cells. Monensin inhibited MMPs and uPA secretion but significantly increased uPAR release, mainly from normal cells. In conclusion, during direct interactions of tumor cells with normal cells, MMPs and the uPA/uPAR system play an important role in the degradation of ECM and tumor development, but as we found, there is a reverse relationship between the concentration and the

  17. Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis

    Science.gov (United States)

    Elsholz, Alexander K. W.; Birk, Marlene S.; Charpentier, Emmanuelle; Turgay, Kürşad

    2017-01-01

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics. PMID:28748186

  18. Mutant HIV-1 protease complexed with tetrapeptide inhibitor

    Czech Academy of Sciences Publication Activity Database

    Skálová, Tereza; Hašek, Jindřich; Dohnálek, Jan; Petroková, Hana; Buchtelová, E.

    2002-01-01

    Roč. 101, - (2002), s. 659-663 ISSN 0587-4246. [Symposium on Synchrotron Crystallography. Krynica, 31.08.2001-04.09.2001] R&D Projects: GA AV ČR IAA4050811; GA ČR GV203/98/K023; GA ČR GA203/00/D117 Institutional research plan: CEZ:AV0Z4050913 Keywords : HIV-1 protease * ethylenamine inhibitor * X-ray diffraction Subject RIV: CD - Macromolecular Chemistry Impact factor: 0.345, year: 2002

  19. 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.

  20. Protective role of E. coli outer membrane vesicles against antibiotics.

    Science.gov (United States)

    Kulkarni, Heramb M; Nagaraj, R; Jagannadham, Medicharla V

    2015-12-01

    The outer membrane vesicles (OMVs) from bacteria are known to posses both defensive and protective functions and thus participate in community related functions. In the present study, outer membrane vesicles have been shown to protect the producer bacterium and two other bacterial species from the growth inhibitory effects of some antibiotics. The OMVs isolated from E. coli MG1655 protected the bacteria against membrane-active antibiotics colistin, melittin. The OMVs of E. coli MG1655 could also protect P. aeruginosa NCTC6751 and A. radiodioresistens MMC5 against these membrane-active antibiotics. However, OMVs could not protect any of these bacteria against the other antibiotics ciprofloxacin, streptomycin and trimethoprim. Hence, OMVs appears to protect the bacterial community against membrane-active antibiotics and not other antibiotics, which have different mechanism of actions. The OMVs of E. coli MG1655 sequester the antibiotic colistin, whereas their protein components degrade the antimicrobial peptide melittin. Proteomic analysis of OMVs revealed the presence of proteases and peptidases which appear to be involved in this process. Thus, the protection of bacteria by OMVs against antibiotics is situation dependent and the mechanism differs for different situations. These studies suggest that OMVs of bacteria form a common defense for the bacterial community against specific antibiotics. Copyright © 2015 Elsevier GmbH. All rights reserved.

  1. Perforin rapidly induces plasma membrane phospholipid flip-flop.

    Directory of Open Access Journals (Sweden)

    Sunil S Metkar

    Full Text Available The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.

  2. Picomolar Inhibition of Plasmepsin V, an Essential Malaria Protease, Achieved Exploiting the Prime Region.

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    Luca Gambini

    Full Text Available Malaria is an infectious disease caused by Plasmodium parasites. It results in an annual death-toll of ~ 600,000. Resistance to all medications currently in use exists, and novel antimalarial drugs are urgently needed. Plasmepsin V (PmV is an essential Plasmodium protease and a highly promising antimalarial target, which still lacks molecular characterization and drug-like inhibitors. PmV, cleaving the PExEl motif, is the key enzyme for PExEl-secretion, an indispensable parasitic process for virulence and infection. Here, we describe the accessibility of PmV catalytic pockets to inhibitors and propose a novel strategy for PmV inhibition. We also provide molecular and structural data suitable for future drug development. Using high-throughput platforms, we identified a novel scaffold that interferes with PmV in-vitro at picomolar ranges (~ 1,000-fold more active than available compounds. Via systematic replacement of P and P' regions, we assayed the physico-chemical requirements for PmV inhibition, achieving an unprecedented IC50 of ~20 pM. The hydroxyethylamine moiety, the hydrogen acceptor group in P2', the lipophilic groups upstream to P3, the arginine and other possible substitutions in position P3 proved to be critically important elements in achieving potent inhibition. In-silico analyses provided essential QSAR information and model validation. Our inhibitors act 'on-target', confirmed by cellular interference of PmV function and biochemical interaction with inhibitors. Our inhibitors are poorly performing against parasite growth, possibly due to poor stability of their peptidic component and trans-membrane permeability. The lowest IC50 for parasite growth inhibition was ~ 15 μM. Analysis of inhibitor internalization revealed important pharmacokinetic features for PExEl-based molecules. Our work disclosed novel pursuable drug design strategies for highly efficient PmV inhibition highlighting novel molecular elements necessary for

  3. ANALISIS POTENSI PROTEASE EKTRASELULER TANAH HUTAN MANGROVE PANTAI SUWUNG KAUH BALI

    Directory of Open Access Journals (Sweden)

    Inten Hardianti Nizar

    2015-10-01

    Full Text Available ABSTRAK: Potensi tanah hutan mangrove pantai Suwung Kauh Bali sebagai sumber protease dapat diketahui dengan melakukan uji aktivitas protease ekstraseluler. Pada penelitian ini telah dilakukan pengukuran aktivitas protease ekstraseluler dan penentuan pengaruh waktu inkubasi serta penambahan toluena terhadap aktivitas protease. Sampel yang digunakan sebagai sumber enzim berupa slurry dan direaksikan dengan substrat kasein 0,3% selama 3,6,9 dan 24 jam dengan dan tanpa penambahan toluena 1% (v/v. Produk reaksi enzimatis diukur dengan metode kolorimetri. Aktivitas protease tertinggi yang diperoleh sebesar 1,9 x 10-4 U/mL dengan penambahan toluena pada waktu inkubasi 6 jam dan sebesar 1,2 x 10-4 U/mL tanpa penambahan toluena pada waktu inkubasi 9 jam. Hasil ini menunjukkan bahwa protease ekstraseluler pada tanah hutan mangrove yang dihasilkan oleh mikroba proteolitik memilki potensi digunakan untuk eksplorasi enzim. Waktu inkubasi dan penambahan toluena tidak berpengaruh signifikan terhadap aktivitas protease.   ABSTRACT: The potency of mangrove soil in Suwung Kauh Bali as a source of protease has been determined by protease activity assay. This research has been done to determine protease activity and the effect of incubation time and the addition of toluene to the protease activity. The slurry of soil was used as a source of extracellular  enzyme for protease assay, which was reacted with casein 0,3% for 3, 6, 9, and 24 hours with and without the addition of toluene 1% (v/v. The enzymatic reaction product was measured by colorimetric method. The highest protease activity with addition of toluene was 1,9 x 10-4 U/mL at 6 hours incubation and without toluene was 1,2 x 10-4 U/mL at 9 hours incubation. These results showed extracelluler protease on mangrove soil produced by proteolytic microorganisms had a potency to be used in enzyme exploration. Furthermore, the incubation time and addition of toluene had no significant effect to protease activity.

  4. Feces derived allergens of Tyrophagus putrescentiae reared on dried dog food and evidence of the strong nutritional interaction between the mite and Bacillus cereus producing protease bacillolysins and exo-chitinases

    Directory of Open Access Journals (Sweden)

    Tomas eErban

    2016-02-01

    Full Text Available Tyrophagus putrescentiae (Schrank, 1781 is an emerging source of allergens in stored products and homes. Feces proteases are the major allergens of astigmatid mites (Acari: Acaridida. In addition, the mites are carriers of microorganisms and microbial adjuvant compounds that stimulate innate signaling pathways. We sought to analyze the mite feces proteome, proteolytic activities and mite-bacterial interaction in dry dog food. Proteomic methods comprising enzymatic and zymographic analysis of proteases and 2D-E-MS/MS were performed. The highest protease activity was assigned to trypsin-like proteases; lower activity was assigned to chymotrypsin-like proteases, and the cysteine protease cathepsin B-like had very low activity. The 2D-E-MS/MS proteomic analysis identified mite trypsin allergen Tyr p3, fatty acid-binding protein Tyr p13 and putative mite allergens ferritin (Grp 30 and (polyubiquitins. Tyr p3 was detected at different positions of the 2D-E. It indicates presence of zymogen at basic pI, and mature-enzyme form and enzyme fragment at acidic pI. Bacillolysins (neutral and alkaline proteases of Bacillus cereus symbiont can contribute to the protease activity of the mite extract. The bacterial exo-chitinases likely contribute to degradation of mite exuviae, mite bodies or food boluses consisting of chitin, including the peritrophic membrane. Thus, the chitinases disrupt the feces and facilitate release of the allergens. B. cereus was isolated and identified based on amplification and sequencing of 16S rRNA and motB genes. B. cereus was added into high-fat, high-protein (dry dog food and low-fat, low-protein (flour diets to 1% and 5% (w/w, and the diets palatability was evaluated in 21-day population growth test. The supplementation of diet with B. cereus significantly suppressed population growth and the suppressive effect was higher in the high-fat, high-protein diet than in the low-fat, low-protein food. Thus, B. cereus has to coexist

  5. An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease

    Directory of Open Access Journals (Sweden)

    Vikram Surendra

    2010-07-01

    Full Text Available Abstract Background Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min. Results Seventy bacterial strains isolated from the soils of Eastern Uttar Pradesh, India were screened for protease production. All of them exhibited protease activity. However, 40% bacterial isolates were found good protease producers as observed by caseinolytic zones on milk agar plates. Among them, culture S-4 was adjudged as the best protease producer, and was identified as Bacillus cereus by morphological, biochemical and 16 S rDNA sequence analyses. The isolate was resistant to heavy metals (As2+, Pb2+, Cs1+ and antibiotics (penicillin, lincomycin, cloxacillin, pefloxacin. Its growth behavior and protease production was studied at 45°C and pH 9.0. The protease units of 88 ml-1 were noted in unoptimized modified glucose yeast extract (GYE medium during early stationary phase at 20 h incubation period. The enzyme was stable in the temperature range of 35°-55°C. Conclusions An antibiotic and heavy metal resistant, halotolerant Bacillus cereus isolate is capable of producing thermoalkaline protease, which is active and stable at pH 9.0 and 35°-55°C. This isolate may be useful in several industrial applications owing to its halotolerance and antibiotic and heavy metal resistance characteristics.

  6. Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, Masashi; Li, Shimo; Itoh, Masanori; Wang, Miao-xing; Hayakawa, Miki; Islam, Saiful; Tana; Nakagawa, Kiyomi [Department of Neurobiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan); Chen, Huayue [Department of Anatomy, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan); Nakagawa, Toshiyuki, E-mail: tnakagaw@gifu-u.ac.jp [Department of Neurobiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194 (Japan)

    2016-05-27

    The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ER luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax. -- Highlights: •We characterized polyglutamine embedded in the ER membrane. •The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. •The ER membrane may be a target of polyglutamine, which triggers cell death.

  7. Microporous silica membranes

    DEFF Research Database (Denmark)

    Boffa, Vittorio; Yue, Yuanzheng

    2012-01-01

    Hydrothermal stability is a crucial factor for the application of microporous silica-based membranes in industrial processes. Indeed, it is well established that steam exposure may cause densification and defect formation in microporous silica membranes, which are detrimental to both membrane...... permeability and selectivity. Numerous previous studies show that microporous transition metal doped-silica membranes are hydrothermally more stable than pure silica membranes, but less permeable. Here we present a quantitative study on the impact of type and concentration of transition metal ions...... on the microporous structure, stability and permeability of amorphous silica-based membranes, providing information on how to design chemical compositions and synthetic paths for the fabrication of silica-based membranes with a well accessible and highly stabile microporous structure....

  8. Clustering on Membranes

    DEFF Research Database (Denmark)

    Johannes, Ludger; Pezeshkian, Weria; Ipsen, John H

    2018-01-01

    Clustering of extracellular ligands and proteins on the plasma membrane is required to perform specific cellular functions, such as signaling and endocytosis. Attractive forces that originate in perturbations of the membrane's physical properties contribute to this clustering, in addition to direct...... protein-protein interactions. However, these membrane-mediated forces have not all been equally considered, despite their importance. In this review, we describe how line tension, lipid depletion, and membrane curvature contribute to membrane-mediated clustering. Additional attractive forces that arise...... from protein-induced perturbation of a membrane's fluctuations are also described. This review aims to provide a survey of the current understanding of membrane-mediated clustering and how this supports precise biological functions....

  9. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Directory of Open Access Journals (Sweden)

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  10. Short hydrogen bonds in the catalytic mechanism of serine proteases

    Directory of Open Access Journals (Sweden)

    VLADIMIR LESKOVAC

    2008-04-01

    Full Text Available The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78–1.28 Å revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 Å long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short “low-barrier” hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the “low-barrier hydrogen bond” hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 Å long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the “low-barrier hydrogen bond” hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

  11. Arginine phosphorylation marks proteins for degradation by a Clp protease.

    Science.gov (United States)

    Trentini, Débora Broch; Suskiewicz, Marcin Józef; Heuck, Alexander; Kurzbauer, Robert; Deszcz, Luiza; Mechtler, Karl; Clausen, Tim

    2016-11-03

    Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC-ClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC-ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC-ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin-proteasome system.

  12. Rapid monitoring of autolysis process of proteases by capillary electrophoresis.

    Science.gov (United States)

    Chen, Xiu-Lan; Shun, Cai-Yun; Zhang, Yu-Zhong; Gao, Pei-Ji

    2003-10-01

    A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 degrees C-50 degrees C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 degrees C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 degrees C. Similar results were obtained when MCP-01 was incubated at 20 degrees C, 40 degrees C and 50 degrees C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.

  13. Specificity and Application of the Lantibiotic Protease NisP

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    Manuel Montalbán-López

    2018-02-01

    Full Text Available Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.

  14. Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jun; Shen, Wei [Laboratory of Structural Biology, Tsinghua University, Beijing 100084 (China); Liao, Ming, E-mail: mliao@scau.edu.cn [Laboratory of Avian Medicine, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642 (China); Bartlam, Mark, E-mail: mliao@scau.edu.cn [Laboratory of Structural Biology, Tsinghua University, Beijing 100084 (China); National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2007-01-01

    The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M{sup pro} was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6{sub 1}22. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.

  15. Epigenetic silencing of serine protease HTRA1 drives polyploidy

    International Nuclear Information System (INIS)

    Schmidt, Nina; Irle, Inga; Ripkens, Kamilla; Lux, Vanda; Nelles, Jasmin; Johannes, Christian; Parry, Lee; Greenow, Kirsty; Amir, Sarah; Campioni, Mara; Baldi, Alfonso; Oka, Chio; Kawaichi, Masashi; Clarke, Alan R.; Ehrmann, Michael

    2016-01-01

    Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation. The online version of this article (doi:10.1186/s12885-016-2425-8) contains supplementary material, which is available to authorized users

  16. Variably protease-sensitive prionopathy in the UK: a retrospective review 1991-2008

    NARCIS (Netherlands)

    Head, M.W.; Yull, H.M.; Ritchie, D.L.; Langeveld, J.P.M.; Fletcher, N.A.; Knight, R.S.; Ironside, J.W.

    2013-01-01

    Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified

  17. Isolation, identification and optimization of alkaline protease production by Candida viswanathii

    Directory of Open Access Journals (Sweden)

    Mandana Lotfi

    2014-03-01

    Conclusion: Due to the high demand for industrial enzymes in the Country and the high activity of alkaline proteases produced by strain. It seems that the native strain can achieve high production of alkaline proteases.These native strains could be resulted in the independence of our country in industrial enzymes production.

  18. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang [Wayne State Univ., Detroit, MI (United States); Case Western Reserve Univ., Cleveland, OH (United States); Harbor Hospital Baltimore, MD (United States); Wang, Yong [Wayne State Univ., Detroit, MI (United States); Yedidi, Ravikiran S. [Wayne State Univ., Detroit, MI (United States); National Institutes of Health, Bethesda, MD (United States); Dewdney, Tamaria G. [Wayne State Univ., Detroit, MI (United States); Reiter, Samuel J. [Wayne State Univ., Detroit, MI (United States); Brunzelle, Joseph S. [Northwestern Univ. Feinberg School of Medicine, Chicago, IL (United States); Kovari, Iulia A. [Wayne State Univ., Detroit, MI (United States); Kovari, Ladislau C. [Wayne State Univ., Detroit, MI (United States)

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  19. Effect of Legionella pneumophila cytotoxic protease on human neutrophil and monocyte function

    DEFF Research Database (Denmark)

    Rechnitzer, C; Kharazmi, A

    1992-01-01

    by the protease in both cell types. Lastly, the protease inhibited the killing of Listeria monocytogenes by neutrophils or monocytes. Inhibition of Listeria killing was concentration-dependent, heat-labile, and did not require the presence of the enzyme in the bactericidal assay. The inhibitory activity of L...

  20. Crystal Structure of a Novel Viral Protease with a Serine/Lysine Catalytic Dyad Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Feldman,A.; Lee, J.; Delmas, B.; Paetzel, M.

    2006-01-01

    The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.

  1. 21 CFR 184.1150 - Bacterially-derived protease enzyme preparation.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bacterially-derived protease enzyme preparation... enzyme preparation. (a) Bacterially-derived protease enzyme preparation is obtained from the culture... subtilis or B. amyloliquefaciens. The preparation is characterized by the presence of the enzymes...

  2. 21 CFR 184.1027 - Mixed carbohydrase and protease enzyme product.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Mixed carbohydrase and protease enzyme product. 184... enzyme product. (a) Mixed carbohydrase and protease enzyme product is an enzyme preparation that includes... current good manufacturing practice conditions of use: (1) The ingredient is used as an enzyme, as defined...

  3. 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...

  4. Recovery of serine protease inhibitor from fish roes by polyethylene glycol precipitation

    Directory of Open Access Journals (Sweden)

    Hyun Ji Lee

    2016-07-01

    Full Text Available Abstract The fractionation of serine protease inhibitor (SPI from fish roe extracts was carried out using polyethylene glycol-4000 (PEG4000 precipitation. The protease inhibitory activity of extracts and PEG fractions from Alaska pollock (AP, bastard halibut (BH, skipjack tuna (ST, and yellowfin tuna (YT roes were determined against target proteases. All of the roe extracts showed inhibitory activity toward bromelain (BR, chymotrypsin (CH, trypsin (TR, papain-EDTA (PED, and alcalase (AL as target proteases. PEG fractions, which have positive inhibitory activity and high recovery (%, were the PEG1 fraction (0–5 %, w/v against cysteine proteases (BR and PA and the PEG4 fraction (20–40 %, w/v against serine proteases (CH and TR. The strongest specific inhibitory activity toward CH and TR of PEG4 fractions was AP (9278 and 1170 U/mg followed by ST (6687 and 2064 U/mg, YT (3951 and 1536 U/mg, and BH (538 and 98 U/mg. The inhibitory activity of serine protease in extracts and PEG fractions from fish roe was stronger than that of cysteine protease toward common casein substrate. Therefore, SPI is mainly distributed in fish roe and PEG fractionation effectively isolated the SPI from fish roes.

  5. EFFECTS OF CHANGING THE INTERACTION BETWEEN SUBDOMAINS ON THE THERMOSTABILITY OF BACILLUS NEUTRAL PROTEASES

    NARCIS (Netherlands)

    EIJSINK, VGH; VRIEND, G; VANDERVINNE, B; HAZES, B; VANDENBURG, B; VENEMA, G

    1992-01-01

    Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to

  6. STABILIZATION OF BACILLUS-STEAROTHERMOPHILUS NEUTRAL PROTEASE BY INTRODUCTION OF PROLINES

    NARCIS (Netherlands)

    HARDY, F; VRIEND, G; VELTMAN, OR; VANDERVINNE, B; VENEMA, G; EIJSINK, VGH

    1993-01-01

    The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by

  7. Kinetics of the dimerization of retroviral proteases: The "fireman's grip" and dimerization

    Czech Academy of Sciences Publication Activity Database

    Ingr, Marek; Kondrová, Taťána; Stříšovský, Kvido; Majerová, E.; Konvalinka, Jan

    2003-01-01

    Roč. 12, - (2003), s. 2173-2182 ISSN 0961-8368 R&D Projects: GA MZd NI6339 Institutional research plan: CEZ:AV0Z4055905 Keywords : retroviral protease * dimerization * HIV protease Subject RIV: CE - Biochemistry Impact factor: 3.787, year: 2003

  8. Continuous-flow protease assay based on fluorescence resonance energy transfer

    NARCIS (Netherlands)

    Hirata, J.; Ariese, F.; Gooijer, C.; Irth, H.

    2003-01-01

    A homogeneous continuous-flow assay using fluorescence resonance energy transfer (FRET) for detection was developed to measure the hydrolysis of HIV Protease Substrate 1 (to which two choromophores, EDANS and DABCYL are covalently attached) by a protease (e.g. Subtilisin Carlsberg) and the influence

  9. A modular system to evaluate the efficacy of protease inhibitors against HIV-2.

    Directory of Open Access Journals (Sweden)

    Mohamed Mahdi

    Full Text Available The human immunodeficiency virus (HIV protease is a homodimeric aspartyl protease that is crucial for the viral life-cycle, cleaving proviral polyproteins, hence creating mature protein components that are required for the formation of an infectious virus. With diagnostic measures and clinically used protease inhibitors focusing on HIV-1, due to its higher virulence and prevalence, studies of the efficacy of those inhibitors on HIV-2 protease remain widely lacking. Utilizing a wild-type HIV-2 vector backbone and cloning techniques we have developed a cassette system where the efficacy of clinically used protease inhibitors can be studied for various serotypes of HIV-2 protease both in enzymatic and cell culture assays. In our experiments, optimization of the expression protocol led to a relatively stable enzyme, for cell culture assays, the efficiency of transfection and transduction capability of the modified vector was tested and was not found to differ from that of the wild-type, moreover, a 2nd generation protease inhibitor was used to demonstrate the usefulness of the system. The combination of assays performed with our cassette system is expected to provide an accurate measure of the efficacy of currently used; as well as experimental protease inhibitors on HIV-2.

  10. A modular system to evaluate the efficacy of protease inhibitors against HIV-2.

    Science.gov (United States)

    Mahdi, Mohamed; Matúz, Krisztina; Tóth, Ferenc; Tőzsér, József

    2014-01-01

    The human immunodeficiency virus (HIV) protease is a homodimeric aspartyl protease that is crucial for the viral life-cycle, cleaving proviral polyproteins, hence creating mature protein components that are required for the formation of an infectious virus. With diagnostic measures and clinically used protease inhibitors focusing on HIV-1, due to its higher virulence and prevalence, studies of the efficacy of those inhibitors on HIV-2 protease remain widely lacking. Utilizing a wild-type HIV-2 vector backbone and cloning techniques we have developed a cassette system where the efficacy of clinically used protease inhibitors can be studied for various serotypes of HIV-2 protease both in enzymatic and cell culture assays. In our experiments, optimization of the expression protocol led to a relatively stable enzyme, for cell culture assays, the efficiency of transfection and transduction capability of the modified vector was tested and was not found to differ from that of the wild-type, moreover, a 2nd generation protease inhibitor was used to demonstrate the usefulness of the system. The combination of assays performed with our cassette system is expected to provide an accurate measure of the efficacy of currently used; as well as experimental protease inhibitors on HIV-2.

  11. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group

    Energy Technology Data Exchange (ETDEWEB)

    Bungard, Christopher J.; Williams, Peter D.; Ballard, Jeanine E.; Bennett, David J.; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S.; Chang, Ronald K.; Dubost, David C.; Fay, John F.; Diamond, Tracy L.; Greshock, Thomas J.; Hao, Li; Holloway, M. Katharine; Felock, Peter J.; Gesell, Jennifer J.; Su, Hua-Poo; Manikowski, Jesse J.; McKay, Daniel J.; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M.; Nantermet, Philippe G.; Nadeau, Christian; Sanchez, Rosa I.; Satyanarayana, Tummanapalli; Shipe, William D.; Singh, Sanjay K.; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M.; Vacca, Joseph P.; Crane, Sheldon N.; McCauley, John A. (Merck); (Albany MR)

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

  12. Activity of the fungus Pleurotus ostreatus and of its proteases on ...

    African Journals Online (AJOL)

    Biological control has been shown to be one of the possible biotechnological applications of fungi and their proteases. The objective of this study was to evaluate the nematicidal activity of the fungus Pleurotus ostreatus and its proteases on Panagrellus sp. larvae. Proteolytic activity of P. ostreatus (PLO 06) was measured ...

  13. Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis

    Czech Academy of Sciences Publication Activity Database

    Kádek, Alan; Tretyachenko, V.; Mrázek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, M.; Schriemer, D. C.; Man, Petr

    2014-01-01

    Roč. 95, MAR 2014 (2014), s. 121-128 ISSN 1046-5928 R&D Projects: GA ČR GAP206/12/0503; GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : Plant aspartic protease * Nepenthesin * Protease characterization Subject RIV: CE - Biochemistry Impact factor: 1.695, year: 2014

  14. Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus

    NARCIS (Netherlands)

    van der Laan, J.M.; Teplyakov, A.V.; Kelders, H.; Kalk, K.H.; Misset, O.; Mulleners, L.J.S.M.; Dijkstra, B.W.

    The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 Å resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has

  15. A novel substrate-based HIV-1 protease inhibitor drug resistance mechanism

    Czech Academy of Sciences Publication Activity Database

    Nijhuis, M.; Maarseveen van, N. M.; Lastere, S.; Schipper, P.; Coakley, E.; Glass, B.; Rovenská, Miroslava; Jong de, D.; Chappey, C.; Goedegebuure, I. W.; Heilek-Snyder, G.; Dulude, D.; Cammack, N.; Brakier-Gingras, L.; Konvalinka, Jan; Parkin, N.; Kräusslich, H. G.; Brun-Vezinet, F.; Boucher, Ch. A. B.

    2007-01-01

    Roč. 4, č. 1 (2007), s. 152-163 ISSN 1549-1277 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV-1 protease * drug resistance * protease inhibitor Subject RIV: CE - Biochemistry Impact factor: 12.601, year: 2007

  16. Serine proteases of the human immune system in health and disease

    NARCIS (Netherlands)

    Heutinck, Kirstin M.; ten Berge, Ineke J. M.; Hack, C. Erik; Hamann, Jörg; Rowshani, Ajda T.

    2010-01-01

    Serine proteases form a large family of protein-cleaving enzymes that play an essential role in processes like blood coagulation, apoptosis and inflammation. Immune cells express a wide variety of serine proteases such as granzymes in cytotoxic lymphocytes, neutrophil elastase, cathepsin G and

  17. Discovery of novel phosphonate derivatives as hepatitis C virus NS3 protease inhibitors.

    Science.gov (United States)

    Sheng, X Christopher; Pyun, Hyung-Jung; Chaudhary, Kleem; Wang, Jianying; Doerffler, Edward; Fleury, Melissa; McMurtrie, Darren; Chen, Xiaowu; Delaney, William E; Kim, Choung U

    2009-07-01

    A novel class of phosphonate derivatives was designed to mimic the interaction of product-like carboxylate based inhibitors of HCV NS3 protease. A phosphonic acid (compound 2) was demonstrated to be a potent HCV NS3 protease inhibitor, and a potential candidate for treating HCV infection. The syntheses and preliminary biological evaluation of this phosphonate class of inhibitor are described.

  18. Gold compounds as cysteine protease inhibitors: perspectives for pharmaceutical application as antiparasitic agents.

    Science.gov (United States)

    Massai, Lara; Messori, Luigi; Micale, Nicola; Schirmeister, Tanja; Maes, Louis; Fregona, Dolores; Cinellu, Maria Agostina; Gabbiani, Chiara

    2017-04-01

    Gold compounds form a new class of promising metal-based drugs with a number of potential therapeutic applications, particularly in the fields of anticancer and antimicrobial treatments. Previous research revealed that a group of structurally diverse gold compounds cause conspicuous inhibition of the protease activities of the human proteasome. Given the pharmacological importance of protease inhibition, the present study further explored whether these gold compounds might inhibit a few other proteases that are accepted druggable targets for disease treatment. In particular, four distinct cysteine proteases were considered here: cathepsin B and L that play a primary role in tumor-cell invasion and metastasis; rhodesain, the major cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense and CPB2.8ΔCTE, a Leishmania mexicana mature cysteine protease. Based on the encouraging results obtained for some of the tested gold compounds on the two parasitic cysteine proteases, especially against CPB2.8ΔCTE, with IC 50s in the micromolar range, we next evaluated whether those gold compounds might contrast effectively the growth of the respective protozoa and indeed important antiprotozoal properties were disclosed; on the other hand a certain lack of selectivity was highlighted. Also, no direct or clear correlation could be established between the in vitro antiprotozoal properties and the level of protease inhibition. The implications of these results are discussed in relation to possible pharmaceutical applications.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Application of dynamic membranes in anaerobic membranes in anaerobic membrane bioreactor systems

    NARCIS (Netherlands)

    Erşahin, M.E.

    2015-01-01

    Anaerobic membrane bioreactors (AnMBRs) physically ensure biomass retention by the application of a membrane filtration process. With growing application experiences from aerobic membrane bioreactors (MBRs), the combination of membrane and anaerobic processes has received much attention and become

  1. Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor.

    Science.gov (United States)

    Erlandson, Martin A; Hegedus, Dwayne D; Baldwin, Douglas; Noakes, Amy; Toprak, Umut

    2010-10-01

    The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources.

  2. Supported ionic liquid membrane in membrane reactor

    Science.gov (United States)

    Makertihartha, I. G. B. N.; Zunita, M.; Dharmawijaya, P. T.; Wenten, I. G.

    2017-01-01

    Membrane reactor is a device that integrates membrane based separation and (catalytic) chemical reaction vessel in a single device. Ionic liquids, considered to be a relatively recent magical chemical due to their unique properties, have a large variety of applications in all areas of chemical industries. Moreover, the ionic liquid can be used as membrane separation layer and/or catalytically active site. This paper will review utilization of ionic liquid in membrane reactor related applications especially Fischer-Tropsch, hydrogenation, and dehydrogenation reaction. This paper also reviews about the capability of ionic liquid in equilibrium reaction that produces CO2 product so that the reaction will move towards the product. Water gas shift reaction in ammonia production also direct Dimethyl Ether (DME) synthesis that produces CO2 product will be discussed. Based on a review of numerous articles on supported ionic liquid membrane (SILM) indicate that ionic liquids have the potential to support the process of chemical reaction and separation in a membrane reactor.

  3. Characterization of a New S8 serine Protease from Marine SedimentaryPhotobacteriumsp. A5-7 and the Function of Its Protease-Associated Domain.

    Science.gov (United States)

    Li, Hui-Juan; Tang, Bai-Lu; Shao, Xuan; Liu, Bai-Xue; Zheng, Xiao-Yu; Han, Xiao-Xu; Li, Ping-Yi; Zhang, Xi-Ying; Song, Xiao-Yan; Chen, Xiu-Lan

    2016-01-01

    Bacterial extracellular proteases are important for bacterial nutrition and marine sedimentary organic nitrogen degradation. However, only a few proteases from marine sedimentary bacteria have been characterized. Some subtilases have a protease-associated (PA) domain inserted in the catalytic domain. Although structural analysis and deletion mutation suggests that the PA domain in subtilases is involved in substrate binding, direct evidence to support this function is still absent. Here, a protease, P57, secreted by Photobacterium sp. A5-7 isolated from marine sediment was characterized. P57 could hydrolyze casein, gelatin and collagen. It showed the highest activity at 40°C and pH 8.0. P57 is a new subtilase, with 63% sequence identity to the closest characterized protease. Mature P57 contains a catalytic domain and an inserted PA domain. The recombinant PA domain from P57 was shown to have collagen-binding ability, and Phe349 and Tyr432 were revealed to be key residues for collagen binding in the PA domain. This study first shows direct evidence that the PA domain of a subtilase can bind substrate, which provides a better understanding of the function of the PA domain of subtilases and bacterial extracellular proteases from marine sediment.

  4. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

    Directory of Open Access Journals (Sweden)

    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.

  5. Phytase and protease supplementation for laying hens in peak egg production

    Directory of Open Access Journals (Sweden)

    Bruno Serpa Vieira

    2016-12-01

    Full Text Available The effects of the combination of enzymes in commercial laying hens need to be more explored in literature. To determine if the type of protease affects performance, egg quality, nutrient intake, and morphometry of intestinal mucosa of laying hens in peak egg production and fed with phytase, 780 25-weeks Hy-Line W36 hens were assigned to a completely randomized design composed of five treatments/diets (one positive control, two negative controls, and negative controls plus protease A or B, with 12 replicates of 13 birds each. There was no effect of treatments (P > 0.05 on egg production, egg mass and feed conversion, even though the nutritional restriction imposed by the negative controls reduced egg weight (P = 0.02, albumen height (P < 0.01, and Haugh unit (P < 0.01. Although inclusion of proteases in negative controls did not cause the calculated intake of protein and amino acids to return to the same amount consumed by positive-control hens, egg quality parameters returned to positive control standards with protease A. Intestinal mucosa responded to treatment only at jejunum, where birds fed with protease B showed greater (P < 0.01 villus height and crypt depth than those treated with protease A. These findings suggest that different proteases and phytases interact distinctly and, in consequence, induce different responses on the birds. Moreover, the behavior of egg quality parameters after protease A inclusion in the diet indicates that the nutritional contribution of the combination of this protease with phytase is greater than the contribution of protease alone.

  6. Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

    Science.gov (United States)

    Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M; Brothers, Kimberly M; Zhang, Liang; Thibodeau, Patrick H

    2015-07-01

    The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Pieper Rembert

    2011-05-01

    Full Text Available Abstract Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3 pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP, SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA and Glutathione S-transferase (GST improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

  8. Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets.

    Science.gov (United States)

    Coupe, Simon A; Sinclair, Ben K; Watson, Lyn M; Heyes, Julian A; Eason, Jocelyn R

    2003-03-01

    Harvest-induced senescence of broccoli results in tissue wilting and sepal chlorosis. As senescence progresses, chlorophyll and protein levels in floret tissues decline and endo-protease activity (measured with azo-casein) increases. Protease activity increased from 24 h after harvest for tissues held in air at 20 degrees C. Activity was lower in floret tissues from branchlets that had been held in solutions of sucrose (2% w/v) or under high carbon dioxide, low oxygen (10% CO(2), 5% O(2)) conditions. Four protease-active protein bands were identified in senescing floret tissue by zymography, and the use of chemical inhibitors of protease action suggests that some 44% of protease activity in senescing floret tissue 72 h after harvest is due to the action of cysteine and serine proteases. Four putative cysteine protease cDNAs have been isolated from broccoli floret tissue (BoCP1, BoCP2, BoCP3, BoCP4). The cDNAs are most similar (73-89% at the amino acid level) to dehydration-responsive cysteine proteases previously isolated from Arabidopsis thaliana (RD19, RD21). The mRNAs encoded by the broccoli cDNAs are expressed in floret tissue during harvest-induced senescence with mRNA accumulating within 6 h of harvest for BoCP1, 12 h of harvest for BoCP4 and within 24 h of harvest for BoCP2 and BoCP3. Induction of the cDNAs is differentially delayed when broccoli branchlets are held in solutions of water or sucrose. In addition, the expression of BoCP1 and BoCP3 is inhibited in tissue held in atmospheres of high carbon dioxide/low oxygen (10% CO(2), 5% O(2)). The putative cysteine protease mRNAs are expressed before measurable increases in endo-protease activity, loss of protein, chlorophyll or tissue chlorosis.

  9. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Science.gov (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  10. Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus.

    Science.gov (United States)

    Mothe, Thirumala; Sultanpuram, Vishnuvardhan Reddy

    2016-06-01

    Alkaline proteases are important enzymes in many industrial applications, especially as additives in laundry detergent industry. Though there are a number of Bacillus species which are reported to be producing proteases, the efficiency of a protease produced by a novel strain has to be studied in comparison to the others. Hence, in this study, an alkaline serine protease produced by a novel species Bacillus caseinilyticus was purified and characterized for its possible usage in detergent industry. Ammonium sulphate, dialysis and DEAE column chromatographic methods were used for purification of the isolated alkaline protease. The molecular weight of the protease was determined by SDS-PAGE and it was found to be 66 kDa. Peptide mass fingerprinting (PMF) was carried out using MALDI-TOF-TOF mass spectrometry and the peptides were found to be similar to that of subtilisin protease. Specific activity of purified protein was found to be 89.2 U/mg. Optimum pH and temperature for enzyme activity were at pH 8 and 60 °C, respectively, showing stability with 10 mM CaCl 2 . Phenyl methyl sulphonyl fluoride (PMSF) at both 5 and 10 mM concentrations completely inhibited the enzyme activity suggesting its serine nature. EDTA, metal ions Mg 2+ and Ca 2+ increased the enzyme activity. The one factor at a time optimisation of the protease production was carried to identify the important factors that affect its production. After optimisation, the protease was produced at lab scale, purified and characterised. This alkali, thermotolerant serine protease was found to be significantly stable in the presence of various surfactants and H 2 O 2. Also, it was successfully able to remove blood stain when used as an additive along with commercial detergent suggesting its potential application in the laundry detergent industry.

  11. Emulsification using microporous membranes

    Directory of Open Access Journals (Sweden)

    Goran T. Vladisavljević

    2011-10-01

    Full Text Available Membrane emulsification is a process of injecting a pure dispersed phase or pre-emulsion through a microporous membrane into the continuous phase. As a result of the immiscibility of the two phases, droplets of the dispersed phase are formed at the outlets of membrane pores. The droplets formed in the process are removed from the membrane surface by applying cross-flow or stirring of the continuous phase or using a dynamic (rotating or vibrating membrane. The most commonly used membrane for emulsification is the Shirasu Porous Glass (SPG membrane, fabricated through spinodal decomposition in a melt consisting of Japanese volcanic ash (Shirasu, boric acid and calcium carbonate. Microsieve membranes are increasingly popular as an alternative to highly tortuous glass and ceramic membranes. Microsieves are usually fabricated from nickel by photolithography and electroplating or they can be manufactured from silicon nitride via Reactive Ion Etching (RIE. An advantage of microsieves compared to the SPG membrane is in much higher transmembrane fluxes and higher tolerance to fouling by the emulsion ingredients due to the existence of short, straight through pores. Unlike conventional emulsification devices such as high-pressure valve homogenisers and rotor-stator devices, membrane emulsification devices permit a precise control over the mean pore size over a wide range and during the process insignificant amount of energy is dissipated as heat. The drop size is primarily determined by the pore size, but it depends also on other parameters, such as membrane wettability, emulsion formulation, shear stress on the membrane surface, transmembrane pressure, etc.

  12. Pathogenic capacity of proteases from Serratia marcescens and Pseudomonas aeruginosa and their suppression by chicken egg white ovomacroglobulin.

    Science.gov (United States)

    Molla, A; Matsumura, Y; Yamamoto, T; Okamura, R; Maeda, H

    1987-01-01

    The pathogenicities of three proteases from Serratia marcescens, two proteases from Pseudomonas aeruginosa, and one thermolysin from Bacillus stearothermophilus were examined. All proteases tested caused acute liquefactive necrosis of the cornea and descemetocele formation in guinea pig eyes after intrastromal injection, with the exception of the 60-kilodalton protease from S. marcescens, which produced only an opaque lesion. When injected into guinea pig skin, the protease also enhanced vascular permeability, which was followed by edema formation. The permeability-enhancing activity of the proteases increased in parallel with the concentration of the enzymes. When tested in vitro for its effect on these bacterial proteases, chicken egg white ovomacroglobulin (ovoM) inhibited the enzymatic activity of all the proteases after a short incubation period at an enzyme/inhibitor ratio (molar) of 1:1 to 1:4 or at a lower concentration after a longer incubation period. Such treatment of the proteases with chicken egg white ovoM before injection intrastromally into the eyes or intradermally into the clipped flanks of guinea pigs protected the cornea from destruction or completely prevented the permeability reaction and edema formation. No inhibitory effects of plasma protease inhibitors against these bacterial proteases were noted. Since the proteases are critical in the pathogenic processes caused by the bacteria, these results suggest a beneficial effect of ovoM against bacterial infections. Images PMID:3115900

  13. Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

    NARCIS (Netherlands)

    N.M. van Maarseveen (Noortje); D. Andersson (Dan); M. Lepšík (Martin); A. Fun (Axel); P.J. Schipper (Pauline); D. de Jong (Dorien); C.A.B. Boucher (Charles); M. Nijhuis (Monique)

    2012-01-01

    textabstractBackground: Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes

  14. Extended virulence genotypes and phylogenetic background of Escherichia coli isolates from patients with cystitis, pyelonephritis, or prostatitis.

    Science.gov (United States)

    Johnson, James R; Kuskowski, Michael A; Gajewski, Abby; Soto, Sara; Horcajada, Juan Pablo; Jimenez de Anta, M Teresa; Vila, Jordi

    2005-01-01

    Molecular analysis of 63 Escherichia coli urine isolates showed that pyelonephritis (n=23) and prostatitis (n=17) isolates exhibited more virulence factors (VFs) among the 35 sought than did cystitis isolates (n=23). Several nontraditional VFs--including bmaE (M fimbriae), gafD (G fimbriae), fyuA (yersiniabactin receptor), ireA and iroN (novel siderophore receptors), cvaC (colicin [microcin] V), traT (serum-resistance associated), ibeA (invasion of brain endothelium), ompT (outer membrane protease T), and malX (pathogenicity island marker)--either differentiated significantly between syndromes (despite small numbers of isolates and possible multiple-comparison artifacts) or were broadly prevalent. Thus, interventions that target conserved uro-VFs may be possible, despite the likely existence of syndrome-specific pathogenetic mechanisms and/or host defense systems.

  15. Ion-conducting membranes

    Energy Technology Data Exchange (ETDEWEB)

    Masel, Richard I.; Sajjad, Syed Dawar; Gao, Yan; Liu, Zengcai; Chen, Qingmei

    2017-12-26

    An anion-conducting polymeric membrane comprises a terpolymer of styrene, vinylbenzyl-R.sub.s and vinylbenzyl-R.sub.x. R.sub.s is a positively charged cyclic amine group. R.sub.x is at least one constituent selected from the group consisting Cl, OH and a reaction product between an OH or Cl and a species other than a simple amine or a cyclic amine. The total weight of the vinylbenzyl-R.sub.x groups is greater than 0.3% of the total weight of the membrane. In a preferred embodiment, the membrane is a Helper Membrane that increases the faradaic efficiency of an electrochemical cell into which the membrane is incorporated, and also allows product formation at lower voltages than in cells without the Helper Membrane.

  16. Gas separation with membranes

    International Nuclear Information System (INIS)

    Schulz, G.; Michele, H.; Werner, U.

    1982-01-01

    Gas separation with membranes has already been tested in numerous fields of application, e.g. uranium enrichment of H 2 separation. In many of these processes the mass transfer units, so-called permeators, have to be connected in tandem in order to achieve high concentrations. A most economical operating method provides for each case an optimization of the cascades with regard to the membrane materials, construction and design of module. By utilization of the concentration gradient along the membrane a new process development has been accomplished - the continuously operating membrane rectification unit. Investment and operating costs can be reduced considerably for a number of separating processes by combining a membrane rectification unit with a conventional recycling cascade. However, the new procedure requires that the specifications for the module construction, flow design, and membrane properties be reconsidered. (orig.) [de

  17. SjAPI, the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

    Science.gov (United States)

    Yang, Weishan; Cao, Zhijian; Zhuo, Renxi; Li, Wenxin; Wu, Yingliang

    2013-01-01

    Background Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. Principal Findings Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues “AAV” and might be a useful template to produce new serine protease inhibitors. Conclusions/Significance To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of

  18. SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms.

    Directory of Open Access Journals (Sweden)

    Zongyun Chen

    Full Text Available BACKGROUND: Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. PRINCIPAL FINDINGS: Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI, Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2, Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI, and Buthus martensii Ascaris-type protease inhibitor (BmAPI. The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors. CONCLUSIONS/SIGNIFICANCE: To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the

  19. Chelating polymeric membranes

    KAUST Repository

    Peinemann, Klaus-Viktor

    2015-01-22

    The present application offers a solution to the current problems associated with recovery and recycling of precious metals from scrap material, discard articles, and other items comprising one or more precious metals. The solution is premised on a microporous chelating polymeric membrane. Embodiments include, but are not limited to, microporous chelating polymeric membranes, device comprising the membranes, and methods of using and making the same.

  20. Polyarylether composition and membrane

    Science.gov (United States)

    Hung, Joyce; Brunelle, Daniel Joseph; Harmon, Marianne Elisabeth; Moore, David Roger; Stone, Joshua James; Zhou, Hongyi; Suriano, Joseph Anthony

    2010-11-09

    A composition including a polyarylether copolymer is provided. The copolymer includes a polyarylether backbone; and a sulfonated oligomeric group bonded to the polyarylether suitable for use as a cation conducting membrane. Method of bonding a sulfonated oligomeric group to the polyarylether backbone to form a polyarylether copolymer. The membrane may be formed from the polyarylether copolymer composition. The chain length of the sulfonated oligomeric group may be controlled to affect or control the ion conductivity of the membrane.

  1. Serine protease EspP from enterohemorrhagic Escherichia coli is sufficient to induce shiga toxin macropinocytosis in intestinal epithelium.

    Science.gov (United States)

    In, Julie; Lukyanenko, Valeriy; Foulke-Abel, Jennifer; Hubbard, Ann L; Delannoy, Michael; Hansen, Anne-Marie; Kaper, James B; Boisen, Nadia; Nataro, James P; Zhu, Chengru; Boedeker, Edgar C; Girón, Jorge A; Kovbasnjuk, Olga

    2013-01-01

    Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells' basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.

  2. Serine protease EspP from enterohemorrhagic Escherichia coli is sufficient to induce shiga toxin macropinocytosis in intestinal epithelium.

    Directory of Open Access Journals (Sweden)

    Julie In

    Full Text Available Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells' basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.

  3. Photoresponsive nanostructured membranes

    KAUST Repository

    Madhavan, Poornima

    2016-07-26

    The perspective of adding stimuli-response to isoporous membranes stimulates the development of separation devices with pores, which would open or close under control of environment chemical composition, temperature or exposure to light. Changes in pH and temperature have been previously investigated. In this work, we demonstrate for the first time the preparation of photoresponsive isoporous membranes, applying self-assembly non-solvent induced phase separation to a new light responsive block copolymer. First, we optimized the membrane formation by using poly(styrene-b-anthracene methyl methacrylate-b-methylmethacrylate) (PS-b-PAnMMA-b-PMMA) copolymer, identifying the most suitable solvent, copolymer block length, and other parameters. The obtained final triblock copolymer membrane morphologies were characterized using atomic force and electron microscopy. The microscopic analysis reveals that the PS-b-PAnMMA-b-PMMA copolymer can form both lamellar and ordered hexagonal nanoporous structures on the membrane top layer in appropriate solvent compositions. The nanostructured membrane emits fluorescence due to the presence of the anthracene mid-block. On irradiation of light the PS-b-PAnMMA-b-PMMA copolymer membranes has an additional stimuli response. The anthracene group undergoes conformational changes by forming [4 + 4] cycloadducts and this alters the membrane\\'s water flux and solute retention. © 2016 The Royal Society of Chemistry.

  4. Gas separation membranes

    Science.gov (United States)

    Schell, William J.

    1979-01-01

    A dry, fabric supported, polymeric gas separation membrane, such as cellulose acetate, is prepared by casting a solution of the polymer onto a shrinkable fabric preferably formed of synthetic polymers such as polyester or polyamide filaments before washing, stretching or calendering (so called griege goods). The supported membrane is then subjected to gelling, annealing, and drying by solvent exchange. During the processing steps, both the fabric support and the membrane shrink a preselected, controlled amount which prevents curling, wrinkling or cracking of the membrane in flat form or when spirally wound into a gas separation element.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Phenolic compounds from Miconia myriantha inhibiting Candida aspartic proteases.

    Science.gov (United States)

    Li, X C; Jacob, M R; Pasco, D S; ElSohly, H N; Nimrod, A C; Walker, L A; Clark, A M

    2001-10-01

    Assay-guided fractionation of the ethanol extract of the twigs and leaves of Miconia myriantha yielded two new compounds, mattucinol-7-O-[4' ',6' '-O-(S)-hexahydroxydiphenoyl]-beta-D-glucopyranoside (1) and mattucinol-7-O-[4' ',6' '-di-O-galloyl]-beta-D-glucopyranoside (2), along with mattucinol-7-O-beta-D-glucopyranoside (3), ellagic acid (4), 3,3'-di-O-methyl ellagic acid-4-O-beta-D-xylopyranoside, and gallic acid. Complete (1)H and (13)C NMR assignments of compound 1, which possesses a hexahydroxydiphenoyl unit, were achieved using the HMBC technique optimized for small couplings to enhance the four-bond and two-bond H/C correlations. Compounds 1 and 4 showed inhibitory effects against Candida albicans secreted aspartic proteases, with IC(50) of 8.4 and 10.5 microM, respectively.

  7. RNA-virus proteases counteracting host innate immunity.

    Science.gov (United States)

    Lei, Jian; Hilgenfeld, Rolf

    2017-10-01

    Virus invasion triggers host immune responses, in particular, innate immune responses. Pathogen-associated molecular patterns of viruses (such as dsRNA, ssRNA, or viral proteins) released during virus replication are detected by the corresponding pattern-recognition receptors of the host, and innate immune responses are induced. Through production of type-I and type-III interferons as well as various other cytokines, the host innate immune system forms the frontline to protect host cells and inhibit virus infection. Not surprisingly, viruses have evolved diverse strategies to counter this antiviral system. In this review, we discuss the multiple strategies used by proteases of positive-sense single-stranded RNA viruses of the families Picornaviridae, Coronaviridae, and Flaviviridae, when counteracting host innate immune responses. © 2017 Federation of European Biochemical Societies.

  8. HIV-1 protease inhibitory substances from Cassia garrettiana

    Directory of Open Access Journals (Sweden)

    Jindaporn Puripattanvong

    2007-01-01

    Full Text Available Cassia garrettiana Craib, a Thai medicinal plant locally known as Samae-sarn, was investigated for its active constituents against HIV-1 protease (HIV-1 PR. Bioassay-guided fractionation of the heart woodof this plant led to the isolation of a stilbene derivative (1, piceatannol and an anthraquinone derivative (2, chrysophanol. Piceatannol exhibited appreciable inhibitory effect against HIV-1 PR with an IC50 value of25.4 μg/ml, whereas that of chrysophanol was 73.5 μg/ml. In addition, other two stilbenoids together with three anthraquinone derivatives were also investigated for their anti-HIV-1 PR activities. The resultindicated that resveratrol possessed anti-HIV-1 PR activity with an IC50 value of 85.0 μg/ml, whereas other stilbenoid (oxyresveratrol and anthraquinone derivatives (emodin, aloe-emodin, rhein were inactive (IC50 > 100 μg/ml.

  9. Calcium and SOL Protease Mediate Temperature Resetting of Circadian Clocks

    Science.gov (United States)

    Tataroglu, Ozgur; Zhao, Xiaohu; Busza, Ania; Ling, Jinli; O’Neill, John S.; Emery, Patrick

    2015-01-01

    Summary Circadian clocks integrate light and temperature input to remain synchronized with the day/night cycle. Although light input to the clock is well studied, the molecular mechanisms by which circadian clocks respond to temperature remain poorly understood. We found that temperature phase shifts Drosophila circadian clocks through degradation of the pacemaker protein TIM. This degradation is mechanistically distinct from photic CRY-dependent TIM degradation. Thermal TIM degradation is triggered by cytosolic calcium increase and CALMODULIN binding to TIM and is mediated by the atypical calpain protease SOL. This thermal input pathway and CRY-dependent light input thus converge on TIM, providing a molecular mechanism for the integration of circadian light and temperature inputs. Mammals use body temperature cycles to keep peripheral clocks synchronized with their brain pacemaker. Interestingly, downregulating the mammalian SOL homolog SOLH blocks thermal mPER2 degradation and phase shifts. Thus, we propose that circadian thermosensation in insects and mammals share common principles. PMID:26590423

  10. Protease activated receptor-2 (PAR2): possible target of phytochemicals.

    Science.gov (United States)

    Kakarala, Kavita Kumari; Jamil, Kaiser

    2015-09-01

    The use of phytochemicals either singly or in combination with other anticancer drugs comes with an advantage of less toxicity and minimal side effects. Signaling pathways play central role in cell cycle, cell growth, metabolism, etc. Thus, the identification of phytochemicals with promising antagonistic effect on the receptor/s playing key role in single transduction may have better therapeutic application. With this background, phytochemicals were screened against protease-activated receptor 2 (PAR2). PAR2 belongs to the superfamily of GPCRs and is an important target for breast cancer. Using in silico methods, this study was able to identify the phytochemicals with promising binding affinity suggesting their therapeutic potential in the treatment of breast cancer. The findings from this study acquires importance as the information on the possible agonists and antagonists of PAR2 is limited due its unique mechanism of activation.

  11. Mapping protease substrates using a biotinylated phage substrate library.

    Energy Technology Data Exchange (ETDEWEB)

    Scholle, M. D.; Kriplani, U.; Pabon, A.; Sishtla, K.; Glucksman, M. J.; Kay, B. K.; Biosciences Division; Chicago Medical School

    2005-05-05

    We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

  12. HIV-protease inhibitors for the treatment of cancer

    DEFF Research Database (Denmark)

    Maksimovic-Ivanic, Danijela; Fagone, Paolo; McCubrey, James

    2017-01-01

    The possible use of HIV protease inhibitors (HIV-PI) as new therapeutic option for the treatment of cancer primarily originated from their success in treating HIV-related Kaposi's sarcoma (KS). While these findings were initially attributed to immune reconstitution and better control of oncogenic...... and nitric oxide (NO) derivatives of HIV-PIs. In this article, we discuss the current preclinical and clinical evidences for the potential use of HIV-PIs, and of novel derivatives, such as saquinavir-NO in the treatment of cancer....... viral infections, the number of reports on solid tumors, KS, lymphoma, fibrosarcoma, multiple myeloma and prostate cancer suggest other mechanisms for the anti-neoplastic activity of PIs. However, a major drawback for the possible adoption of HIV-PIs in the therapy of cancer relies on their relatively...

  13. Development of potent inhibitors of the coxsackievirus 3C protease

    International Nuclear Information System (INIS)

    Lee, Eui Seung; Lee, Won Gil; Yun, Soo-Hyeon; Rho, Seong Hwan; Im, Isak; Yang, Sung Tae; Sellamuthu, Saravanan; Lee, Yong Jae; Kwon, Sun Jae; Park, Ohkmae K.; Jeon, Eun-Seok; Park, Woo Jin; Kim, Yong-Chul

    2007-01-01

    Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Recognition of misfolded proteins by Lon, a AAA(+) protease.

    Science.gov (United States)

    Gur, Eyal; Sauer, Robert T

    2008-08-15

    Proteins unfold constantly in cells, especially under stress conditions. Degradation of denatured polypeptides by Lon and related ATP-dependent AAA(+) proteases helps prevent toxic aggregates formation and other deleterious consequences, but how these destructive enzymatic machines distinguish between damaged and properly folded proteins is poorly understood. Here, we show that Escherichia coli Lon recognizes specific sequences -- rich in aromatic residues -- that are accessible in unfolded polypeptides but hidden in most native structures. Denatured polypeptides lacking such sequences are poor substrates. Lon also unfolds and degrades stably folded proteins with accessible recognition tags. Thus, protein architecture and the positioning of appropriate targeting sequences allow Lon degradation to be dependent or independent of the folding status of a protein. Our results suggest that Lon can recognize multiple signals in unfolded polypeptides synergistically, resulting in nanomolar binding and a mechanism for discriminating irreversibly damaged proteins from transiently unfolded elements of structure.

  16. Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling

    Directory of Open Access Journals (Sweden)

    Morrill Gene A

    2013-01-01

    Full Text Available Abstract Background Insulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions. Results [125I]Insulin binding (Kd = 54 ± 6 nM at the oocyte plasma membrane activates membrane serine protease(s, followed by the loss of low affinity ouabain binding sites, with a concomitant 3–4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na+/Ca2+ exchange, increased endocytosis, decreased Na+ conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi. Hyperpolarization is largely due to Na+-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na+/glucose cotransport. The Na+ sym- and antiporter systems are driven by the Na+ free energy gradient generated by Na+/K+-ATPase. Shifts in α and/or β Na+-pump subunits to caveolar (lipid raft membrane regions may activate Na/K-ATPase and contribute to the Na+ free energy gradient and the increase in both Na+/glucose co-transport and pHi. Conclusions Under physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na+ channels and mobilizes fully functional Na+-pumps, generating a Na+ free energy gradient which serves as the energy source for several membrane anti- and symporter systems.

  17. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    Science.gov (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

    Directory of Open Access Journals (Sweden)

    Zareena Mushtaq

    2015-04-01

    Full Text Available In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35ºC for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40ºC, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The Km , Kcat , Vmax and Kcat/Km values of purified protease were 7.0 mg/mL, 3.8 x102S-1, 54.30 µmol/min and 54.28 s-1mg -1.mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

  19. Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Michael B Butterworth

    Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  20. Optimization of protease extraction from horse mango (Mangifera foetida Lour) kernels by a response surface methodology.

    Science.gov (United States)

    Ahmad, Mohammad Norazmi; Liew, Siew Ling; Yarmo, Mohd Ambar; Said, Mamot

    2012-01-01

    Protease is one of the most important industrial enzymes with a multitude of applications in both food and non-food sectors. Although most commercial proteases are microbial proteases, the potential of non-conventional protease sources, especially plants, should not be overlooked. In this study, horse mango (Mangifera foetida Lour) fruit, known to produce latex with a blistering effect upon contact with human skin, was chosen as a source of protease, and the effect of the extraction process on its protease activity evaluated. The crude enzyme was extracted from the kernels and extraction was optimized by a response surface methodology (RSM) using a central composite rotatable design (CCRD). The variables studied were pH (x(1)), CaCl(2) (x(2)), Triton X-100 (x(3)), and 1,4-dithryeitol (x(4)). The results obtained indicate that the quadratic model is significant for all the variables tested. Based on the RSM model generated, optimal extraction conditions were obtained at pH 6.0, 8.16 mM CaCl(2), 5.0% Triton X-100, and 10.0 mM DTT, and the estimated response was 95.5% (w/w). Verification test results showed that the difference between the calculated and the experimental protease activity value was only 2%. Based on the t-value, the effects of the variables arranged in ascending order of strength were CaCl(2) < pH < DTT < Triton X-100.