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Sample records for 3c protease complexed

  1. Poliovirus protease 3C(pro) kills cells by apoptosis.

    Science.gov (United States)

    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation. Copyright 2000 Academic Press.

  2. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    NARCIS (Netherlands)

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M

    2014-01-01

    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  3. A Novel Enterovirus 71 (EV71) Virulence Determinant: The 69th Residue of 3C Protease Modulates Pathogenicity

    Science.gov (United States)

    Li, Bingqing; Yue, Yingying; Zhang, Yajie; Yuan, Zenglin; Li, Peng; Song, Nannan; Lin, Wei; Liu, Yan; Gu, Lichuan; Meng, Hong

    2017-01-01

    Human enterovirus type 71 (EV71), the major causative agent of hand-foot-and-mouth disease, has been known to cause fatal neurological complications. Unfortunately, the reason for neurological complications that have been seen in fatal cases of the disease and the relationship between EV71 virulence and viral genetic sequences remains largely undefined. The 3C protease (3Cpro) of EV71 plays an irreplaceable role in segmenting the precursor polyprotein during viral replication, and intervening with host life activity during viral infection. In this study, for the first time, the 69th residue of 3C protease has been identified as a novel virulence determinant of EV71. The recombinant virus with single point variation, in the 69th of 3Cpro, exhibited obvious decline in replication, and virulence. We further determined the crystal structure of 3C N69D at 1.39 Ǻ resolution and found that conformation of 3C N69D demonstrated significant changes compared with a normal 3C protein, in the substrate-binding site and catalytic active site. Strikingly, one of the switch loops, essential in fixing substrates, adopts an open conformation in the 3C N69D-rupintrivir complex. Consistent with this apparent structural disruption, the catalytic activity of 3C N69D decreased sharply for host derived and viral derived substrates, detected for both in vitro and in vivo. Interestingly, in addition to EV71, Asp69 was also found in 3C proteases of other virus strains, such as CAV16, and was conserved in nearly all C type human rhinovirus. Overall, we identified a natural virulence determinant of 3C protease and revealed the mechanism of attenuated virulence is mediated by N69D substitution. Our data provides new insight into the enzymatic mechanism of a subdued 3C protease and suggests a theoretical basis for virulence determinantion of picornaviridae. PMID:28217559

  4. Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease.

    Science.gov (United States)

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C; Chang, Kyeong-Ok

    2013-02-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC(50) in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.

  5. Human rhinovirus 3C protease as a potential target for the development of antiviral agents.

    Science.gov (United States)

    Wanga, Q May; Chen, Shu-Hui

    2007-02-01

    As the major cause of the common cold in children and adults, human rhinoviruses (HRVs) are a group of small single-stranded positive-sense RNA viruses. HRVs translate their genetic information into a polyprotein precursor that is mainly processed by a virally encoded 3C protease (3Cpro) to generate functional viral proteins and enzymes. It has been shown that the enzymatic activity of HRV 3Cpro is essential to viral replication. The 3Cpro is distinguished from most other proteases by the fact that it has a cysteine nucleophile but with a chymotrypsin-like serine protease folding. This unique protein structure together with its essential role in viral replication made the 3Cpro an excellent target for antiviral intervention. In recent years, considerable efforts have been made in the development of antiviral compounds targeting this enzyme. To further facilitate the design of potent 3C protease inhibitors for therapeutic use, this review summarizes the biochemical and structural characterization conducted on HRV 3C protease along with the recent progress on the development of 3C protease inhibitors.

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

    Science.gov (United States)

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

    2009-07-01

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

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

    OpenAIRE

    2016-01-01

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

  8. Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

    Science.gov (United States)

    Matthews, D A; Dragovich, P S; Webber, S E; Fuhrman, S A; Patick, A K; Zalman, L S; Hendrickson, T F; Love, R A; Prins, T J; Marakovits, J T; Zhou, R; Tikhe, J; Ford, C E; Meador, J W; Ferre, R A; Brown, E L; Binford, S L; Brothers, M A; DeLisle, D M; Worland, S T

    1999-09-28

    Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

  9. Structure-based design of ketone-containing, tripeptidyl human rhinovirus 3C protease inhibitors.

    Science.gov (United States)

    Dragovich, P S; Zhou, R; Webber, S E; Prins, T J; Kwok, A K; Okano, K; Fuhrman, S A; Zalman, L S; Maldonado, F C; Brown, E L; Meador, J W; Patick, A K; Ford, C E; Brothers, M A; Binford, S L; Matthews, D A; Ferre, R A; Worland, S T

    2000-01-03

    Tripeptide-derived molecules incorporating C-terminal ketone electrophiles were evaluated as reversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). An optimized example of such compounds displayed potent 3CP inhibition activity (K = 0.0045 microM) and in vitro antiviral properties (EC50=0.34 microM) when tested against HRV serotype-14.

  10. The coxsackievirus B 3C protease cleaves MAVS and TRIF to attenuate host type I interferon and apoptotic signaling.

    Directory of Open Access Journals (Sweden)

    Amitava Mukherjee

    2011-03-01

    Full Text Available The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR pathways that converge on the induction of type I interferons (IFNs. Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3C(pro cysteine protease of coxsackievirus B3 (CVB3 cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3C(pro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3C(pro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3C(pro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.

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

    Directory of Open Access Journals (Sweden)

    Raheem Ullah

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-07-15

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its str...

  15. Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments

    Science.gov (United States)

    Becker, Daniel; Kaczmarska, Zuzanna; Arkona, Christoph; Schulz, Robert; Tauber, Carolin; Wolber, Gerhard; Hilgenfeld, Rolf; Coll, Miquel; Rademann, Jörg

    2016-09-01

    Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme-inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  17. Crystal structure of foot-and-mouth disease virus 3C protease. New insights into catalytic mechanism and cleavage specificity.

    Science.gov (United States)

    Birtley, James R; Knox, Stephen R; Jaulent, Agnès M; Brick, Peter; Leatherbarrow, Robin J; Curry, Stephen

    2005-03-25

    Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C(pro)), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C(pro), determined the crystal structure to 1.9-angstroms resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C(pro) adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4-P4') and that FMDV 3C(pro) discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C(pro) enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C(pro) of an extended beta-ribbon that folds over the substrate binding cleft in other picornavirus 3C(pro) structures. Collectively, these results establish a valuable framework for the development of FMDV 3C(pro) inhibitors.

  18. Substituted benzamide inhibitors of human rhinovirus 3C protease: structure-based design, synthesis, and biological evaluation.

    Science.gov (United States)

    Reich, S H; Johnson, T; Wallace, M B; Kephart, S E; Fuhrman, S A; Worland, S T; Matthews, D A; Hendrickson, T F; Chan, F; Meador, J; Ferre, R A; Brown, E L; DeLisle, D M; Patick, A K; Binford, S L; Ford, C E

    2000-05-04

    A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate. alpha,beta-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC(50) 0.60 microM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

  19. Structure-based design of irreversible, tripeptidyl human rhinovirus 3C protease inhibitors containing N-methyl amino acids.

    Science.gov (United States)

    Dragovich, P S; Webber, S E; Prins, T J; Zhou, R; Marakovits, J T; Tikhe, J G; Fuhrman, S A; Patick, A K; Matthews, D A; Ford, C E; Brown, E L; Binford, S L; Meador, J W; Ferre, R A; Worland, S T

    1999-08-02

    Tripeptide-derived molecules incorporating N-methyl amino acid residues and C-terminal Michael acceptor moieties were evaluated as irreversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). Such compounds displayed good 3CP inhibition activity (k(obs)/[I] up to 610,000 M(-1) s(-1)) and potent in vitro antiviral properties (EC50 approaching 0.03 microM) when tested against HRV serotype-14.

  20. Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation.

    Directory of Open Access Journals (Sweden)

    Kuo-Feng Weng

    2009-09-01

    Full Text Available Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell-virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71 3C protease (3C(pro cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3' pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C(pro. CstF-64 was cleaved in vitro by 3C(pro but neither by mutant 3C(pro (in which the catalytic site was inactivated nor by another EV71 protease 2A(pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C(pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500. An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3'-end pre-mRNA processing and polyadenylation in 3C(pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C(pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.

  1. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Jon [University of Southampton, England; Coates, Leighton [ORNL; Hussey, Robert [University of Southampton, England

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  2. Human rhinovirus 3C protease: generation of pharmacophore models for peptidic and nonpeptidic inhibitors and their application in virtual screening.

    Science.gov (United States)

    Steindl, Theodora; Laggner, Christian; Langer, Thierry

    2005-01-01

    Three-dimensional pharmacophore models for peptidic and small organic nonpeptidic inhibitors of the human rhinovirus 3C protease were generated in a structure-based as well as in a ligand-based approach, using the software package Catalyst. The inhibitors possess an electrophilic moiety, often a Michael acceptor function, which covalently binds to a cysteine in the active site of the enzyme. Since this process presents the key step for virus inactivation, the creation of a new function in Catalyst was required in order to include this decisive functionality into the pharmacophore models. In the present study we focus on this feature definition process because it presents an innovative strategy to expand the pharmacophore description ability of the Catalyst software to also include covalent bonds between ligand and binding site. The resulting hypotheses were then used for virtual screening of 3D databases in order to verify their quality and to search for structurally diverse, possible new lead substances.

  3. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 1. Michael acceptor structure-activity studies.

    Science.gov (United States)

    Dragovich, P S; Webber, S E; Babine, R E; Fuhrman, S A; Patick, A K; Matthews, D A; Lee, C A; Reich, S H; Prins, T J; Marakovits, J T; Littlefield, E S; Zhou, R; Tikhe, J; Ford, C E; Wallace, M B; Meador, J W; Ferre, R A; Brown, E L; Binford, S L; Harr, J E; DeLisle, D M; Worland, S T

    1998-07-16

    The structure-based design, chemical synthesis, and biological evaluation of peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds incorporate various Michael acceptor moieties and are shown to irreversibly bind to HRV serotype 14 3CP with inhibition activities (kobs/[I]) ranging from 100 to 600 000 M-1 s-1. These inhibitors are also shown to exhibit antiviral activity when tested against HRV-14-infected H1-HeLa cells with EC50's approaching 0.50 microM. Extensive structure-activity relationships developed by Michael acceptor alteration are reported along with the evaluation of several compounds against HRV serotypes other than 14. A 2.0 A crystal structure of a peptide-derived inhibitor complexed with HRV-2 3CP is also detailed.

  4. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus infected cells

    DEFF Research Database (Denmark)

    Kristensen, Thea; Normann, Preben; Gullberg, Maria

    2016-01-01

    The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been des...... have implications for the testing of potential antiviral agents targeting the FMDV 3C protease....

  5. Inhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3'-digallate (TF3

    Directory of Open Access Journals (Sweden)

    Chia-Nan Chen

    2005-01-01

    Full Text Available SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS. The virally encoded 3C-like protease (3CLPro has been presumed critical for the viral replication of SARS-CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CLPro. Two compounds in the library were found to be inhibitive: tannic acid (IC50 = 3 µM and 3-isotheaflavin-3-gallate (TF2B (IC50 = 7 µM. These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CLPro-inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CLPro. Several other known compositions in teas were also evaluated for their activities in inhibiting 3CLPro. We found that caffeine, (—-epigallocatechin gallte (EGCg, epicatechin (EC, theophylline (TP, catechin (C, epicatechin gallate (ECg and epigallocatechin (EGC did not inhibit 3CLPro activity. Only theaflavin-3,3′-digallate (TF3 was found to be a 3CLPro inhibitor. This study has resulted in the identification of new compounds that are effective 3CLPro inhibitors.

  6. Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique

    Institute of Scientific and Technical Information of China (English)

    Shuai CHEN; Hua-liang JIANG; Li-li CHEN; Hai-bin LUO; Tao SUN; Jing CHEN; Fei YE; Jian-hua CAI; Jing-kang SHEN; Xu SHEN

    2005-01-01

    Aim: To characterize enzymatic activity of severe acute respiratory syndrome(SARS) coronavirus (CoV) 3C-like protease (3CLpro) and its four site-directed mutants. Methods: Based on the fluorescence resonance energy transfer (FRET)principle using 5-[(2'-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and 4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV 3CLpro proteolytic activity. Results: The kinetic parameters of the fluorogenic substrate have been determined as Km=404 μmol.L-1, kcat=1.08 min-1, and kcat/Km=2.7 gered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CLpro revealed that substitutions of His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity. Conclusion: This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CLpro. This FRET-based assay might supply an ideal approach for the exploration SARSCoV 3CLpro putative inhibitors.

  7. NMR solution structures of the apo and peptide-inhibited human rhinovirus 3C protease (Serotype 14): structural and dynamic comparison.

    Science.gov (United States)

    Bjorndahl, Trent C; Andrew, Lena C; Semenchenko, Valentyna; Wishart, David S

    2007-11-13

    The human rhinovirus (HRV) is a positive sense RNA virus responsible for about 30% of "common colds". It relies on a 182 residue cysteine protease (3C) to proteolytically process its single gene product. Inhibition of this enzyme in vitro and in vivo has consistently demonstrated cessation of viral replication. This suggests that 3C protease inhibitors could serve as good drug candidates. However, significant proteolytic substrate diversity exists within the 110+ known rhinovirus serotypes. To investigate this variability we used NMR to solve the structure of the rhinovirus serotype 14 3C protease (subgenus B) covalently bound to a peptide (acetyl-LEALFQ-ethylpropionate) inhibitor. The inhibitor-bound structure was determined to an overall rmsd of 0.82 A (backbone atoms) and 1.49 A (all heavy atoms). Comparison with the X-ray structure of the serotype 2 HRV 3C protease from subgenus A (51% sequence identity) bound to the inhibitor ruprintrivir allowed the identification of conserved intermolecular interactions involved in proximal substrate binding as well as subgenus differences that might account for the variability observed in SAR studies. To better characterize the 3C protease and investigate the structural and dynamic differences between the apo and bound states we also solved the solution structure of the apo form. The apo structure has an overall rmsd of 1.07 +/- 0.17 A over backbone atoms, which is greater by 0.25 A than what is seen for the inhibited enzyme (2B0F.pdb). This increase is localized to the enzyme's C-terminal beta-barrel domain, which is responsible for recognizing and binding proteolytic substrates. Amide hydrogen exchange dynamics revealed dramatic differences between the two enzyme states. Furthermore, a number of residues exhibited exchange-broadened amide NMR signals in the apo state compared to the inhibited state. The majority of these residues are associated with proteolytic substrate interaction.

  8. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 3. Structure-activity studies of ketomethylene-containing peptidomimetics.

    Science.gov (United States)

    Dragovich, P S; Prins, T J; Zhou, R; Fuhrman, S A; Patick, A K; Matthews, D A; Ford, C E; Meador, J W; Ferre, R A; Worland, S T

    1999-04-08

    The structure-based design, chemical synthesis, and biological evaluation of various ketomethylene-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The ketomethylene-containing inhibitors typically display slightly reduced 3CP inhibition activity relative to the corresponding peptide-derived molecules, but they also exhibit significantly improved antiviral properties. Optimization of the ketomethylene-containing compounds is shown to provide several highly active 3C protease inhibitors which function as potent antirhinoviral agents (EC90 = <1 microM) against multiple virus serotypes in cell culture.

  9. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 2. Peptide structure-activity studies.

    Science.gov (United States)

    Dragovich, P S; Webber, S E; Babine, R E; Fuhrman, S A; Patick, A K; Matthews, D A; Reich, S H; Marakovits, J T; Prins, T J; Zhou, R; Tikhe, J; Littlefield, E S; Bleckman, T M; Wallace, M B; Little, T L; Ford, C E; Meador, J W; Ferre, R A; Brown, E L; Binford, S L; DeLisle, D M; Worland, S T

    1998-07-16

    The structure-based design, chemical synthesis, and biological evaluation of various peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of an ethyl propenoate Michael acceptor moiety and a tripeptidyl binding determinant. The systematic modification of each amino acid residue present in the binding determinant as well as the N-terminal functionality is described. Such modifications are shown to provide irreversible HRV-14 3CP inhibitors with anti-3CP activities (kobs/[I]) ranging from 60 to 280 000 M-1 s-1 and antiviral EC50's which approach 0.15 microM. An optimized inhibitor which incorporates several improvements identified by the structure-activity studies is also described. This molecule displays very rapid irreversible inhibition of HRV-14 3CP (kobs/[I] = 800 000 M-1 s-1) and potent antiviral activity against HRV-14 in cell culture (EC50 = 0.056 microM). A 1.9 A crystal structure of an S-alkylthiocarbamate-containing inhibitor complexed with HRV-2 3CP is also detailed.

  10. Sequence adaptations affecting cleavage of the VP1/2A junction by the 3C protease in foot-and-mouth disease virus-infected cells

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    2014-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited clea...

  11. Low levels of foot-and-mouth disease virus 3C protease expression are required to achieve optimal capsid protein expression and processing in mammalian cells

    DEFF Research Database (Denmark)

    Polacek, Charlotta; Gullberg, Maria; Li, Jiong;

    2013-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor (P1-2A) is processed by the virus-encoded 3C protease (3Cpro) to produce VP0, VP3, VP1 and 2A. Within the virus-encoded polyprotein, the P1-2A and 3Cpro can be expected to be produced at equivalent concentrations. However, using...... production of diagnostic reagents and improved vaccines against foot-and-mouth disease....

  12. Design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus 3C-like proteases.

    Science.gov (United States)

    Chuck, Chi-Pang; Chen, Chao; Ke, Zhihai; Wan, David Chi-Cheong; Chow, Hak-Fun; Wong, Kam-Bo

    2013-01-01

    Coronaviral infection is associated with up to 5% of respiratory tract diseases. The 3C-like protease (3CL(pro)) of coronaviruses is required for proteolytic processing of polyproteins and viral replication, and is a promising target for the development of drugs against coronaviral infection. We designed and synthesized four nitrile-based peptidomimetic inhibitors with different N-terminal protective groups and different peptide length, and examined their inhibitory effect on the in-vitro enzymatic activity of 3CL(pro) of severe-acute-respiratory-syndrome-coronavirus. The IC(50) values of the inhibitors were in the range of 4.6-49 μM, demonstrating that the nitrile warhead can effectively inactivate the 3CL(pro) autocleavage process. The best inhibitor, Cbz-AVLQ-CN with an N-terminal carbobenzyloxy group, was ~10x more potent than the other inhibitors tested. Crystal structures of the enzyme-inhibitor complexes showed that the nitrile warhead inhibits 3CL(pro) by forming a covalent bond with the catalytic Cys145 residue, while the AVLQ peptide forms a number of favourable interactions with the S1-S4 substrate-binding pockets. We have further showed that the peptidomimetic inhibitor, Cbz-AVLQ-CN, has broad-spectrum inhibition against 3CL(pro) from human coronavirus strains 229E, NL63, OC43, HKU1, and infectious bronchitis virus, with IC(50) values ranging from 1.3 to 3.7 μM, but no detectable inhibition against caspase-3. In summary, we have shown that the nitrile-based peptidomimetic inhibitors are effective against 3CL(pro), and they inhibit 3CL(pro) from a broad range of coronaviruses. Our results provide further insights into the future design of drugs that could serve as a first line defence against coronaviral infection. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  13. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements.

    Science.gov (United States)

    Dragovich, P S; Prins, T J; Zhou, R; Webber, S E; Marakovits, J T; Fuhrman, S A; Patick, A K; Matthews, D A; Lee, C A; Ford, C E; Burke, B J; Rejto, P A; Hendrickson, T F; Tuntland, T; Brown, E L; Meador, J W; Ferre, R A; Harr, J E; Kosa, M B; Worland, S T

    1999-04-08

    The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

  14. Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain.

    Science.gov (United States)

    Shi, Jiahai; Han, Nanyu; Lim, Liangzhong; Lua, Shixiong; Sivaraman, J; Wang, Lushan; Mu, Yuguang; Song, Jianxing

    2011-02-01

    Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro.

  15. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 8. Pharmacological optimization of orally bioavailable 2-pyridone-containing peptidomimetics.

    Science.gov (United States)

    Dragovich, Peter S; Prins, Thomas J; Zhou, Ru; Johnson, Theodore O; Hua, Ye; Luu, Hiep T; Sakata, Sylvie K; Brown, Edward L; Maldonado, Fausto C; Tuntland, Tove; Lee, Caroline A; Fuhrman, Shella A; Zalman, Leora S; Patick, Amy K; Matthews, David A; Wu, Ellen Y; Guo, Ming; Borer, Bennett C; Nayyar, Naresh K; Moran, Terence; Chen, Lijian; Rejto, Paul A; Rose, Peter W; Guzman, Mark C; Dovalsantos, Elena Z; Lee, Steven; McGee, Kevin; Mohajeri, Michael; Liese, Andreas; Tao, Junhua; Kosa, Maha B; Liu, Bo; Batugo, Minerva R; Gleeson, Jean-Paul R; Wu, Zhen Ping; Liu, Jia; Meador, James W; Ferre, Rose Ann

    2003-10-09

    The optimization of the pharmacokinetic performance of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors following oral administration to either beagle dogs or CM-monkeys is described. The molecules described in this work are composed of a 2-pyridone-containing peptidomimetic binding determinant and an alpha,beta-unsaturated ester Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. Modification of the ester contained within these compounds is detailed along with alteration of the P(2) substituent present in the peptidomimetic portion of the inhibitors. The pharmacokinetics of several inhibitors in both dogs and monkeys are described (7 h plasma concentrations after oral administration) along with their human plasma stabilities, stabilities in incubations with human, dog, and monkey microsomes and hepatocytes, Caco-2 permeabilities, and aqueous solubilities. Compounds containing an alpha,beta-unsaturated ethyl ester fragment and either an ethyl or propargyl P(2) moiety displayed the most promising combination of 3C enzyme inhibition (k(obs)/[I] 170 000-223 000 M(-1) s(-1)), antiviral activity (EC(50) = 0.047-0.058 microM, mean vs seven HRV serotypes), and pharmacokinetics following oral administration (7 h dog plasma levels = 0.248-0.682 microM; 7 h CM-monkey plasma levels = 0.057-0.896 microM).

  16. Structure-based design of a parallel synthetic array directed toward the discovery of irreversible inhibitors of human rhinovirus 3C protease.

    Science.gov (United States)

    Johnson, Theodore O; Hua, Ye; Luu, Hiep T; Brown, Edward L; Chan, Fora; Chu, Shao Song; Dragovich, Peter S; Eastman, Brian W; Ferre, Rose Ann; Fuhrman, Shella A; Hendrickson, Thomas F; Maldonado, Fausto C; Matthews, David A; Meador, James W; Patick, Amy K; Reich, Siegfried H; Skalitzky, Donald J; Worland, Stephen T; Yang, Michelle; Zalman, Leora S

    2002-05-09

    Utilizing the tools of parallel synthesis and structure-based design, a new class of Michael acceptor-containing, irreversible inhibitors of human rhinovirus 3C protease (HRV 3CP) was discovered. These inhibitors are shown to inhibit HRV-14 3CP with rates of inactivation ranging from 886 to 31 400 M(-1) sec(-1). These inhibitors exhibit antiviral activity when tested against HRV-14 infected H1-HeLa cells, with EC(50) values ranging from 1.94 to 0.15 microM. No cytotoxicity was observed at the limits of the assay concentration. A crystal structure of one of the more potent inhibitors covalently bound to HRV-2 3CP is detailed. These compounds were also tested against HRV serotypes other than type 14 and were found to have highly variable activities.

  17. Crystallization and preliminary crystallographic study of Feline infectious peritonitis virus main protease in complex with an inhibitor.

    Science.gov (United States)

    Wang, Jinshan; Wang, Fenghua; Tan, Yusheng; Chen, Xia; Zhao, Qi; Fu, Sheng; Li, Shuang; Chen, Cheng; Yang, Haitao

    2014-12-01

    Feline infectious peritonitis virus (FIPV) causes a lethal systemic granulomatous disease in wild and domestic cats around the world. Currently, no effective vaccines or drugs have been developed against it. As a member of the genus Alphacoronavirus, FIPV encodes two polyprotein precursors required for genome replication and transcription. Each polyprotein undergoes extensive proteolytic processing, resulting in functional subunits. This process is mainly mediated by its genome-encoded main protease, which is an attractive target for antiviral drug design. In this study, the main protease of FIPV in complex with a Michael acceptor-type inhibitor was crystallized. The complex crystals diffracted to 2.5 Å resolution and belonged to space group I422, with unit-cell parameters a = 112.3, b = 112.3, c = 102.1 Å. There is one molecule per asymmetric unit.

  18. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 6. Structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics.

    Science.gov (United States)

    Dragovich, Peter S; Prins, Thomas J; Zhou, Ru; Brown, Edward L; Maldonado, Fausto C; Fuhrman, Shella A; Zalman, Leora S; Tuntland, Tove; Lee, Caroline A; Patick, Amy K; Matthews, David A; Hendrickson, Thomas F; Kosa, Maha B; Liu, Bo; Batugo, Minerva R; Gleeson, Jean-Paul R; Sakata, Sylvie K; Chen, Lijian; Guzman, Mark C; Meador, James W; Ferre, Rose Ann; Worland, Stephen T

    2002-04-11

    The structure-based design, chemical synthesis, and biological evaluation of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and a Michael acceptor moiety, which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The 2-pyridone-containing inhibitors typically display improved 3CP inhibition properties relative to related peptide-derived molecules along with more favorable antiviral properties. The cocrystal structure of one pyridone-derived 3CP inhibitor complexed with HRV-2 3CP is also described along with certain ab initio conformation analyses. Optimization of the 2-pyridone-containing compounds is shown to provide several highly active 3CP inhibitors (k(obs)/[I] > 500,00 M(-1) s(-1)) that function as potent antirhinoviral agents (EC(50) = <0.05 microM) against multiple virus serotypes in cell culture. One 2-pyridone-containing 3CP inhibitor is shown to be bioavailable in the dog after oral dosing (F = 48%).

  19. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. Part 7: structure-activity studies of bicyclic 2-pyridone-containing peptidomimetics.

    Science.gov (United States)

    Dragovich, Peter S; Prins, Thomas J; Zhou, Ru; Johnson, Theodore O; Brown, Edward L; Maldonado, Fausto C; Fuhrman, Shella A; Zalman, Leora S; Patick, Amy K; Matthews, David A; Hou, Xinjun; Meador, James W; Ferre, Rose Ann; Worland, Stephen T

    2002-03-11

    The structure-based design, chemical synthesis, and biological evaluation of bicyclic 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. An optimized compound is shown to exhibit antiviral activity when tested against a variety of HRV serotypes (EC(50)'s ranging from 0.037 to 0.162 microM).

  20. Understanding the specificity of serpin-protease complexes through interface analysis.

    Science.gov (United States)

    Rashid, Qudsia; Kapil, Charu; Singh, Poonam; Kumari, Vineeta; Jairajpuri, Mohamad Aman

    2015-01-01

    Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.

  1. Crystallographic Refinement by Incorporation of Molecular Dynamics : Thermostable Serine Protease Thermitase Complexed with Eglin c

    NARCIS (Netherlands)

    Gros, Piet; Fujinaga, Masao; Dijkstra, Bauke W.; Kalk, Kor H.; Hol, W G J

    1989-01-01

    In order to investigate the principles of protein thermostability, the crystal structure of thermitase from Thermoactinomyces vulgaris, a thermostable member of the subtilisin family of serine proteases, has been determined in a complex with eglin c. Eglin c is a serine protease inhibitor from the l

  2. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

    Science.gov (United States)

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-09-01

    Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

  3. 3C和3CL蛋白酶及广谱抑制剂的研究进展%Broad spectrum inhibitors of 3C and 3CL proteases:research advances

    Institute of Scientific and Technical Information of China (English)

    张启燕; 张文会; 肖军海; 李松

    2016-01-01

    Picornaviruses(PV)and coronaviruses(CoV) are positive-stranded RNA viruses. Pathogens in the family can cause hand,foot and mouth disease,myocarditis, common cold ,severe respiratory and intestinal diseases. 3C and 3CL proteases, belonging to cysteine proteases,are required to process polyproteins into mature proteins for viral replication,which plays an impor⁃tant role in viral replication because substrate binding sites are highly conservative and have similar catalytic mechanism. 3C and 3CL proteases are different from protease in the human body ,which represents a promising anti-viral drug target. Using 3C and 3CL pro⁃teinase structural similarities,broad spectrum protease inhibitors have been found successfully. This review describes recent develop⁃ments of broad spectrum protease inhibitors targeting on 3C and 3CL proteases,and briefly illustrates the mechanism of the inhibitors, which may benefit to the development of virus therapy.%小RNA病毒和冠状病毒属于单正链RNA病毒,其家族中的病原体易导致手足口病、心肌炎、普通感冒以及严重的呼吸道和肠道疾病。3C和3CL蛋白酶都属于半胱氨酸蛋白酶,底物结合位点高度保守且具有相似的催化机制,是催化单正链RNA病毒前体蛋白裂解的关键蛋白酶,对病毒的复制有重要作用。人体中没有与其相似的蛋白酶,是目前广谱抗单正链RNA病毒研究的重要靶点。利用3C和3CL蛋白酶结构的相同点,成功发现了具有广谱作用的蛋白酶抑制剂。本文简要概述3C和3CL蛋白酶的结构、功能和广谱抑制剂的研究进展,并简要阐释抑制剂的作用机制,对该类酶的广谱抑制剂研究和相关病毒的治疗具有指导意义。

  4. 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.

  5. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.

    Science.gov (United States)

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-07

    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

  6. Synthesis, modification and docking studies of 5-sulfonyl isatin derivatives as SARS-CoV 3C-like protease inhibitors.

    Science.gov (United States)

    Liu, Wei; Zhu, He-Min; Niu, Guo-Jun; Shi, En-Zhi; Chen, Jie; Sun, Bo; Chen, Wei-Qiang; Zhou, Hong-Gang; Yang, Cheng

    2014-01-01

    The Severe Acute Respiratory Syndrome (SARS) is a serious life-threatening and strikingly mortal respiratory illness caused by SARS-CoV. SARS-CoV which contains a chymotrypsin-like main protease analogous to that of the main picornavirus protease, 3CL(pro). 3CL(pro) plays a pivotal role in the viral replication cycle and is a potential target for SARS inhibitor development. A series of isatin derivatives as possible SARS-CoV 3CL(pro) inhibitors was designed, synthesized, and evaluated by in vitro protease assay using fluorogenic substrate peptide, in which several showed potent inhibition against the 3CL(pro). Structure-activity relationship was analyzed, and possible binding interaction modes were proposed by molecular docking studies. Among all compounds, 8k₁ showed most potent inhibitory activity against 3CL(pro) (IC₅₀=1.04 μM). These results indicated that these inhibitors could be potentially developed into anti-SARS drugs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Biophysical characterization of in vitro bound Streptomyces peucetius daunorubicin-serine protease complex.

    Science.gov (United States)

    Dubey, Rashmi; Prasad, Ranjan

    2014-03-01

    A serine protease of Streptomyces peucetius is found in association with daunorubicin in the culture filtrate and co-purifies as a complex as reported earlier by us (Dubey et al., 2013). The same protease was purified without drug attachment from dpsA(-) mutant of S. peucetius, which does not produce daunorubicin. Drug-protein complex was made in vitro by mixing daunorubicin and the protease. Spectral analysis and circular dichroism (CD) analysis were employed to determine the interaction between daunorubicin and the protease. Our study showed that interaction of daunorubicin with the protease affects the spectral characteristics of the drug and changes the secondary structure of the protein. Thin layer chromatography (TLC) analysis showed that the drug-protein interaction results in partial conversion of the drug to aglyconic form. The complex formation implies sequestration of the drug when it attains potentially lethal level in the extracellular milieu of S. peucetius culture. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication.

    Science.gov (United States)

    Kundu, Pallob; Raychaudhuri, Santanu; Tsai, Weimin; Dasgupta, Asim

    2005-08-01

    The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

  9. Structures of Enterovirus 71 3C proteinase (strain E2004104-TW-CDC) and its complex with rupintrivir.

    Science.gov (United States)

    Wu, Caiming; Cai, Qixu; Chen, Chen; Li, Ning; Peng, Xuanjia; Cai, Yaxian; Yin, Ke; Chen, Xinsheng; Wang, Xiaolong; Zhang, Rongfu; Liu, Lijie; Chen, Shuhui; Li, Jian; Lin, Tianwei

    2013-05-01

    The crystal structure of 3C proteinase (3C(pro)) from Enterovirus 71 (EV71) was determined in space group C2221 to 2.2 Å resolution. The fold was similar to that of 3C(pro) from other picornaviruses, but the difference in the β-ribbon reported in a previous structure was not observed. This β-ribbon was folded over the substrate-binding cleft and constituted part of the essential binding sites for interaction with the substrate. The structure of its complex with rupintrivir (AG7088), a peptidomimetic inhibitor, was also characterized in space group P212121 to 1.96 Å resolution. The inhibitor was accommodated without any spatial hindrance despite the more constricted binding site; this was confirmed by functional assays, in which the inhibitor showed comparable potency towards EV71 3C(pro) and human rhinovirus 3C(pro), which is the target that rupintrivir was designed against.

  10. A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

    Science.gov (United States)

    Schiering, Nikolaus; D’Arcy, Allan; Villard, Frederic; Simić, Oliver; Kamke, Marion; Monnet, Gaby; Hassiepen, Ulrich; Svergun, Dmitri I.; Pulfer, Ruth; Eder, Jörg; Raman, Prakash; Bodendorf, Ursula

    2011-01-01

    Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed protease-helicase domain assembly in solution. PMID:22160684

  11. A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target.

    Science.gov (United States)

    Schiering, Nikolaus; D'Arcy, Allan; Villard, Frederic; Simic, Oliver; Kamke, Marion; Monnet, Gaby; Hassiepen, Ulrich; Svergun, Dmitri I; Pulfer, Ruth; Eder, Jörg; Raman, Prakash; Bodendorf, Ursula

    2011-12-27

    Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed protease-helicase domain assembly in solution.

  12. Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease.

    Science.gov (United States)

    Lu, Zengjun; Bao, Huifang; Cao, Yimei; Sun, Pu; Guo, Jianhun; Li, Pinghua; Bai, Xingwen; Chen, Yingli; Xie, Baoxia; Li, Dong; Liu, Zaixin; Xie, Qingge

    2008-12-19

    Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine.

  13. Synthesis and Characterization of Quaternary Complex of Sm-CH3C(CH2OH)2COOH-Phen

    Institute of Scientific and Technical Information of China (English)

    Xi Peng; Wang Lianjun; Huang Xiangan

    2005-01-01

    A new quaternary complex with 2,2-dihydroxymethyl propionic acid and 1,10-phenanthroline, Sm(CH3C(CH2OH)2COO)(NO3)2(phen), was synthesized in ethanol solution and characterized by elemental analysis, IR and solid-state 13C NMR. The result of molar electrolytic conductivity of the complex indicates that it is non-electrolyte. Solution and thermal properties of the complex further verifies that the complex and ligant are different. In addition, the analytical results also imply that the two hydroxyl groups of 2,2-dihydroxymethyl propionic acid are not involved in the coordinate reaction. This will provide a new way for producing new multifunctional polymer materials with excellent fluorescent property. The fluorescence spectra of the complex were also studied. The main fluorescence emission peak of the complex appeared at 555, 604 and 646 nm. These fluorescent emission peaks correspond to transitions from 4G5/2 to 6H5/2, 6H7/2 and 6H9/2 energy levels of samarium(Ⅲ) ion, respectively. The most intense peak is at 646 nm, which lies in the range of absorption wavelength of chlorophyll a and chlorophyll b (600~680 nm), and it is beneficial to the crops to grow up.

  14. Computational titration analysis of a multiprotic HIV-1 protease-ligand complex.

    Science.gov (United States)

    Spyrakis, Francesca; Fornabaio, Micaela; Cozzini, Pietro; Mozzarelli, Andrea; Abraham, Donald J; Kellogg, Glen E

    2004-09-29

    A new computational method for analyzing the protonation states of protein-ligand complexes with multiple ionizable groups is applied to the structurally characterized complex between the peptide Glu-Asp-Leu and HIV-1 protease. This complex has eight ionizable groups at the active site: four from the ligand and four Asp residues on the protein. Correlation, with an error of ca. 0.6 kcal mol-1, is made between the calculated titration curve and the experimental titration curve. The analysis suggests that between four and five of the eight ionizable groups are protonated at the pH of crystallization.

  15. Crystal structures of multidrug-resistant HIV-1 protease in complex with two potent anti-malarial compounds

    Energy Technology Data Exchange (ETDEWEB)

    Yedidi, Ravikiran S.; Liu, Zhigang; Wang, Yong; Brunzelle, Joseph S.; Kovari, Iulia A.; Woster, Patrick M.; Kovari, Ladislau C.; Gupta, Deepak (LECOM); (WSI); (NWU); (MUSC); (WSU)

    2012-06-19

    Two potent inhibitors (compounds 1 and 2) of malarial aspartyl protease, plasmepsin-II, were evaluated against wild type (NL4-3) and multidrug-resistant clinical isolate 769 (MDR) variants of human immunodeficiency virus type-1 (HIV-1) aspartyl protease. Enzyme inhibition assays showed that both 1 and 2 have better potency against NL4-3 than against MDR protease. Crystal structures of MDR protease in complex with 1 and 2 were solved and analyzed. Crystallographic analysis revealed that the MDR protease exhibits a typical wide-open conformation of the flaps (Gly48 to Gly52) causing an overall expansion in the active site cavity, which, in turn caused unstable binding of the inhibitors. Due to the expansion of the active site cavity, both compounds showed loss of direct contacts with the MDR protease compared to the docking models of NL4-3. Multiple water molecules showed a rich network of hydrogen bonds contributing to the stability of the ligand binding in the distorted binding pockets of the MDR protease in both crystal structures. Docking analysis of 1 and 2 showed a decrease in the binding affinity for both compounds against MDR supporting our structure-function studies. Thus, compounds 1 and 2 show promising inhibitory activity against HIV-1 protease variants and hence are good candidates for further development to enhance their potency against NL4-3 as well as MDR HIV-1 protease variants.

  16. Serotype-Specific Structural Differences in the Protease-Cofactor Complexes of the Dengue Virus Family

    Energy Technology Data Exchange (ETDEWEB)

    Chandramouli, Sumana; Joseph, Jeremiah S.; Daudenarde, Sophie; Gatchalian, Jovylyn; Cornillez-Ty, Cromwell; Kuhn, Peter (Scripps)

    2010-03-04

    With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-{angstrom} crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.

  17. X-ray structure and inhibition of the feline infectious peritonitis virus 3C-like protease: Structural implications for drug design.

    Science.gov (United States)

    St John, Sarah E; Therkelsen, Matthew D; Nyalapatla, Prasanth R; Osswald, Heather L; Ghosh, Arun K; Mesecar, Andrew D

    2015-11-15

    Feline infectious peritonitis (FIP) is a deadly disease that effects both domestic and wild cats and is caused by a mutation in feline coronavirus (FCoV) that allows the virus to replicate in macrophages. Currently, there are no treatments or vaccines available for the treatment of FIP even though it kills approximately 5% of cats in multi-cat households per year. In an effort to develop small molecule drugs targeting FIP for the treatment of cats, we screened a small set of designed peptidomimetic inhibitors for inhibition of FIPV-3CL(pro), identifying two compounds with low to sub-micromolar inhibition, compound 6 (IC50=0.59±0.06 μM) and compound 7 (IC50=1.3±0.1 μM). We determined the first X-ray crystal structure of FIPV-3CL(pro) in complex with the best inhibitor identified, compound 6, to a resolution of 2.10 Å to better understand the structural basis for inhibitor specificity. Our study provides important insights into the structural requirements for the inhibition of FIPV-3CL(pro) by peptidomimetic inhibitors and expands the current structural knowledge of coronaviral 3CL(pro) architecture.

  18. Insights into the mechanism of drug resistance. X-ray structure analysis of multi-drug resistant HIV-1 protease ritonavir complex

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang [Wayne State Univ., Detroit, MI (United States); Harbor Hospital Baltimore, MD (United States); Yedidi, Ravikiran S. [Wayne State Univ., Detroit, MI (United States); National Inst. of Health (NIH), Bethesda, MD (United States); Wang, Yong [Wayne State Univ., Detroit, MI (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., Chicago, IL (United States); Kovari, Iulia A. [Wayne State Univ., Detroit, MI (United States); Kovari, Ladislau C. [Wayne State Univ., Detroit, MI (United States)

    2013-01-08

    Ritonavir (RTV) is a first generation HIV-1 protease inhibitor with rapidly emerging drug resistance. Mutations at residues 46, 54, 82 and 84 render the HIV-1 protease drug resistant against RTV. We report the crystal structure of multi-drug resistant (MDR) 769 HIV-1 protease (carrying resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84 and 90) complexed with RTV and the in vitro enzymatic IC50 of RTV against MDR HIV-1 protease. The structural and functional studies demonstrate significant drug resistance of MDR HIV-1 protease against RTV, arising from reduced hydrogen bonds and Van der Waals interactions between RTV and MDR HIV-1 protease.

  19. Complex formation between human prostate-specific antigen and protease inhibitors in mouse plasma.

    Science.gov (United States)

    Hekim, Can; Riipi, Tero; Zhu, Lei; Laakkonen, Pirjo; Stenman, Ulf-Håkan; Koistinen, Hannu

    2010-04-01

    When secreted from the prostate, most of prostate-specific antigen (PSA) is free and enzymatically active. Upon reaching circulation, active PSA is inactivated by complex formation with protease inhibitors. To justify the use of mouse models for evaluation of the function of PSA and for studies on therapeutic modalities based on modulation of PSA activity, it is important to know whether PSA complexation is similar in mouse and man. To characterize the circulating forms of PSA in mouse, we used subcutaneous LNCaP and 22RV1 human prostate cancer cell xenograft tumor models. We also added PSA directly to mouse serum. Free and total PSA were measured by immunoassay, and PSA complexes were extracted by immunopurification followed by SDS-PAGE, in-gel trypsin digestion and identification of signature peptides by mass spectrometry. In mice bearing xenograft tumors, 68% of the immunoreactive PSA occurred in complex, and when added to mouse serum, over 70% of PSA forms complexes that comprises alpha(2)-macroglobulin and members of the alpha(1)-antitrypsin (AAT) family. In mouse plasma, PSA forms complexes similar to those in man, but the major immunoreactive complex contains AAT rather than alpha(1)-antichymotrypsin, which is the main complex forming serpin in man. The complex formation of PSA produced by xenograft tumor models in mice is similar to that of human prostate tumors with respect to the complexation of PSA. (c) 2009 Wiley-Liss, Inc.

  20. The complex circumnuclear environment of the broad-line radio galaxy 3C 390.3 revealed by Chandra HETG

    CERN Document Server

    Tombesi, F; Kallman, T; Reynolds, C S; Mushotzky, R F; Braito, V; Behar, E; Leutenegger, M A; Cappi, M

    2016-01-01

    We present the first high spectral resolution X-ray observation of the broad-line radio galaxy 3C 390.3 obtained with the high energy transmission grating (HETG) spectrometer on board the Chandra X-ray Observatory. The spectrum shows complex emission and absorption features in both the soft X-rays and Fe K band. We detect emission and absorption lines in the energy range between E = 700-1000 eV associated with ionized Fe L transitions (Fe XVII-XX). An emission line at the energy of E=6.4 keV consistent with the Fe K\\alpha is also observed. Our best-fit model requires at least three different components: (i) a hot emission component likely associated with the hot interstellar medium in this elliptical galaxy with temperature kT=0.5+/-0.1 keV; (ii) a warm absorber with ionization parameter log\\xi=2.3+/-0.5 erg s^{-1} cm, column density logN_H=20.7+/-0.1 cm^{-2}, and outflow velocity of v_{out}<150 km s^{-1}; (iii) a lowly ionized reflection component in the Fe K band likely associated with the optical broad ...

  1. Effects of nanosuspension and inclusion complex techniques on the in vitro protease inhibitory activity of naproxen

    Energy Technology Data Exchange (ETDEWEB)

    Dharmalingam, Senthil Rajan; Chidambaram, Kumarappan; Srinivasan, Ramamurthy; Nadaraju, Shamala, E-mail: dsenthilrajan@yahoo.co.in [School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur (Malaysia)

    2014-01-15

    This study investigated the effects of nanosuspension and inclusion complex techniques on in vitro trypsin inhibitory activity of naproxen—a member of the propionic acid derivatives, which are a group of antipyretic, analgesic, and non-steroidal anti-inflammatory drugs. Nanosuspension and inclusion complex techniques were used to increase the solubility and anti-inflammatory efficacy of naproxen. The evaporative precipitation into aqueous solution (EPAS) technique and the kneading methods were used to prepare the nanosuspension and inclusion complex of naproxen, respectively. We also used an in vitro protease inhibitory assay to investigate the anti-inflammatory effect of modified naproxen formulations. Physiochemical properties of modified naproxen formulations were analyzed using UV, IR spectra, and solubility studies. Beta-cyclodextrin inclusion complex of naproxen was found to have a lower percentage of antitryptic activity than a pure nanosuspension of naproxen did. In conclusion, nanosuspension of naproxen has a greater anti-inflammatory effect than the other two tested formulations. This is because the nanosuspension formulation reduces the particle size of naproxen. Based on these results, the antitryptic activity of naproxen nanosuspension was noteworthy; therefore, this formulation can be used for the management of inflammatory disorders. (author)

  2. Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

    Directory of Open Access Journals (Sweden)

    Little Tom J

    2009-06-01

    Full Text Available Abstract Background Mosquitoes of the Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution. Methods Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the An. gambiae species complex in both East and West Africa. Results Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes. Conclusion It is well known that phylogenetic and population history in the An. gambiae complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the An. gambiae genome are discussed.

  3. Dynamical analysis of the complex radio structure in 3C 293: Clues on a rapid jet realignment in X-shaped radio galaxies

    CERN Document Server

    Machalski, J; Stawarz, L; Wezgowiec, M

    2016-01-01

    Radio galaxies classified as X-shaped/winged, are characterised by two pairs of extended and misaligned lobes, which suggest a rapid realignment of the jet axis, for which a potential cause is still under debate. Here we analyse the complex radio structure of 3C 293 winged source hosted by the post-merger galaxy, which uniquely displays a significant asymmetry between the sizes of the two pairs of lobes, indicating that an episode of jet realignment took place only very recently. Based on all the available radio data for 3C 293, we have performed a detailed spectral modelling for the older and younger lobes in the system. In this way we derived the lobes' ages and jet energetics, which we then compared to the accretion power in the source. We found that the 200 kpc-scale outer lobes of 3C 293 are ~60 Myr old and that jet activity related to the formation of the outer lobes ceased within the last Myr. Meanwhile, the inner 4 kpc-scale lobes, tilted by ~40 deg with respect to the outer ones, are only about ~0.3 ...

  4. The crystal structure of the secreted aspartic protease 1 from Candida parapsilosis in complex with pepstatin A

    Energy Technology Data Exchange (ETDEWEB)

    Dostál, Ji& #345; í; Brynda, Ji& #345; í; Hrušková-Heidingsfeldová, Olga; Sieglová, Irena; Pichová, Iva; & #344; ezá& #269; ová, Pavlína; (ASCR-ICP)

    2010-09-01

    Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-term survival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by production of extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied as potential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is less invasive than C. albicans, however, it is one of the leading causative agents of yeast infections. We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatin A, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein isolated from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold and topology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family and known structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparison revealed noticeable differences in the structure of loops surrounding the active site. This resulted in differential character, shape, and size of the substrate binding site explaining divergent substrate specificities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species might contribute to the development of new Sap-specific inhibitors.

  5. Thermokinetics on the Reaction of Formation of the Ternary Complex Nd[(C5H8NS2)3(C12H8N2)

    Institute of Scientific and Technical Information of China (English)

    FAN,Xue-Zhong(樊学忠); MENG,Xiang-Xin(孟祥鑫); CHEN,San-Ping(陈三平); JIAO,Bao-Juan(焦宝娟); REN,Yi-Xia(任宜霞); GAO,Sheng-Li(高胜利); SHI,Qi-Zhen(史启祯)

    2004-01-01

    The title ternary complex Nd[(C5H8NS2)3(C12H8N2)] has been synthesized in absolute ethanol by the reaction of atmosphere without any cautions against moisture. The bonding characteristics of the complex were characterized by IR, showing that Nd3+ is bonded with sulfur atom in the APDC and coordinated with nitrogen atom in the phen.TG-DTG investigation indicates that the title complex was decomposed into Nd2S3 and deposited carbon in one step where Nd2S3 predominated in the final products. The enthalpy change of formation of the reaction on the title complex in liquid-phase has been determined by a microcalorimeter. Thermodynamic parameters (the activation enthalpy, the activation entropy and the activation free energy) and kinetics parameters (the rate constant, the apparent activation energy, the pre-exponential constant and the reaction order) of the title reaction have been calculated. The enthalpy change of the solid-phase reaction has been obtained by a thermochemistry cycle.

  6. Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

    DEFF Research Database (Denmark)

    Zunszain, Patricia A; Knox, Stephen R; Sweeney, Trevor R;

    2010-01-01

    Foot-and-mouth disease (FMD) is a serious, widespread viral disease of cloven-hoofed animals, including important agricultural species such as cattle, sheep, pigs and goats (19, 45). The virus spreads rapidly and, although endemic and epidemic situations can be controlled using vaccines...

  7. Epstein-Barr virus nuclear antigen 3A partially coincides with EBNA3C genome-wide and is tethered to DNA through BATF complexes.

    Science.gov (United States)

    Schmidt, Stefanie C S; Jiang, Sizun; Zhou, Hufeng; Willox, Bradford; Holthaus, Amy M; Kharchenko, Peter V; Johannsen, Eric C; Kieff, Elliott; Zhao, Bo

    2015-01-13

    Epstein-Barr Virus (EBV) conversion of B-lymphocytes to Lymphoblastoid Cell Lines (LCLs) requires four EBV nuclear antigen (EBNA) oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and EBNALP associate with EBV and cell enhancers, up-regulate the EBNA promoter, MYC, and EBV Latent infection Membrane Proteins (LMPs), which up-regulate BCL2 to protect EBV-infected B-cells from MYC proliferation-induced cell death. LCL proliferation induces p16(INK4A) and p14(ARF)-mediated cell senescence. EBNA3A and EBNA3C jointly suppress p16(INK4A) and p14(ARF), enabling continuous cell proliferation. Analyses of the EBNA3A human genome-wide ChIP-seq landscape revealed 37% of 10,000 EBNA3A sites to be at strong enhancers; 28% to be at weak enhancers; 4.4% to be at active promoters; and 6.9% to be at weak and poised promoters. EBNA3A colocalized with BATF-IRF4, ETS-IRF4, RUNX3, and other B-cell Transcription Factors (TFs). EBNA3A sites clustered into seven unique groups, with differing B-cell TFs and epigenetic marks. EBNA3A coincidence with BATF-IRF4 or RUNX3 was associated with stronger EBNA3A ChIP-Seq signals. EBNA3A was at MYC, CDKN2A/B, CCND2, CXCL9/10, and BCL2, together with RUNX3, BATF, IRF4, and SPI1. ChIP-re-ChIP revealed complexes of EBNA3A on DNA with BATF. These data strongly support a model in which EBNA3A is tethered to DNA through a BATF-containing protein complexes to enable continuous cell proliferation.

  8. Structural and functional characterization of complex formation between two Kunitz-type serine protease inhibitors from Russell's Viper venom.

    Science.gov (United States)

    Mukherjee, Ashis K; Dutta, Sumita; Kalita, Bhargab; Jha, Deepak K; Deb, Pritam; Mackessy, Stephen P

    2016-01-01

    Snake venom Kunitz-type serine protease inhibitors (KSPIs) exhibit various biological functions including anticoagulant activity. This study elucidates the occurrence and subunit stoichiometry of a putative complex formed between two KSPIs (Rusvikunin and Rusvikunin-II) purified from the native Rusvikunin complex of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV). The protein components of the Rusvikunin complex were identified by LC-MS/MS analysis. The non-covalent interaction between two major components of the complex (Rusvikunin and Rusvikunin-II) at 1:2 stoichiometric ratio to form a stable complex was demonstrated by biophysical techniques such as spectrofluorometric, classical gel-filtration, equilibrium gel-filtration, circular dichroism (CD), dynamic light scattering (DLS), RP-HPLC and SDS-PAGE analyses. CD measurement showed that interaction between Rusvikunin and Rusvikunin-II did not change their overall secondary structure; however, the protein complex exhibited enhanced hydrodynamic diameter and anticoagulant activity as compared to the individual components of the complex. This study may lay the foundation for understanding the basis of protein complexes in snake venoms and their role in pathophysiology of snakebite.

  9. Radio Polarimetry of 3C119, 3C318, and 3C343 at milliarcsecond resolution

    CERN Document Server

    Mantovani, F; Junor, W; Saikia, D J; Salter, C J

    2010-01-01

    We report VLBA polarimetric observations of the CSS sources 3C119, 3C318, and 3C343 at 5 and 8.4 GHz. The CSS source 3C119 has source rest-frame RM values up to ~10200 rad/m**2 in a region which coincides with a change in the direction of the inner jet. This component is located ~325 pc from the core, which is variable and has a peaked radio spectrum. In the case of 3C318, a rest-frame RM of ~3030 rad/m**2 has been estimated for the brightest component which contributes almost all of the polarised emission. Further, two more extended components have been detected, clearly showing "wiggles" in the jet towards the southern side of the source. The CSS source 3C343 contains two peaks of emission and a curved jet embedded in more diffuse emission. It exhibits complex field directions near the emission peaks, which indicate rest-frame RM values in excess of ~6000 rad/m**2. The locations of the cores in 3C318 and 3C343 are not clear. The available data on mas-scale rest-frame RM estimates for CSS sources show that t...

  10. Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease

    Energy Technology Data Exchange (ETDEWEB)

    Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E.; Dunn, Ben M.; Wlodawer, Alexander (Debrecen); (NCI); (Florida); (Suan Sunandha)

    2012-10-23

    Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K{sub i}, depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place.

  11. Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain.

    Directory of Open Access Journals (Sweden)

    Liangzhong Lim

    Full Text Available Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT, except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our

  12. Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain.

    Science.gov (United States)

    Lim, Liangzhong; Shi, Jiahai; Mu, Yuguang; Song, Jianxing

    2014-01-01

    Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A) with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT), except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD) simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our results thus imply a

  13. Synthesis, Crystal Structure and Properties of Complex VO(C10H9NO3)(C13H10NO2)

    Institute of Scientific and Technical Information of China (English)

    MA Dong-sheng; GAO Shan; HUO Li-hua; GAO Jin-sheng; ZHAO Jin-gui

    2005-01-01

    Complex VO(C10H9NO3)(C13H10NO2)(C10H9NO2-3=N-salicylidene-L-alaninate, C13H10NO-2=N-phenylbenzohydroxamate) was synthesized and characterized by means of elemental analysis, IR, UV, 1H NMR spectroscopies, cyclic voltammetry and single crystal X-ray diffraction. The complex crystallized in a monoclinic system with space group P21 and crystal cell parameters a=0.9720(1) nm, b=1.8274(2) nm, c=1.2542(1) nm, β=104.868(9)°, V=2.1532(4) nm3, Mr=470.34, Z=2. The two oxygen atoms and the one nitrogen atom of the tridentate Schiff base ligand and the one oxime oxygen atom of the hydroxamate ligand coordinate to the vanadium atom, forming an equatorial plane, the two axial positions are respectively occupied by the oxygen atom of the oxovanadium and the carbonyl oxygen atom of the hydroxamate and the vanadium atom exhibits a distorted octahedral VO(ONO)(OO) coordination sphere. The 1H NMR spectrum suggests that the two isomers, endo and exo in a molar ratio of 1/1.7, coexist in the solution of the title complex in CDCl3. There exists a quasi-reversible one-electron redox reaction corresponding to VⅤ/VⅥ couple in the three non-aqueous solvents, and the redox potential E1/2 of the title complex substantially shifts in the direction of the positive voltage increase in the order: CH2Cl2<CH3CN<DMF.

  14. Dynamical analysis of the complex radio structure in 3C 293: clues on a rapid jet realignment in X-shaped radio galaxies

    Science.gov (United States)

    Machalski, J.; Jamrozy, M.; Stawarz, Ł.; Weżgowiec, M.

    2016-10-01

    Context. Radio galaxies classified as X-shaped/winged, are characterised by two pairs of extended and misaligned lobes, which suggest a rapid realignment of the jet axis, for which a potential cause (including binary supermassive black holes, a black hole merger, or a Lense-Thirring precession) is still under debate. Aims: Here we analyse the complex radio structure of 3C 293 winged source hosted by the post-merger galaxy UGC 8782, which uniquely displays a significant asymmetry between the sizes (and therefore the ages) of the two pairs of lobes, indicating that an episode of jet realignment took place only very recently. This allows us to tightly constrain the corresponding timescales, and therefore to discriminate between different models proposed for the formation of X-shaped radio galaxies in general. Methods: Based on all the available and carefully re-analysed radio data for 3C 293, we have performed a detailed spectral modelling for the older and younger lobes in the system, using the existing evolutionary DYNAGE algorithm. In this way we derived the lobes' ages and jet energetics, which we then compared to the accretion power in the source. Results: We found that the 200 kpc-scale outer lobes of 3C 293 are 60 Myr old and, until very recently, have been supplied with fresh electrons and magnetic field by the jets, i.e., jet activity related to the formation of the outer lobes ceased within the last Myr. Meanwhile, the inner 4 kpc-scale lobes, tilted by 40° with respect to the outer ones, are only about 0.3 Myr old. Interestingly, the best model fits also return identical values of the jet power supplying the outer and the inner structures. This power, moreover, is of the order of the maximum kinetic luminosity of a Blandford-Znajek jet for a given black hole mass and accretion rate, but only in the case of relatively low values of a black hole spin, a 0.2. Conclusions: The derived jet energetics and timescales, along with the presence of two optical nuclei

  15. Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway.

    Science.gov (United States)

    Hsu, Shih-Che; Huang, Shih-Ming; Chen, Ann; Sun, Chiao-Yin; Lin, Shih-Hua; Chen, Jin-Shuen; Liu, Shu-Ting; Hsu, Yu-Juei

    2014-08-01

    The Klotho gene functions as an aging suppressor gene. Evidence from animal models suggests that induction of Klotho expression may be a potential treatment for age-associated diseases. However, the molecular mechanism involved in regulating renal Klotho gene expression remains unclear. In this study, we determined that resveratrol, a natural polyphenol, induced renal Klotho expression both in vivo and in vitro. In the mouse kidney, resveratrol administration markedly increased both Klotho mRNA and protein expression. In resveratrol-treated NRK-52E cells, increased Klotho expression was accompanied by the upregulation and nuclear translocation of activating transcription factor 3 (ATF3) and c-Jun. ATF3 or c-Jun overexpression enhanced the transcriptional activation of Klotho. Conversely, resveratrol-induced Klotho expression was attenuated in the presence of dominant-negative ATF3 or c-Jun. Coimmunoprecipitation and a chromatin immunoprecipitation assay revealed that ATF3 physically interacted with c-Jun and that the ATF3/c-Jun complex directly bound to the Klotho promoter through ATF3- and AP-1-binding elements. c-Jun cotransfection augmented the effects of ATF3 on Klotho transcription in vitro. Although Sirtuin 1 mRNA expression was induced by resveratrol and involved in regulating Klotho mRNA expression, it was not the primary cause for the aforementioned ATF3/c-Jun pathway. In summary, resveratrol enhances the renal expression of the anti-aging Klotho gene, and the transcriptional factors ATF3 and c-Jun functionally interact and coordinately regulate the resveratrol-mediated transcriptional activation of Klotho.

  16. Pharmacological properties and pathophysiological significance of a Kunitz-type protease inhibitor (Rusvikunin-II) and its protein complex (Rusvikunin complex) purified from Daboia russelii russelii venom.

    Science.gov (United States)

    Mukherjee, Ashis K; Mackessy, Stephen P

    2014-10-01

    A 7.1 kDa basic peptide (Rusvikunin-II) was purified from a previously described protein complex (Rusvikunin complex, consists of Rusvikunin and Rusvikunin-II) of Daboia russelii russelii venom. The N-terminal sequence of Rusvikunin-II was found to be blocked, but peptide mass fingerprinting analysis indicated its identity as Kunitz-type basic protease inhibitor 2, previously reported from Russell's Viper venom. A tryptic peptide sequence of Rusvikunin-II containing the N-terminal sequence HDRPTFCNLFPESGR demonstrated significant sequence homology to venom basic protease inhibitors, Kunitz-type protease inhibitors and trypsin inhibitors. The secondary structure of Rusvikunin-II was dominated by β-sheets (60.4%), followed by random coil (38.2%), whereas α-helix (1.4%) contributes the least to its secondary structure. Both Rusvikunin-II and the Rusvikunin complex demonstrated dose-dependent anticoagulant activity; however, the anticoagulant potency of latter was found to be higher. Both inhibited the amidolytic activity of trypsin > plasmin > FXa, fibrinogen clotting activity of thrombin, and, to a lesser extent, the prothrombin activation property of FXa; however, the inhibitory effect of the Rusvikunin complex was more pronounced. Neither Rusvikunin-II nor Rusvikunin complex inhibited the amidolytic activity of chymotrypsin and thrombin. Rusvikunin-II at 10 μg/ml was not cytotoxic to Colo-205, MCF-7 or 3T3 cancer cells; conversely, Rusvikunin complex showed ∼30% reduction of MCF-7 cells under identical experimental conditions. Rusvikunin-II (5.0 mg/kg body weight, i.p. injection) was not lethal to mice or House Geckos; nevertheless, it showed in vivo anticoagulant action in mice. However, the Rusvikunin complex (at 5.0 mg/kg) was toxic to NSA mice, but not to House Geckos, suggesting it has prey-specific toxicity. Rusvikunin complex-treated mice exhibited dyspnea and hind-limb paresis prior to death. The present study indicates that the Kunitz

  17. Crystal structure and properties of terbium o-methylbenzoate complex with 1,10-phenanthroline [Tb(o-CH3C6H4COO)3(C12H8N2)]2

    Institute of Scientific and Technical Information of China (English)

    WANG Ruifen; WANG Shuping; SHI Shikao; ZHANG Jianjun

    2004-01-01

    A terbium o-methylbenzoate complex with 1,10-phenanthroline, Tb(o-MBA)3phen (where o-MBA=o-methylbenzoate and phen = 1,10-phenanthroline) was prepared from ethanol solution and its crystal structure was determined by X-ray diffraction. The crystal of the complex Th(o-MBA)3phen belongs to triclinic crystal system and P 1 (#2)space group. The crystal data are as follows: a = 1.4371(4) nm, b = 1.7387(2) nm, c = 1.3109(2) nm, α = 96.37(1)°, β =107.21 (2)°, γ= 82.78(2)°, V = 3.094(1) nm3, Z = 2, Mr = 1489.12, Dc = 1.598 g.cm-3,μ = 2.330 rnm-1 and F(000) = 1488.00.The final R and Rw are 0.038 and 0.047 for 8668 [Ⅰ>2σ(Ⅰ)] unique reflections, respectively. In the complex each Tb3+ ion is eight-coordinated by one 1,10-phenanthroline molecule, one bidentate carboxylate group and four bridging carboxylate groups. The carboxylate groups are bonded to the terbium ion in two modes: the chelating bidentate and the bridging bidentate. Excitation and luminescence data observed at room temperature show that the title complex emits strong green fluorescence under ultraviolet light. The results of thermal analysis indicate that the complex Tb(o-MBA)3phen is quite stable to heat.

  18. Protein phosphatase complex PP5/PPP2R3C dephosphorylates P-glycoprotein/ABCB1 and down-regulates the expression and function.

    Science.gov (United States)

    Katayama, Kazuhiro; Yamaguchi, Miho; Noguchi, Kohji; Sugimoto, Yoshikazu

    2014-04-01

    P-glycoprotein (P-gp)/ABCB1 is a key molecule of multidrug resistance in cancer. Protein phosphatase (PP) 2A, regulatory subunit B, gamma (PPP2R3C), which is a regulatory subunit of PP2A and PP5, was identified as a binding candidate to P-gp. Immunoprecipitation-western blotting revealed that PP5 and PPP2R3C were coprecipitated with P-gp, while PP2A was not. PP5/PPP2R3C dephosphorylated protein kinase A/protein kinase C-phosphorylation of P-gp. Knockdown of PP5 and/or PPP2R3C increased P-gp expression and lowered the sensitivity to vincristine and doxorubicin. Consequently, our results indicate that PP5/PPP2R3C negatively regulates P-gp expression and function.

  19. Fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus aspartic protease, an Aspergillus aspartic protease per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a protease of the aspartic proteinase-type,

  20. Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2

    DEFF Research Database (Denmark)

    Vorup-Jensen, T; Petersen, Steen Vang; Hansen, A G;

    2000-01-01

    Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine...

  1. The Levels of the Lectin Pathway Serine Protease MASP-1 and Its Complex Formation with C1 Inhibitor Are Linked to the Severity of Hereditary Angioedema

    DEFF Research Database (Denmark)

    Hansen, Cecilie Bo; Csuka, Dorottya; Munthe-Fog, Lea

    2015-01-01

    C1 inhibitor (C1-INH) is known to form complexes with the lectin complement pathway serine proteases MASP-1 and MASP-2. Deficiency of C1-INH is associated with hereditary angioedema (HAE), an autosomal inherited disease characterized by swelling attacks caused by elevated levels of bradykinin. MASP...

  2. Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

    Energy Technology Data Exchange (ETDEWEB)

    Shenoy, Rajesh T. [National Univ. of Singapore (Singapore); Thangamani, Saravanan [National Univ. of Singapore (Singapore); Univ. of Texas Medical Branch, Galveston, TX (United States); Velazquez-Campoy, Adrian [Univ. of Zaragoza (Spain); Ho, Bow [National Univ. of Singapore (Singapore); Ding, Jeak Ling [National Univ. of Singapore (Singapore); Sivaraman, J. [National Univ. of Singapore (Singapore); Kursula, Petri [Univ. of Oulu (Germany)

    2011-04-26

    Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki=1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1:2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.

  3. Crystallisation and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael-acceptor inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Coates, Leighton [ORNL; Cooper, Jon [University of Southampton, England; Hussey, Robert [University of Southampton, England

    2008-01-01

    Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. While the native crystals were found to diffract only to medium resolution (2.9 {angstrom}), cocrystals of an inhibitor complex diffracted X-rays to 1.7 {angstrom} resolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

  4. Crystal Structure of the VP4 Protease from Infectious Pancreatic Necrosis Virus Reveals the acyl-enzyme Complex for an Intermolecular Self-Cleavage Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Lee,J.; Feldman, A.; Delmas, B.; Paetzel, M.

    2007-01-01

    Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH{sub 2}-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-{angstrom} resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser{sup 633}O{gamma} forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala{sup 716}, of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-{angstrom} resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity ({approx}19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.

  5. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused...... to the protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N...... of few known proteases to have substrate specificity for the C-terminal side of the scissile bond. LysN exhibits specificity for lysine, and has primarily been used to complement trypsin in to proteomic studies. A working hypothesis during this study was the potential of LysN as a processing protease...

  6. Effects of nanosuspension and inclusion complex techniques on the in vitro protease inhibitory activity of naproxen

    OpenAIRE

    Dharmalingam, Senthil Rajan; Chidambaram, Kumarappan; Ramamurthy, Srinivasan; Nadaraju,Shamala

    2014-01-01

    This study investigated the effects of nanosuspension and inclusion complex techniques on in vitro trypsin inhibitory activity of naproxen—a member of the propionic acid derivatives, which are a group of antipyretic, analgesic, and non-steroidal anti-inflammatory drugs. Nanosuspension and inclusion complex techniques were used to increase the solubility and anti-inflammatory efficacy of naproxen. The evaporative precipitation into aqueous solution (EPAS) technique and the kneading metho...

  7. Crystal structure of an FIV/HIV chimeric protease complexed with the broad-based inhibitor, TL-3

    Directory of Open Access Journals (Sweden)

    Elder John H

    2007-01-01

    Full Text Available Abstract We have obtained the 1.7 Å crystal structure of FIV protease (PR in which 12 critical residues around the active site have been substituted with the structurally equivalent residues of HIV PR (12X FIV PR. The chimeric PR was crystallized in complex with the broad-based inhibitor TL-3, which inhibits wild type FIV and HIV PRs, as well as 12X FIV PR and several drug-resistant HIV mutants 1234. Biochemical analyses have demonstrated that TL-3 inhibits these PRs in the order HIV PR > 12X FIV PR > FIV PR, with Ki values of 1.5 nM, 10 nM, and 41 nM, respectively 234. Comparison of the crystal structures of the TL-3 complexes of 12X FIV and wild-typeFIV PR revealed theformation of additinal van der Waals interactions between the enzyme inhibitor in the mutant PR. The 12X FIV PR retained the hydrogen bonding interactions between residues in the flap regions and active site involving the enzyme and the TL-3 inhibitor in comparison to both FIV PR and HIV PR. However, the flap regions of the 12X FIV PR more closely resemble those of HIV PR, having gained several stabilizing intra-flap interactions not present in wild type FIV PR. These findings offer a structural explanation for the observed inhibitor/substrate binding properties of the chimeric PR.

  8. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

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

  9. Selective modulation of the CD4 molecular complex by Pseudomonas aeruginosa alkaline protease and elastase

    DEFF Research Database (Denmark)

    Pedersen, B K; Kharazmi, A; Theander, T G

    1987-01-01

    The binding of monoclonal antibodies against CD4 was specifically inhibited by treatment of human CD4+ cells with either alkaline protease (AP) or elastase (Ela), purified from Pseudomonas aeruginosa. Binding of antibodies against CD3 (pan T), CD5 (pan T), CD8 (T suppressor/cytotoxic), HLA-ABC, HLA......-DR, HLA-DQ, HLA-DP/DR, and beta 2 microglobulin was not inhibited by AP or Ela. Heat-inactivation of the proteases at 65 degrees C for 20 min or treatment with the metal chelator EDTA abolished the inhibitory activity of both proteases. These findings may serve to develop novel immunological methods...

  10. Relationship between angiotensinogen, alpha 1-protease inhibitor elastase complex, antithrombin III and C-reactive protein in septic ARDS.

    Science.gov (United States)

    Hilgenfeldt, U; Kellermann, W; Kienapfel, G; Jochum, M

    1990-01-01

    The time-course of plasma angiotensinogen (Ao), elastase-alpha 1-protease inhibitor complex (EL alpha 1PI), antithrombin III (AT III) and C-reactive protein (CRP) have been investigated of six patients suffering from adult respiratory distress syndrome (ARDS). The total plasma Ao level (active and inactive Ao) varied in individuals but was increased up to five-fold. An increasing amount of inactive Ao is found. From the beginning of their stay in the intensive care unit up to five days half of the patients displayed a positive correlation between the plasma CRP and Ao level. The CRP and Ao values were either not or were negatively correlated with the AT III values. In contrast plasma Ao and AT III levels in all patients were positively correlated during a particular period in the subsequent phase of the disease, where there was no or a negative correlation with CRP. The two acute phase reactants CRP and EL alpha 1PI were only correlated in two patients at the beginning of the disease. The markedly increased plasma level at the beginning of the inflammatory disease indicates that Ao is an acute phase reactant, and this is supported by the parallel changes in plasma CRP and Ao levels during the early days of ARDS. The relationship between the plasma levels of Ao and AT III for more than fourteen days suggests similar regulation of these members of the serpin family after termination of the acute-phase.

  11. Protease inhibitor

    DEFF Research Database (Denmark)

    2009-01-01

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

  12. Synthesis and crystal structure of a new homoleptic tetraarylruthenium(IV) complex Ru(2,4,5-Me{sub 3}C{sub 6}H{sub 2}){sub 4}

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chang-Jiu; Wu, Xiu-Li; Ma, Xiu-Fang; Jia, Ai-Quan; Zhang, Qian-Feng [Anhui Univ. of Technology, Anhui (China). Inst. of Molecular Engineering and Applied Chemistry and Anhui Province Key Lab. of Metallurgy Engineering and Resources Recycling

    2017-08-01

    Treatment of [Ru(acac){sub 3}] (acac-=acetylacetonate) with (2,4,5-Me{sub 3}C{sub 6}H{sub 2})MgBr, followed by column chromatography in air, afforded the homoleptic tetraaryl-ruthenium(IV) complex [Ru(2,4,5-Me{sub 3}C{sub 6}H{sub 2}){sub 4}] (1) in moderate yield. The product was characterized by proton NMR spectroscopy and microanalyses. Its crystal structure has also been established by X-ray crystallography.

  13. Supermarket Proteases.

    Science.gov (United States)

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

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

  14. Insights into the structural function of the complex of HIV-1 protease with TMC-126: molecular dynamics simulations and free-energy calculations

    Energy Technology Data Exchange (ETDEWEB)

    Li, Dan; Han, Ju-Guang; Chen, Hang; Li, Liang; Zhao, Run-Ning Zhao; Liu, Guang; Duan, Yuhua

    2012-05-01

    The binding properties of the protein-inhibitor complex of human immunodeficiency virus type 1 (HIV-1) protease with the inhibitor TMC-126 are investigated by combining computational alanine scanning (CAS) mutagenesis with binding free-energy decomposition (BFED). The calculated results demonstrate that the flap region (residues 38-58) and the active site region (residues 23-32) in HIV-1 protease contribute 63.72% of the protease to the binding of the inhibitor. In particular, the mechanisms for the interactions of key residues of these species are fully explored and analyzed. Interestingly, the regression analyses show that both CAS and BFED based on the generalized Born model yield similar results, with a correlation coefficient of 0.94. However, compared to CAS, BFED is faster and can decompose the per-residue binding free-energy contributions into backbone and sidechain contributions. The results obtained in this study are useful for studying the binding mechanism between receptor and ligand and for designing potent inhibitors that can combat diseases.

  15. Molecular modeling and molecular dynamics simulation study of the human Rab9 and RhoBTB3 C-terminus complex

    Science.gov (United States)

    Junaid, Muhammad; Muhseen, Ziyad Tariq; Ullah, Ata; Wadood, Abdul; Liu, Junjun; Zhang, Houjin

    2014-01-01

    Rab9 is required for the transport of mannose 6-phosphate receptors to the trans-Golgi network from late endosomes through the interaction with its effector: RhoBTB3. Earlier research indicates the C-terminus of RhoBTB3 (Rho_Cterm) is used for the interaction with Rab9. We used the homology modeling along with the molecular dynamics (MD) simulation to study the binding pattern of Rho_Cterm and Rab9 at atomic level. Both modeled structures, Rab9 and Rho_Cterm, are of high quality as suggested by the Ramachandran plot and ProCheck. The complex of Rab9-Rho_Cterm was generated by unrestrained pairwise docking using ZDOCK server. The interface of complex is consistent with the previous experimental data. The results of MD simulation indicate that the binding interface is stable along the simulation process. PMID:25670879

  16. Peritoneal lavage efficiently eliminates protease-alpha-2-macroglobulin complexes and components of the contact system from the peritoneal cavity in patients with severe acute pancreatitis.

    Science.gov (United States)

    Aasen, A O; Ruud, T E; Roeise, O; Bouma, B N; Stadaas, J O

    1989-01-01

    Trypsin (Try), plasma kallikrein (KK) and plasmin activities together with coagulation factor XII (F XII, Hageman factor), high-molecular-weight kininogen (HMWK), plasma prekallikrein (PKK), alpha 2-macroglobulin (alpha 2-M), C1 inhibitor (C1Inh), and functional plasma kallikrein inhibition (KKI) values were studied in peritoneal fluid and lavage taps of 9 patients with severe acute pancreatitis treated with peritoneal lavage. Both immunochemical methods and functional techniques based on chromogenic peptide substrate assays were used. In the exudate obtained before peritoneal lavage was performed, F XII was 52%, HMWK was 30%, PKK was 40%, alpha 2-M was 29% and C1Inh was 57% of standard plasma pool values, determined by immunochemical technique. Functional plasma KKI values were zero, whereas Try activities determined by chromogenic peptide substrate technique were markedly elevated in the exudate. Using a prepacked HR 10/30 Superose Tm 12 column (Pharmacia, Uppsala, Sweden) and chromogenic peptide substrate assays, Try and KK activities were detected in the alpha 2-M containing fractions of the peritoneal exudate demonstrating KK-alpha 2-M and Try-alpha 2-M complex formation. The peritoneal lavage procedure efficiently eliminated components of the contact system and protease activities. In the first lavage tap, Try activities were markedly reduced compared to values found in the exudate and concentrations of F XII, HMWK, PKK, alpha 2-M and C1Inh were all zero. In consecutive lavage taps Try values were also zero. The study shows that the lavage procedures efficiently clears the peritoneal cavity for protease-alpha 2-M complexes generated during acute pancreatitis. Also, components of the contact system found in peritoneal exudate, and which might serve as substrates for the protease-alpha 2-M complexes, are rapidly eliminated by the procedure.

  17. Crystallization of a Nonclassical Kazal-type Carcinoscorpius Rotundicauda Serine Protease Inhibitor, CrSPI-1, Complexed with Subtilisin

    Energy Technology Data Exchange (ETDEWEB)

    Tulsidas, S.; Thangamani, S; Ho, B; Sivaraman, J; Ding, J

    2009-01-01

    Serine proteases play a major role in host-pathogen interactions. The innate immune system is known to respond to invading pathogens in a nonspecific manner. The serine protease cascade is an essential component of the innate immune system of the horseshoe crab. The serine protease inhibitor CrSPI isoform 1 (CrSPI-1), a unique nonclassical Kazal-type inhibitor of molecular weight 9.3 kDa, was identified from the hepatopancreas of the horseshoe crab Carcinoscorpius rotundicauda. It potently inhibits subtilisin and constitutes a powerful innate immune defence against invading microbes. Here, the cloning, expression, purification and cocrystallization of CrSPI-1 with subtilisin are reported. The crystals diffracted to 2.6 {angstrom}resolution and belonged to space group P2{sub 1}, with unit-cell parameters a = 73.8, b = 65.0, c = 111.9 {angstrom}, {beta} = 95.4. The Matthews coefficient (VM = 2.64 {angstrom}3 Da-1, corresponding to 53% solvent content) and analysis of the preliminary structure solution indicated the presence of one heterotrimer (1:2 ratio of CrSPI-1:subtilisin) and one free subtilisin molecule in the asymmetric unit.

  18. Hydrothermal Synthesis and Electrochemical Properties of Complex Cu(CH3C6H4COOH)2(2,2'-bipy)·(H2O)

    Institute of Scientific and Technical Information of China (English)

    LI Wei; LI Chang-Hong; YANG Ying-Qun; KUANG Dai-Zhi; XU Wei-Jian

    2006-01-01

    The title complex has been synthesized by 4-methylbenzoic acid and 2,2'-bipyridine (bipy) in the mixed solvent of water and methanol. It crystallizes in the triclinic system, space group (P1-) with a = 0.7047(3), b = 1.1217(5), c = 1.6718(7) nm, α = 103.826(7), β = 90.772(6), γ = 104.195(6)°, C26H25CuN2O5.50, Mr = 517.02, V = 1.2404(9) nm3, Dc = 1.384 g/cm3, Z = 2, F(000) = 536, μ(MoKα= 0.921 mm-1, R = 0.0782 and wR = 0.2172. Structural analysis shows that the copper atom is coordinated with three oxygen atoms from two 4-methylbenzoic acids and one water molecule together with two nitrogen atoms from 2,2'-bipyridine, giving a distorted square-pyramid coordination geometry. The cyclic voltametric behavior of the complex has also been described.

  19. Synthesis, characterization, and DNA binding, photocleavage, cytotoxicity, cellular uptake, apoptosis, and on-off light switching studies of Ru(II) mixed-ligand complexes containing 7-fluorodipyrido[3,2-a:2',3'-c]phenazine.

    Science.gov (United States)

    Deepika, Nancherla; Kumar, Yata Praveen; Shobha Devi, Chittimalli; Reddy, Putta Venkat; Srishailam, Avudoddi; Satyanarayana, Sirasani

    2013-10-01

    Four new ruthenium(II) polypyridyl complexes-[Ru(phen)2(7-F-dppz)](2+) (7-F-dppz is 7-fluorodipyrido[3,2-a:2',3'-c]phenazine, phen is 1,10-phenanthroline), [Ru(bpy)2(7-F-dppz)](2+)(2) (bpy is 2,2'-bipyridine), [Ru(dmb)2(7-F-dppz)](2+) (dmb is 4,4'-dimethyl-2,2'-bipyridine), and [Ru(hdpa)2(7-F-dppz)](2+) (hdpa is 2,2'-dipyridylamine)-have been synthesized and characterized. Their DNA binding behavior has been explored by various spectroscopic titrations and viscosity measurements, which indicated that all the complexes bind to calf thymus DNA by means of intercalation with different binding strengths. The light switching properties of these complexes have been evaluated, and their antimicrobial activities have been investigated. Photoinduced DNA cleavage studies have been performed. All the complexes exhibited efficient photocleavage of pBR322 DNA on irradiation. The cytotoxicity of these complexes has been evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with various tumor cell lines. Cellular uptake was studied by flow cytometry and confocal microscopy. Flow cytometry experiments showed that these complexes induced apoptosis of HeLa cell lines.

  20. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Hurkman William J

    2011-02-01

    Full Text Available Abstract Background Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences. Results Grain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%. Conclusions This is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar

  1. 3C DUCT DESIGN METHOD

    Institute of Scientific and Technical Information of China (English)

    Huan-RueiShiu; Feng-ChuOu; Sih-LiChen

    2002-01-01

    A new 3C duct design method is proposed for designing a high quality, energy-efficiency cost-effective air duct system. It not only considers the demand of volume flow rate, but also takes into consideration a number of issues including system pressure balance, noise, vibration, space limitation and total system cost. This new method comprises three major calculation procedures:initial computer-aided design (CAD), computer-aided simulation (CAS) and correction processes (CP). An example is presented in this study to understand the characteristics of 3C method. It shows that 3C duct design method provides a simple computation procedure for an optimum air duct system. It also shortens the design schedule, prevents human calculation errors, and reduces the dependence on designer experience. In addition to apply in a new duct system design, 3C duct design method is also a powerful design tool for the expansion of an existing duct system.

  2. Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy

    Science.gov (United States)

    Wang, Li; Li, Xueying; Shen, Hongchun; Mao, Nan; Wang, Honglian; Cui, Luke; Cheng, Yuan; Fan, Junming

    2016-01-01

    Mesangial deposition of aberrantly glycosylated IgA1 (agIgA1) and its immune complexes is a key pathogenic mechanism of IgA nephropathy (IgAN). However, treatment of IgAN remains ineffective. We report here that bacteria-derived IgA proteases are capable of degrading these pathogenic agIgA1 and derived immune complexes in vitro and in vivo. By screening 14 different bacterial strains (6 species), we found that 4 bacterial IgA proteases from H. influenzae, N. gonorrhoeae and N. meningitidis exhibited high cleaving activities on serum agIgA1 and artificial galactose-depleted IgA1 in vitro and the deposited agIgA1-containing immune complexes in the mesangium of renal biopsy from IgAN patients and in a passive mouse model of IgAN in vitro. In the modified mouse model of passive IgAN with abundant in situ mesangial deposition of the agIgA-IgG immune complexes, a single intravenous delivery of IgA protease from H. influenzae was able to effectively degrade the deposited agIgA-IgG immune complexes within the glomerulus, demonstrating a therapeutic potential for IgAN. In conclusion, the bacteria-derived IgA proteases are biologically active enzymes capable of cleaving the circulating agIgA and the deposited agIgA-IgG immune complexes within the kidney of IgAN. Thus, the use of such IgA proteases may represent a novel therapy for IgAN. PMID:27485391

  3. Protease gene families in Populus and Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jansson Stefan

    2006-12-01

    Full Text Available Abstract Background Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Results We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. Conclusion Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

  4. Insight to structural subsite recognition in plant thiol protease-inhibitor complexes : Understanding the basis of differential inhibition and the role of water

    Directory of Open Access Journals (Sweden)

    Mukhopadhayay Bishnu P

    2001-09-01

    Full Text Available Abstract Background This work represents an extensive MD simulation / water-dynamics studies on a series of complexes of inhibitors (leupeptin, E-64, E-64-C, ZPACK and plant cysteine proteases (actinidin, caricain, chymopapain, calotropin DI of papain family to understand the various interactions, water binding mode, factors influencing it and the structural basis of differential inhibition. Results The tertiary structure of the enzyme-inhibitor complexes were built by visual interactive modeling and energy minimization followed by dynamic simulation of 120 ps in water environment. DASA study with and without the inhibitor revealed the potential subsite residues involved in inhibition. Though the interaction involving main chain atoms are similar, critical inspection of the complexes reveal significant differences in the side chain interactions in S2-P2 and S3-P3 pairs due to sequence differences in the equivalent positions of respective subsites leading to differential inhibition. Conclusion The key finding of the study is a conserved site of a water molecule near oxyanion hole of the enzyme active site, which is found in all the modeled complexes and in most crystal structures of papain family either native or complexed. Conserved water molecules at the ligand binding sites of these homologous proteins suggest the structural importance of the water, which changes the conventional definition of chemical geometry of inhibitor binding domain, its shape and complimentarity. The water mediated recognition of inhibitor to enzyme subsites (Pn...H2O....Sn of leupeptin acetyl oxygen to caricain, chymopapain and calotropinDI is an additional information and offer valuable insight to potent inhibitor design.

  5. Structure and mechanism of rhomboid protease.

    Science.gov (United States)

    Ha, Ya; Akiyama, Yoshinori; Xue, Yi

    2013-05-31

    Rhomboid protease was first discovered in Drosophila. Mutation of the fly gene interfered with growth factor signaling and produced a characteristic phenotype of a pointed head skeleton. The name rhomboid has since been widely used to describe a large family of related membrane proteins that have diverse biological functions but share a common catalytic core domain composed of six membrane-spanning segments. Most rhomboid proteases cleave membrane protein substrates near the N terminus of their transmembrane domains. How these proteases function within the confines of the membrane is not completely understood. Recent progress in crystallographic analysis of the Escherichia coli rhomboid protease GlpG in complex with inhibitors has provided new insights into the catalytic mechanism of the protease and its conformational change. Improved biochemical assays have also identified a substrate sequence motif that is specifically recognized by many rhomboid proteases.

  6. Structure of Psb29/Thf1 and its association with the FtsH protease complex involved in photosystem II repair in cyanobacteria.

    Science.gov (United States)

    Bec Ková, Martina; Yu, Jianfeng; Krynická, Vendula; Kozlo, Amanda; Shao, Shengxi; Koník, Peter; Komenda, Josef; Murray, James W; Nixon, Peter J

    2017-09-26

    One strategy for enhancing photosynthesis in crop plants is to improve their ability to repair photosystem II (PSII) in response to irreversible damage by light. Despite the pivotal role of thylakoid-embedded FtsH protease complexes in the selective degradation of PSII subunits during repair, little is known about the factors involved in regulating FtsH expression. Here we show using the cyanobacterium Synechocystis sp. PCC 6803 that the Psb29 subunit, originally identified as a minor component of His-tagged PSII preparations, physically interacts with FtsH complexes in vivo and is required for normal accumulation of the FtsH2/FtsH3 hetero-oligomeric complex involved in PSII repair. We show using X-ray crystallography that Psb29 from Thermosynechococcus elongatus has a unique fold consisting of a helical bundle and an extended C-terminal helix and contains a highly conserved region that might be involved in binding to FtsH. A similar interaction is likely to occur in Arabidopsis chloroplasts between the Psb29 homologue, termed THF1, and the FTSH2/FTSH5 complex. The direct involvement of Psb29/THF1 in FtsH accumulation helps explain why THF1 is a target during the hypersensitive response in plants induced by pathogen infection. Downregulating FtsH function and the PSII repair cycle via THF1 would contribute to the production of reactive oxygen species, the loss of chloroplast function and cell death.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'. © 2017 The Authors.

  7. SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex.

    Science.gov (United States)

    Chen, Xiaojuan; Yang, Xingxing; Zheng, Yang; Yang, Yudong; Xing, Yaling; Chen, Zhongbin

    2014-05-01

    SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

  8. Proteases as Insecticidal Agents

    OpenAIRE

    Robert L. Harrison; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic...

  9. Mouse Ficolin B Has an Ability to Form Complexes with Mannose-Binding Lectin-Associated Serine Proteases and Activate Complement through the Lectin Pathway

    Directory of Open Access Journals (Sweden)

    Yuichi Endo

    2012-01-01

    Full Text Available Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP- recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs, most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs, leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.

  10. The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation.

    Science.gov (United States)

    Bao, Rui; Zhou, Cong-Zhao; Jiang, Chunhui; Lin, Sheng-Xiang; Chi, Cheng-Wu; Chen, Yuxing

    2009-09-25

    The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 A resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 A apart. The overall fold of API-A belongs to the beta-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu(87) and Lys(145), and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met(83) to P5' Ala(92), adopts a novel conformation with the Leu(87) completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys(141) to P5' Glu(150), binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.

  11. 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.

  12. 复合蛋白酶在卤鹅类潮式卤水生产中的应用%Application of Protease Complex in the Production of Chaozhou Brine Goose

    Institute of Scientific and Technical Information of China (English)

    陈宇; 郭卓钊; 郭美媛; 郭奕纯; 杨曼; 黄妙云

    2012-01-01

    针对蛋白酶在潮式卤鹅传统熟肉食品生产中的应用,以剪切力的变化来研究木瓜蛋白酶、胰蛋白酶和复合蛋白酶对卤鹅肉质的影响.通过单因素试验和正交试验确定了最佳的工艺生产条件为:复合酶液的胰蛋白酶用量为2000 U/kg原料、木瓜蛋白酶用量为1000 U/kg原料,温度为20℃,处理时间为30 min,所得到的卤鹅颜色酱红有光泽、肉质细嫩有弹性,滋味浓郁.%This paper described the application of protease complex in the traditional Chaozhou brine goose, by analysis of the change of shearing force after dealing by papain, trypsin and mixed proteases. The optional conditions were obtained by orthogonal experiments as follows: trypsin in mixed proteases 2000 U/kg, papain in mixed proteases 1000 U/kg, temperature 20 ℃and time 30 min. The brine goose had a glossy garnet red color, and the meat showed a delicious and elastic taste with rich flavor.

  13. Mixed ligand μ-phenoxo-bridged dinuclear copper(II) complexes with diimine co-ligands: efficient chemical nuclease and protease activities and cytotoxicity.

    Science.gov (United States)

    Loganathan, Rangasamy; Ramakrishnan, Sethu; Suresh, Eringathodi; Palaniandavar, Mallayan; Riyasdeen, Anvarbatcha; Akbarsha, Mohamad Abdulkadhar

    2014-04-28

    a DNA cleavage activity more prominent than 1 and 2. The ability of the complexes to bind and cleave the protein BSA varies in the order, 4 > 3 > 5 > 2 > 1. Interestingly, 3 and 4 cleave the protein in the presence of H2O2 as an activator in a non-specific manner suggesting that they can act as chemical proteases. It is remarkable that all the complexes exhibit cytotoxicity against human breast cancer cell lines (MCF-7) with a potency more than the widely used drug cisplatin indicating that they have the potential to act as effective anticancer drugs in a time dependent manner. The morphological assessment data obtained by using Hoechst 33258 staining reveal that 3 and 4 induce apoptosis much more effectively than the other complexes. Also, the alkaline single-cell gel electrophoresis study (comet assay) suggests that the same complexes induce DNA fragmentation more efficiently than others.

  14. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis

    Science.gov (United States)

    Rey, Félix A.

    2017-01-01

    The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. PMID:28151973

  15. Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

    Science.gov (United States)

    Leong, L. E.-C.; Walker, P. A.; Porter, A. G.

    1992-08-01

    The protease (3C pro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3C pro has been achieved by fusing the protein to the car☐y-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3C pro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3C pro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3C pro. Sufficient protease 3C pro has been purified for initial attempts at crystallization.

  16. Theoretical study of the structure, bonding and electronic behaviour of sandwich complexes [M3(C7H7)2X3]- (M = Ni, Pd, Pt; X = F, Cl)

    Science.gov (United States)

    Zhou, Ke; Min, Suotian; Xue, Ganglin; Huang, Wendeng

    2014-08-01

    The correlations between the structural and electronic properties of the clusters [M3X3]3- and sandwich complexes [M3(C7H7)2X3]- (where M = Ni, Pd, Pt; X = F, Cl) were studied with density functional theory (B3PW91). All of the sandwich complexes are donating and back-donating metal-ligand bonding structures. The influence of the ligand, significant variations in the Msbnd C, Msbnd X, Msbnd M, Csbnd C bond lengths and binding energies were examined to obtain qualitative and quantitative pictures of the intramolecular C7R7+-M3X33- interactions. Our theoretical investigations show that the binding energies of the sandwich complexes gradually reduce from Ni to Pt, as well as from F to Cl. Meanwhile, the geometric and electronic structures and the relative stabilities have a strong relation to each other. S1). 7-B-(H-10) is a delocalised σ orbital, which has contributions from Pt (15% s orbital of each Pt, 8% d orbital of each Pt), and there is little contribution from Cl (Table S2). 7-C-(H-11) is a delocalised π orbital, which mainly involves contribution from Pt (25% dxz and dyz of each Pt) and Cl (8.5% pz of each Cl) (Table S3). 7-D-(H-14) is a delocalised σ orbital, which is mostly composed of d orbitals (14% for each Pt) and p orbitals (16% for each Cl) (Table S4). Pt s (10%) and d (10%) orbitals mainly participate in the formations of 7-E-(H-23), and there is a 13% p orbital contribution from each Cl (Table S5). For structure 13, the LUMO is mainly s orbitals of C, and there is some contribution from the s and d orbitals of Pt (Table S6), whereas the HOMO is mainly s orbitals of C (Table S7). 13-c-(H-14) is mainly the dxz and dyz orbitals of Pt (Table S8).NICS can be used to predict and understand some of the properties of a molecule, especially its stability due to aromatic stabilisation, which is based on the negative of the magnetic shielding computed at or above the geometrical centres of rings or clusters. Systems with negative NICS values are aromatic

  17. Poliovirus 2Apro induces the nucleic translocation of poliovirus 3CD and 3C' proteins

    Institute of Scientific and Technical Information of China (English)

    Wenwu Tian; Zongqiang Cui; Zhiping Zhang; Hongping Wei; XianEn Zhang

    2011-01-01

    Poliovirus genomic RNA replication, protein translation, and virion assembly are performed in the cytoplasm of host cells. However, this does not mean that there is no relationship between poliovirus infection and the cellular nucleus. In this study, recombinant fluorescence-tagged poliovirus 3CD and 3C' proteins were shown to be expressed mainly in the cytoplasm of Vero cells in the absence of other viral proteins. However, upon poliovirus infection, many of these proteins redistributed to the nucleus, as well as to the cytoplasm. A series of transfec-tion experiments revealed that the poliovirus 2Apro was responsible for the same redistribution of 3CD and 3C' proteins to the nucleus. Furthermore, a mutant 2Apr0 protein lacking protease activity abrogated this effect The poliovirus 2Apro protein was also found to co-localize with the IN up 153 protein, a component of the nuclear pore complexes on the nuclear envelope. These data provide further evidence that there are intrinsic interactions between poliovirus proteins and the cell nucleus, despite that many processes in the poliovirus replication cycle occur in the cytoplasm.

  18. Calpain 8/nCL-2 and calpain 9/nCL-4 constitute an active protease complex, G-calpain, involved in gastric mucosal defense.

    Directory of Open Access Journals (Sweden)

    Shoji Hata

    2010-07-01

    Full Text Available Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8(-/- and Capn9(-/- mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8(-/- stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9(-/-. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed "G-calpain," in which both were essential for activity. This is the first example of a "hybrid" calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S "knock-in" (Capn8(CS/CS mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8(-/- stomach, that of the Capn8(CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric

  19. Degradation of phycobilisomes in Synechocystis sp. PCC6803: evidence for essential formation of an NblA1/NblA2 heterodimer and its codegradation by A Clp protease complex.

    Science.gov (United States)

    Baier, Antje; Winkler, Wiebke; Korte, Thomas; Lockau, Wolfgang; Karradt, Anne

    2014-04-25

    When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3-5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ~7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used "model organism" Synechocystis sp. PCC6803 expresses two such genes, nblA16803 and nblA26803, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Förster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N2-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.

  20. 口蹄疫病毒株OA/58 3C蛋白酶的结构模拟和功能分析%Modeling and Analysis of 3C Protease Structure and Function from A Foot-and-month Disease Virus Strain OA/58

    Institute of Scientific and Technical Information of China (English)

    周建华; 丛国正; 高闪电; 常惠芸

    2007-01-01

    以口蹄疫病毒株OA/58 RNA为模板,反转录并扩增目的cDNA,然后与pGEM-T Easy载体连接并转化JM109菌株,提取的重组质粒用凝胶电泳、PCR和EcoRⅠ酶切法鉴定.该毒株与Poliovirus,Hepatitis A virus 和 Human rhinovirus 89毒株 3C 序列对比分析发现核苷酸序列一致性分别为38.8%,37.1%和36.5%,氨基酸序列一致性分别为23.7%,19.2%和17.6% .通过Swiss-pdbViewer软件模拟出FMDV OA/58 3C蛋白酶的3D结构和表面模型.并鉴定出此毒株3C蛋白酶的活性中心为Cys31-His46-Asp84.

  1. Analysis of structural water and CH···π interactions in HIV-1 protease and PTP1B complexes using a hydrogen bond prediction tool, HBPredicT.

    Science.gov (United States)

    Yesudas, Joshy P; Sayyed, Fareed Bhasha; Suresh, Cherumuttathu H

    2011-02-01

    A hydrogen bond prediction tool HBPredicT is developed for detecting structural water molecules and CH···π interactions in PDB files of protein-ligand complexes. The program adds the missing hydrogen atoms to the protein, ligands, and oxygen atoms of water molecules and subsequently all the hydrogen bonds in the complex are located using specific geometrical criteria. Hydrogen bonds are classified into various types based on (i) donor and acceptor atoms, and interactions such as (ii) protein-protein, (iii) protein-ligand, (iv) protein-water, (v) ligand-water, (vi) water-water, and (vii) protein-water-ligand. Using the information in category (vii), the water molecules which form hydrogen bonds with the ligand and the protein simultaneously-the structural water-is identified and retrieved along with the associated ligand and protein residues. For CH···π interactions, the relevant portions of the corresponding structures are also extracted in the output. The application potential of this program is tested using 19 HIV-1 protease and 11 PTP1B inhibitor complexes. All the systems showed presence of structural water molecules and in several cases, the CH···π interaction between ligand and protein are detected. A rare occurrence of CH···π interactions emanating from both faces of a phenyl ring of the inhibitor is identified in HIV-1 protease 1D4L.

  2. Novel fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus serine protease of the subtilisin-type, an Aspergillus serine protease of the subtilisin-type per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a

  3. Structural determinants of tobacco vein mottling virus protease substrate specificity.

    Science.gov (United States)

    Sun, Ping; Austin, Brian P; Tözsér, József; Waugh, David S

    2010-11-01

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1' position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k(cat) and K(m) for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

  4. Comparative study of the catalytic activity of the complexes Cp{sup *}RuCl(PAr{sub 3}){sub 2} [Ar = -C{sub 6H}5 and 4-CF{sub 3}-C{sub 6}H{sub 4}] in the ATRP of styrene

    Energy Technology Data Exchange (ETDEWEB)

    Villa-Hernandez, Alejandro M.; Rosales-Velazquez, Claudia P.; Torres-Lubian, Jose R., E-mail: rtorres@ciqa.mx [Departamento de Sintesis de Polimeros, Centro de Investigacion en Quimica Aplicada, Coah. (Mexico); Saldivar-Guerra, Enrique [Departamento de Procesos de Polimerizacion, Centro de Investigacion en Quimica Aplicada, Coah. (Mexico)

    2011-09-15

    Styrene polymerization by ATRP was conducted independently using the complexes Cp{sup *}RuCl(PPh{sub 3}){sub 2}, and Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2} as catalysts, in order to evaluate the influence of the electronic properties of the phosphine ligands on the rate and control of the polymerization. The kinetic data for polymerizations carried out with Cp{sup *}RuCl(PPh{sub 3}){sub 2}, show that molecular weights increase linearly with conversion with an average initiation efficiency of 0.77. The molecular weights obtained in the kinetic study with Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2} also increase with conversion but show a marked deviation below the theoretical molecular weights. This behavior was explained by the gradual, irreversible, oxidation of catalyst Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2} as confirmed by {sup 31}P-NMR spectroscopy. Catalyst Cp{sup *}RuCl(PPh{sub 3}){sub 2} promotes the polymerization with a rate of polymerization higher than that obtained using Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2}; this is consistent with the better electron donating properties of PPh{sub 3} versus P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}. Preliminary studies of styrene polymerization by ATRP in supercritical CO{sub 2}, shows that only catalyst Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2}, with fluorinated ligands, was active. (author)

  5. Structural Analysis of a Viral Ovarian Tumor Domain Protease from the Crimean-Congo Hemorrhagic Fever Virus in Complex with Covalently Bonded Ubiquitin

    Energy Technology Data Exchange (ETDEWEB)

    Capodagli, Glenn C.; McKercher, Marissa A.; Baker, Erica A.; Masters, Emily M.; Brunzelle, Joseph S.; Pegan, Scott D. (Denver); (NWU)

    2014-10-02

    Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne, negative-sense, single-stranded RNA [ssRNA(-)] nairovirus that produces fever, prostration, and severe hemorrhages in humans. With fatality rates for CCHF ranging up to 70% based on several factors, CCHF is considered a dangerous emerging disease. Originally identified in the former Soviet Union and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recent reports have identified a viral homologue of the ovarian tumor protease superfamily (vOTU) within its L protein. This protease has subsequently been implicated in downregulation of the type I interferon immune response through cleavage of posttranslational modifying proteins ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of vOTU have been suggested to perform similar roles in the positive-sense, single-stranded RNA [ssRNA(+)] arteriviruses. By utilizing X-ray crystallographic techniques, the structure of vOTU covalently bound to ubiquitin propylamine, a suicide substrate of the enzyme, was elucidated to 1.7 {angstrom}, revealing unique structural elements that define this new subclass of the OTU superfamily. In addition, kinetic studies were carried out with aminomethylcoumarin (AMC) conjugates of monomeric Ub, ISG15, and NEDD8 (neural precursor cell expressed, developmentally downregulated 8) substrates in order to provide quantitative insights into vOTU's preference for Ub and Ub-like substrates.

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

    Science.gov (United States)

    2012-10-14

    crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor. Biochemistry 42, 6709-6718...deacylation in cleavage of physiological sequences by the processing protease Kex2. Biochemistry 40, 3657-3665. (26) Rockwell, N. C., and Fuller, R. S

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

    Science.gov (United States)

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

    2007-07-01

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

  8. 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.

  9. Contact factor proteases and the complexes formed with alpha 2-macroglobulin can interfere in protein C assays by cleaving amidolytic substrates.

    Science.gov (United States)

    Mackie, I J; Gallimore, M; Machin, S J

    1992-10-01

    Plasma from women taking combined oral contraceptives and cold-activated plasma contain proteases which cleave chromogenic substrates in protein C assays in the absence of protein C activators such as Protac. This spontaneous activity makes a background substraction necessary and makes protein C (PC) assays less accurate. We investigated two commonly used substrates PKK)-deficient plasmas led to cleavage of chromogenic substrate for protein C. The protein C substrates were cleaved by purified kallikrein and alpha- and beta-FXIIa. Immunoabsorption with alpha 2-macroglobulin (alpha 2M) antibodies removed 60% of the alpha 2M and 70% of the activity on PC Substrate. Gel filtration of normal plasma on Sephadex G-150 gave a single peak of protein C activity and antigen in the included volume. After cold activation of the fractions, a second protein C-like peak appeared in the void volume, but with no detectable protein C antigen. This peak coincided with alpha 2M (chromogenic and ELISA) and plasma kallikrein (S-2302), but FXII (measured with a substrate insensitive to kallikrein) eluted separately.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Well-defined mono(η3-allyl)nickel complex MONi(η3-C3H5) (M = Si or Al) grafted onto silica or alumina: A molecularly dispersed nickel precursor for syntheses of supported small size nickel nanoparticles

    KAUST Repository

    Li, Lidong

    2014-01-01

    Preparing evenly-dispersed small size nickel nanoparticles over inert oxides remains a challenge today. In this context, a versatile method to prepare supported small size nickel nanoparticles (ca. 1-3 nm) with narrow size distribution via a surface organometallic chemistry (SOMC) route is described. The grafted mono(η3-allyl)nickel complexes MONi(η 3-C3H5) (M = Si or Al) as precursors are synthesized and fully characterized by elemental analysis, FTIR spectroscopy and paramagnetic solid-state NMR. © 2014 the Partner Organisations.

  11. FMDV-induced stress granules are disrupted by the viral L-protease

    DEFF Research Database (Denmark)

    Polacek, Charlotta; Belsham, Graham; McInerney, Gerald

    2014-01-01

    as a general cellular defense mechanism. For picornaviruses, poliovirus have been shown to disrupt SGs by the 3C-protease dependent cleavage of G3BP (3) and for cardioviruses (Theiler’s murine encephomyelitis virus and mengovirus), SG formation is inhibited by the presence of the viral L-protein (1, 2). We......Eukaryotic cells respond to environmental stress by entering a state of reduced protein synthesis, redirecting resources to damage control and defense. This reduced translation is closely linked to the formation of cytoplasmic stress granules (SGs). SGs are multicomponent foci, which contain...... stalled translation preinitiation complexes, including polyadenylated mRNAs, and several aggregation-prone RNA binding factors, such as the Ras-GAP SH3 domain-binding protein (G3BP) that enable their formation. Once the stress is lifted, the stalled complexes from the SGs are believed to re...

  12. Triangular oxalate clusters [W(3)(mu(3)-S)(mu(2)-S(2))(3)(C(2)O(4))(3)](2)(-) as building blocks for coordination polymers and nanosized complexes.

    Science.gov (United States)

    Sokolov, Maxim N; Gushchin, Artem L; Kovalenko, Konstantin A; Peresypkina, Eugenia V; Virovets, Alexander V; Sanchiz, Joaquin; Fedin, Vladimir P

    2007-03-19

    The reaction of aqueous [W3S7(C2O4)3](2-) with Ln(3+) and Th(4+) in a 1:1 molar ratio leads to oxalate-bridged heteropolynuclear molecular complexes and coordination polymers. La(3+) and Ce(3+) give a layered structure with big (about 1.8 nm) honeycomb pores which are filled with water molecules and lanthanide ions, in {[Ln(H2O)6]3[W3S7(C2O4)3]4}Br x xH2O (Ia and Ib). The smaller Pr(3+), Nd(3+), Sm(3+), Eu(3+), and Gd(3+) ions give discrete nanomolecules [(W3S7(C2O4)3Ln(H2O)5)2(mu-C2O4)] (with a separation of about 3.2 nm between the most distant parts of the molecule), which are further united into zigzag chains by specific S2...Br- contacts to achieve the overall stoichiometry K[(W3S7(C2O4)3Ln(H2O)5)2(mu-C2O4)]Br.xH2O (IIa-IId). Th(4+) gives K2[(W3S7(C2O4)3)4Th2(OH)2(H2O)10] x 14.33H2O (III) with a nanosized discrete anion (with a separation of about 2.7 nm between the most distant parts of the molecule), in which two thorium atoms are bound via two hydroxide groups into the Th2(OH)2(6+) unit, and each Th is further coordinated by five water molecules and two monodentate [W3S7(C2O4)](2-) cluster ligands. All compounds were characterized by X-ray structure analysis and IR spectroscopy. Magnetic susceptibility measurements in the temperature range of 2-300 K show weak antiferromagnetic interactions between two lanthanides atoms for compounds IIa, IIb, and IId. The thermal decomposition of Ia, Ib, and IIb was studied by thermogravimetry.

  13. 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.

  14. Proteolysis in Plastids of Arabidopsis Thaliana: Functional Analysis of ClpS1,2,T and their Physical and Genetic Interactions with the ClpPR Protease Core Complex and Clp Chaperones

    Energy Technology Data Exchange (ETDEWEB)

    van Wijk, Klaas

    2009-01-12

    Chloroplasts are essential organelles required for plant growth and biomass production. They synthesize many essential secondary metabolites (e.g. hormones, isoprenoids, amino acids, etc.) and house the photosynthetic apparatus needed for conversion of light energy and CO2 into chemical energy [in the form of reduced carbohydrates, ATP and NADPH]. Thus chloroplasts are essential for life on earth and essential for production of bioenergy. Formation and maintenance of a functional chloroplast requires an extensive investment in the biogenesis and homeostasis apparatus. Protease and proteolysis play a critical role in these processes, with the Clp gene family being particularly central. Proteolysis of proteins and protein complexes in plastids is poorly understood, and is not only critical for biogenesis, adaptation and maintenance but is also important for plant development. Several years ago, the vanWijk lab identified a large and relatively abundant ClpP/R/S complex, along with ClpC1,C2 and ClpD chaperones and a putative Clp affinity modulator in plastids. So far, no substrate recognition mechanism has been determined for any Clp complex in plants. The purpose of this grant was to initiate functional analysis of three members of the Clp family.

  15. Structure-Function of Falcipains: Malarial Cysteine Proteases

    Directory of Open Access Journals (Sweden)

    Kailash C. Pandey

    2012-01-01

    Full Text Available Evidence indicates that cysteine proteases play essential role in malaria parasites; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria. Studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis. The best characterized Plasmodium cysteine proteases are falcipains, which are papain family enzymes. Falcipain-2 and falcipain-3 are major hemoglobinases of P. falciparum. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. Overall, the complexes of falcipain-2 and falcipain-3 with small and macromolecular inhibitors provide structural insight to facilitate the design or modification of effective drug treatment against malaria. Drug development targeting falcipains should be aided by a strong foundation of biochemical and structural studies.

  16. Synthesis of novel p-tert-butylcalix[4]arene Schiff bases and their complexes with C60, potential HIV-Protease inhibitors

    Science.gov (United States)

    Khadra, Khalid Abu; Mizyed, Shehadeh; Marji, Deeb; Haddad, Salim F.; Ashram, Muhammad; Foudeh, Ayat

    2015-02-01

    Some p-tert-butylcalix[4]arene Schiff base crown ethers were synthesized, characterized using 1H, 13C-NMR, DEPT 135 and Mass spectrometry. Their complexes with C60 were isolated and characterized. The inhibition effect of these complexes on HIVP was studied and found that complexes of 9 and 10 have comparable Ki values to Pepstatine which is known as HIVP inhibitor and used as a control. The synthesis of the ligands, complexes and the inhibition behavior are discussed in this article.

  17. Cysteine Proteases Hydrolysis of Protein/pigment Complex Film%巯基蛋白酶水解蛋白质/色素复合膜

    Institute of Scientific and Technical Information of China (English)

    张怡; 左起亮; 姚江武

    2013-01-01

    目的:采用茶黄素(theaflavin,TF)和去磷酸化牛β-酪蛋白(dephosphorylated bovine β-casein,Dβ-CN)在芯片表面形成蛋白质/色素复合膜的模型,评估3种巯基蛋白酶(cysteine proteases,CPs),木瓜蛋白酶、菠萝蛋白酶和无花果蛋白酶,对Dβ-C/TF复合膜的清除作用.方法:在消散石英晶体微天平(quartz crystal mi-crobalance with dissipation,QCM-D)上建立Dβ-C/TF复合膜,Boltzman方程评估CPs水解复合膜的质量,水解前后膜变化通过掠角傅里叶变换红外光谱(grazing angle fourier transform infrared spectroscopy spectra,GA-FTIR).结果:3种CPs水解复合膜的功效由大到小依次为无花果蛋白酶、木瓜蛋白酶、菠萝蛋白酶(P<0.05).红外光谱表征结果证实,CPs能够有效地清除结合在Dβ-CN表面的TF.结论:本研究表明,CPs具有在牙色渍清除中潜在的应用价值.在开发口腔保健产品中该类酶值得进一步研究.

  18. Proteases in Periodontal Disease

    Directory of Open Access Journals (Sweden)

    Ana Rita Sokolonski ANTON

    2006-09-01

    Full Text Available Introduction: The caries and the periodontal disease (PD are the most frequent alterations in the oral cavity. The PD presents two stages: gengivitis and periodontitis. The destruction of collagenous fibers which encases the tooth onto the alveolar bone is characteristic of the pariodontitis. The inclusion loss caused by this pathology is due to the presence of bacteria and their products, besides the tissue destruction. This process is caused by excessive discharge of cells of the organism defence which reach the damaged area, and among these cells are neutrophils. These cells free lysosomal granule, where enzymes known as proteases (elastase, colagenasis and catepsin G are present. When excessively delivered, they cause extensive tissue destruction. The organism innate defence respond to this process activating anti-proteases, such as alfa-1-antitripsin e alfa-2-macrogoblulin, and, as consequence, the inflammatory process is subdued. Objective: Revision of the literature on periodontitis and its markers. In periodontitis, the balance between protease and anti-protese seems to be altered and lead to the appearance of these ones. There is an increase of prevalence of PD in the world population. In recent times, it has been associated to systemic conditions that lead to tissue destruction. Perhaps, the cause is based on an exacerbated tissue reaction, more than on the bacterial aggression. Conclusion: The predisposition of the organism is an important factor for the disease development. At reading different studies, it was observed that the discharged protease during the neutrophils degranulation process has internal, not bacterial, origin.

  19. 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 p

  20. NATO-3C/Delta launch

    Science.gov (United States)

    1978-01-01

    NATO-3C, the third in a series of NATO defense-related communication satellites, is scheduled to be launched on a delta vehicle from the Eastern Test Range no earlier than November 15, 1978. NATO-3A and -3B were successfully launched by Delta vehicles in April 1976 and January 1977, respectively. The NATO-3C spacecraft will be capable of transmitting voice, data, facsimile, and telex messages among military ground stations. The launch vehicle for the NATO-3C mission will be the Delta 2914 configuration. The launch vehicle is to place the spacecraft in a synchronous transfer orbit. The spacecraft Apogee Kick motor is to be fired at fifth transfer orbit apogee to circularize its orbit at geosynchronous altitude of 35,900 km(22,260 miles) above the equator over the Atlantic Ocean somewhere between 45 and 50 degrees W longitude.

  1. Analyzing polarization swings in 3C 279

    CERN Document Server

    Kiehlmann, S; Jorstad, S G; Sokolovsky, K V; Schinzel, F K; Agudo, I; Arkharov, A A; Benitez, E; Berdyugin, A; Blinov, D A; Bochkarev, N G; Borman, G A; Burenkov, A N; Casadio, C; Doroshenko, V T; Efimova, N V; Fukazawa, Y; Gomez, J L; Hagen-Thorn, V A; Heidt, J; Hiriart, D; Itoh, R; Joshi, M; Kimeridze, G N; Konstantinova, T S; Kopatskaya, E N; Korobtsev, I V; Kovalev, Y Y; Krajci, T; Kurtanidze, O; Kurtanidze, S O; Larionov, V M; Larionova, E G; Larionova, L V; Lindfors, E; Lopez, J M; Marscher, A P; McHardy, I M; Molina, S N; Morozova, D A; Nazarov, S V; Nikolashvili, M G; Nilsson, K; Pulatova, N G; Reinthal, R; Sadun, A; Sergeev, S G; Sigua, L A; Sorcia, M; Spiridonova, O I; Takalo, L O; Taylor, B; Troitsky, I S; Ugolkova, L S; Zensus, J A; Zhdanova, V E

    2013-01-01

    Quasar 3C 279 is known to exhibit episodes of optical polarization angle rotation. We present new, well-sampled optical polarization data for 3C 279 and introduce a method to distinguish between random and deterministic electric vector position angle (EVPA) variations. We observe EVPA rotations in both directions with different amplitudes and find that the EVPA variation shows characteristics of both random and deterministic cases. Our analysis indicates that the EVPA variation is likely dominated by a random process in the low brightness state of the jet and by a deterministic process in the flaring state.

  2. Analyzing polarization swings in 3C 279

    Directory of Open Access Journals (Sweden)

    Kiehlmann S.

    2013-12-01

    Full Text Available Quasar 3C 279 is known to exhibit episodes of optical polarization angle rotation. We present new, well-sampled optical polarization data for 3C 279 and introduce a method to distinguish between random and deterministic electric vector position angle (EVPA variations. We observe EVPA rotations in both directions with different amplitudes and find that the EVPA variation shows characteristics of both random and deterministic cases. Our analysis indicates that the EVPA variation is likely dominated by a random process in the low brightness state of the jet and by a deterministic process in the flaring state.

  3. Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii.

    Science.gov (United States)

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

    2006-01-01

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

  4. L3+C air shower array

    CERN Multimedia

    Laurent Guiraud

    2000-01-01

    Photo 01: a view of the L3+C air shower array; 50 scintillators on the roof of the SX-hall above L3. Photo 02: view of one of the detectors of the array.Photo 04: detectors seen against the background of the LEP Point 2 facilities.

  5. PROTEASES AND PROTEASE INHIBITORS INTERACTION: DEFENCE STRATEGY AGAINST

    Directory of Open Access Journals (Sweden)

    R.S.DHANDE 1 N.J.CHIKHALE 2

    2014-12-01

    Full Text Available ABSTRACT: An increase in crop yield, its management and preservation are among the main challenges standing before the human population that exceed 10 billion by the mid of 21 st  century.  Every year, considerable agricultural losses occur due to repeated practices of cultivation of large genetically similar populations.  Such cultivation practices favors incidence of more insect pests (Hilder and Boulter, 1999;  Oerke  et  al.,  1994;  Smith,  1999.  To  solve  these  problems,  current approaches  rely  on  use  of  synthetic  chemicals  like  fertilizers,  insecticides, herbicides,  fungicides  etc.  But  this  exerts  excessively  high  pressure  on environment  and  destabilizes  the  ecological  balance.  The  traditional  pest control method involves the use of conventional pesticides, most of which are non-specific and wipe out the entire community, pollutes the agro-ecosystem, and  increases  the  cost  of  production.  The  emergence  of  gene  transfer technology  has  solved  some  problems  regarding  overuse  of  chemical pesticides.  The  delta  endotoxin  encoding  gene  from  Bacillus  thuringiensis,  a gram positive soil borne bacteria transferred in crops has given little relief from coleopterans and lepidopterans attack.  Whereas, the insects belonging to these orders like Helicoverpa Sps. have developed resistance against Bt toxins. The other approach takes advantage of use of plant genes encoding defense proteins like protease inhibitors which is more appealing, simpler and safer (Dunaevsky et.  al.,  2005.  Proteinase  inhibitors  (PIs  are  naturally  occurring  proteins  in living organisms and are able to inhibit & control the activity of proteases. PIs act  on  an  active  site  of  digestive  proteolytic  enzymes  and  form  a  stable complex  unlike  enzyme-substrate  or  enzyme-product  weak  complexes  which

  6. Sumo-dependent substrate targeting of the SUMO protease Ulp1

    Directory of Open Access Journals (Sweden)

    Westerbeck Jason W

    2011-10-01

    Full Text Available Abstract Background In the yeast Saccharomyces cerevisiae, the essential small ubiquitin-like modifier (SUMO protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood. Results Using a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3(C580S, that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3(C580S to purify Smt3-modified proteins from cell extracts. Conclusions Our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates in vivo and in vitro. Furthermore, we found that a substrate-trapping Ulp1(3(C580S interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.

  7. From proteases to proteomics.

    Science.gov (United States)

    Neurath, H

    2001-04-01

    This personal and professional autobiography covers the 50-yr period of 1950-2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments).

  8. Multifrequency observations of extended radio galaxies V - 3C 31, 3C 33.1, 3C 35, 3C 66B, 3C 129, 3C 130, 3C 223, 3C 310, 3C 390.3 and 4C 48.29

    Science.gov (United States)

    van Breugel, W.; Jagers, W.

    1982-08-01

    A sample of 3C radio sources of large angular size has been observed in total and polarized intensity at several wavelengths with the Westerbork Synthesis Radio Telescope. The sources were selected such that their largest angular size was greater than about 200 arcsec and their declination greater than about 25 degrees. Some additional sources with radio jets or peculiar morphology were also included. The name of each source, its structural type classification, wavelength of observation, and data references are given.

  9. Lack of FTSH4 Protease Affects Protein Carbonylation, Mitochondrial Morphology, and Phospholipid Content in Mitochondria of Arabidopsis: New Insights into a Complex Interplay.

    Science.gov (United States)

    Smakowska, Elwira; Skibior-Blaszczyk, Renata; Czarna, Malgorzata; Kolodziejczak, Marta; Kwasniak-Owczarek, Malgorzata; Parys, Katarzyna; Funk, Christiane; Janska, Hanna

    2016-08-01

    FTSH4 is one of the inner membrane-embedded ATP-dependent metalloproteases in mitochondria of Arabidopsis (Arabidopsis thaliana). In mutants impaired to express FTSH4, carbonylated proteins accumulated and leaf morphology was altered when grown under a short-day photoperiod, at 22°C, and a long-day photoperiod, at 30°C. To provide better insight into the function of FTSH4, we compared the mitochondrial proteomes and oxyproteomes of two ftsh4 mutants and wild-type plants grown under conditions inducing the phenotypic alterations. Numerous proteins from various submitochondrial compartments were observed to be carbonylated in the ftsh4 mutants, indicating a widespread oxidative stress. One of the reasons for the accumulation of carbonylated proteins in ftsh4 was the limited ATP-dependent proteolytic capacity of ftsh4 mitochondria, arising from insufficient ATP amount, probably as a result of an impaired oxidative phosphorylation (OXPHOS), especially complex V. In ftsh4, we further observed giant, spherical mitochondria coexisting among normal ones. Both effects, the increased number of abnormal mitochondria and the decreased stability/activity of the OXPHOS complexes, were probably caused by the lower amount of the mitochondrial membrane phospholipid cardiolipin. We postulate that the reduced cardiolipin content in ftsh4 mitochondria leads to perturbations within the OXPHOS complexes, generating more reactive oxygen species and less ATP, and to the deregulation of mitochondrial dynamics, causing in consequence the accumulation of oxidative damage.

  10. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  11. Cathepsin proteases in Toxoplasma gondii

    OpenAIRE

    Dou, Zhicheng; Carruthers, Vern B.

    2011-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and n...

  12. Protease-mediated drug delivery

    Science.gov (United States)

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

    2003-12-01

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

  13. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

    -specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers......, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection...

  14. ALMA Polarization Science Verification: 3C 286

    Science.gov (United States)

    Nagai, H.; Nakanishi, K.; Paladino, R.; Moellenbrock, G.; Fomalont, E.; Amigano, A.; Vlahakis, C.; Remijan, A.; ALMA Polarization Team

    2015-12-01

    The ALMA polarization science verification results on 3C 286 are presented. The measured polarization percentage and polarization position angle of the continuum emission at 1.3 mm are about 16% and 39 degrees, respectively. They are quite similar to those at longer wavelength, but seem to increase slightly. Similar trends were also found in the previous measurement using the IRAM 30-m telescope (Agudo et al. 2012). The final image rms on the polarization image is better than 0.1% of the total intensity, demonstrating the very high polarization sensitivity of ALMA.

  15. Application of protease-hydrolyzed whey as a complex nitrogen source to increase poly(3-hydroxybutyrate) production from oils by Cupriavidus necator.

    Science.gov (United States)

    Obruca, Stanislav; Benesova, Pavla; Oborna, Jana; Marova, Ivana

    2014-04-01

    Whole whey hydrolyzed by Alcalase (WWH) was tested as a complex nitrogen source for the production of poly(3-hydroxybutyrate) (PHB) from waste frying oils by Cupriavidus necator H16. Addition of WWH (10 % (v/v) of cultivation media) supported the growth and PHB accumulation; PHB yields in Erlenmeyer flasks were more than 3.5-fold higher than in control cultivations. The positive influence of WWH on PHB production was confirmed in experiments performed in laboratory fermentor. C. necator cultivated with WWH produced 28.1 g PHB l(-1) resulting in a very high product yield coefficient of 0.94 g PHB per g oil. Since PHB yields were ~40 % higher than in the control cultivation, WWH can be considered as an excellent inexpensive nitrogen source for PHB production by C. necator.

  16. The multiwavelength variability of 3C 273

    CERN Document Server

    Soldi, S; Paltani, S; Aller, H D; Aller, M F; Bürki, G; Chernyakova, M; Lähteenmäki, A; McHardy, I M; Robson, E I; Staubert, R; Tornikoski, M; Walter, R; Courvoisier, T J -L

    2008-01-01

    We present an update of 3C 273's database hosted by the ISDC, completed with data from radio to gamma-ray observations over the last 10 years. We use this large data set to study the multiwavelength properties of this quasar,especially focussing on its variability behaviour. We study the amplitude of the variations and the maximum variability time scales across the broad-band spectrum and correlate the light curves in different bands, specifically with the X-rays, to search for possible connections between the emission at different energies. 3C 273 shows variability at all frequencies, with amplitudes and time scales strongly depending on the energy and being the signatures of the different emission mechanisms. The variability properties of the X-ray band imply the presence of either two separate components (possibly a Seyfert-like and a blazar-like) or at least two parameters with distinct timing properties to account for the X-ray emission below and above ~20 keV. The dominant hard X-ray emission is most pr...

  17. Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites.

    Science.gov (United States)

    Polonais, Valerie; Shea, Michael; Soldati-Favre, Dominique

    2011-08-01

    Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.

  18. Metal-based antimicrobial protease inhibitors.

    Science.gov (United States)

    Kellett, A; Prisecaru, A; Slator, C; Molphy, Z; McCann, M

    2013-01-01

    Limitations associated with the production cost, metabolic instability, side-effects, resistance and poor pharmacokinetics of organic protease inhibitors (PIs), which form an essential component of the front line HAART treatment for HIV, have fuelled efforts into finding novel, transition metal-based alternatives. Some of the attractive features of metalbased therapeutics include synthetic simplicity, solubility control, redox capability, expansion of coordination number and topography matching of the complex to the protein's active site. Building asymmetry into the complex, which may offer better discrimination between host and rogue cell, can readily be achieved through coordination of chiral ligands to the metal centre. Although the scope of this review has been limited to metal-based agents that have been reported to bind/inhibit HIV-1 and parasitic proteases, some desirables, such as high activity, low dosage, minimal toxicity, crossinhibition, unique binding modes and selectivity, have already been delivered. The variability of the d-block metals, coupled with the availability of designer organic ligands, augers well for the future development of clinical metallo-drugs for deployment against protease-associated, fatal diseases.

  19. 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.

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

    Science.gov (United States)

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

    2006-06-01

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

  1. 汞和金属离子及多硫化物对木瓜蛋白酶活性的抑制作用%Inhibition of cysteine protease papain by metal ions and polysulfide complexes, especially mercuric ion

    Institute of Scientific and Technical Information of China (English)

    姜军; 杨晓达; 王夔

    2007-01-01

    Abstract: Aim Cysteine proteases are closely associated with many human and non-human pathological processes and are potential targets for metal ions especially Hg2+ and the related species. In the present work, on the basis of to the general study on the effects of some metal ions on the activity of papain, a well-known representative of cysteine protease family, the inhibitory effects of Hg2+ and polysulfide complexes were studied. Results All the metal ions tested (Hg2+, Cu2+, Ag+, Au3+, Zn2+, Cd2+, Fe3+, Mn2+, Pb2+, Yb3+) inhibit the activity of papain anda good correlation between the inhibitory potency and softness-and-hardness was observed. Among the metals, Hg2+ was shown to be a potent inhibitor of papain with a Ki of 2 × 10-7 mol· L-1 among. Excessive amounts of glutathione and cysteine could reactivate the enzyme activity of papain deactivated by Hg2+. These evidences supported that Hg2+ might bind to the catalytic site of papain. Interestingly,Hg (Ⅱ) polysulfide complexes were for the first time found to inhibit papain with a Ki of 7 × 10-6 mol ·L-1, whose potency is close to a well known mercury compound, thimerosal (Ki=2.7 × 10-6). In addition, Hg (Ⅱ) polysulfide complexes exhibit good permeability (1.9 × 10-5cm·s-1) to caco-2 monolayer. Conclusion These results suggested that mercury polysulfide complexes might be potential bioactive species in the interaction with cysteine proteases and other- SH-content proteins, providing a new clue to understand the mechanism of the toxicological and pharmacological actions of cinnabar and other insoluble mercury compounds.%目的 半胱氨酸蛋白酶参与了很多动植物生理过程和病理过程,是Hg2+及其化合物作用的潜在靶点.木瓜蛋白酶是半胱氨酸蛋白酶家族中最具代表性、研究最为广泛深入的一种蛋白酶,本文以木瓜蛋白酶为模型,研究了汞等金属离子及其化合物对半胱氨酸蛋白酶活性的抑制作用.结果 Hg2+对木瓜蛋白酶

  2. A 3d-4f complex constructed by the assembly of a cationic template, [Cu(en){sub 2}]{sup 2+}, and a 3D anionic coordination polymer, [Sm{sub 2}(C{sub 2}O{sub 4}){sub 3}(C{sub 5}O{sub 5})(H{sub 2}O){sub 2}]{sup 2-}

    Energy Technology Data Exchange (ETDEWEB)

    Ke, Szu-Yu; Yeh, Chang-Tsung; Wang, Chih-Chieh [Department of Chemistry, Soochow University, Taipei, Taiwan (China); Lee, Gene-Hsiang [Instrumentation Center, National Taiwan University, Taipei, Taiwan (China); Sheu, Hwo-Shuenn [National Synchrotron Radiation Research Center, Hsinchu, Taiwan (China)

    2017-05-18

    A three-dimensional (3D) 3d-4f complex, [Cu(en){sub 2}][Sm{sub 2}(C{sub 5}O{sub 5})(C{sub 2}O{sub 4}){sub 3}(H{sub 2}O){sub 2}].8H{sub 2}O (1) (en = ethylenediamine, C{sub 5}O{sub 5}{sup 2-} = dianion of 4,5-dihydroxycyclopent-4-ene-1,2,3-trione), were prepared via the in-situ ring-opening oxidation reaction of croconate in the presence of the template-directed complex, [Cu(en){sub 2}]{sup 2+} cation. The structural characterization determined by X-ray diffraction determination reveals that the 3D anionic coordination polymer of [Sm{sub 2}(C{sub 2}O{sub 4}){sub 3}(C{sub 5}O{sub 5})(H{sub 2}O){sub 2}]{sup 2-} in 1 can be describe in terms of in-plane 2D honeycomb-like [Sm{sub 2}(C{sub 2}O{sub 4}){sub 3}] layered frameworks bridged by oxalate with bis-chelating mode, being mutually interlinked via the bridge of μ{sub 1,2,3,4}-croconate ligands with bis-chelating coordination mode to complete the 3D open framework, which gives rise to 1D channels with pore size of 14.023 x 11.893 Aa (longest atom-atom contact distances) along the b axis. The structure-directing complex, [Cu(en){sub 2}]{sup 2+}, and solvated water molecules are resided into these honeycomb-type hexagonal channels. The thermal stability of 1 was further studied by TGA and in-situ powder X-ray diffraction measurement. (copyright 2017 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  3. Photometric reverberation mapping of 3C120

    CERN Document Server

    Nuñez, F Pozo; Westhues, C; Bruckmann, C; Haas, M; Chini, R; Steenbrugge, K; Murphy, M; 10.1051/0004-6361/201219107

    2013-01-01

    We present the results of a five month monitoring campaign of the local active galactic nuclei (AGN) 3C120. Observations with a median sampling of two days were conducted with the robotic 15cm telescope VYSOS-6 located near Cerro Armazones in Chile. Broad band (B,V) and narrow band (NB) filters were used in order to measure fluxes of the AGN and the H_beta broad line region (BLR) emission line. The NB flux is constituted by about 50% continuum and 50% H_beta emission line. To disentangle line and continuum flux, a synthetic H_beta light curve was created by subtracting a scaled V-band light curve from the NB light curve. Here we show that the H_beta emission line responds to continuum variations with a rest frame lag of 23.6 +/- 1.69 days. We estimate a virial mass of the central black hole M_BH = 57 +/- 27 * 10^6 solar masses, by combining the obtained lag with the velocity dispersion of a single contemporaneous spectrum. Using the flux variation gradient (FVG) method, we determined the host galaxy subtracte...

  4. Low-frequency study of two giant radio galaxies: 3C 35 and 3C 223

    Science.gov (United States)

    Orrù, E.; Murgia, M.; Feretti, L.; Govoni, F.; Giovannini, G.; Lane, W.; Kassim, N.; Paladino, R.

    2010-06-01

    Aims: Radio galaxies with a projected linear size ⪆1 Mpc are classified as giant radio sources. According to the current interpretation these are old sources which have evolved in a low-density ambient medium. Because radiative losses are negligible at low frequency, extending spectral aging studies in this frequency range will allow us to determine the zero-age electron spectrum injected and then to improve the estimate of the synchrotron age of the source. Methods: We present Very Large Array images at 74 MHz and 327 MHz of two giant radio sources: 3C 35 and 3C 223. We performed a spectral study using 74, 327, 608 and 1400 GHz images. The spectral shape is estimated in different positions along the source. Results: The radio spectrum follows a power-law in the hotspots, while in the inner region of the lobe the shape of the spectrum shows a curvature at high frequencies. This steepening agrees with synchrotron aging of the emitting relativistic electrons. In order to estimate the synchrotron age of the sources, the spectra were fitted with a synchrotron model of emission. They show that 3C 35 is an old source of 143 ± 20 Myr, while 3C 223 is a younger source of 72 ± 4 Myr.

  5. Direct visualization of protease action on collagen triple helical structure.

    Directory of Open Access Journals (Sweden)

    Gabriel Rosenblum

    Full Text Available Enzymatic processing of extracellular matrix (ECM macromolecules by matrix metalloproteases (MMPs is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen (3/4 fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.

  6. 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.

  7. 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

    2017-01-01

    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. PMID:28067849

  8. Cathepsin proteases in Toxoplasma gondii

    Science.gov (United States)

    Dou, Zhicheng; Carruthers, Vern B.

    2014-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and nutrient acquisition.. Here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development. PMID:21660658

  9. Hydrothermal synthesis and study of an inorganic-organic hybrid vanadate of a nickel(II) coordination complex with pyrazine, Ni{sub 3}(C{sub 4}H{sub 4}N{sub 2}){sub 3}(V{sub 8}O{sub 23})

    Energy Technology Data Exchange (ETDEWEB)

    Larrea, Edurne S. [Dpto. de Mineralogia y Petrologia, Facultad de Ciencia y Tecnologia, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, UPV/EHU, Apdo. 644, E-48080 Bilbao (Spain); Mesa, Jose L., E-mail: joseluis.mesa@ehu.es [Dpto. de Quimica Inorganica, Facultad de Ciencia y Tecnologia, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, UPV/EHU, Apdo. 644, E-48080 Bilbao (Spain); Arriortua, Maria I. [Dpto. de Mineralogia y Petrologia, Facultad de Ciencia y Tecnologia, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, UPV/EHU, Apdo. 644, E-48080 Bilbao (Spain)

    2011-06-15

    Highlights: {yields} A novel inorganic-organic hybrid vanadate of nickel(II) coordination complex with pyrazine has been synthesized hydrothermally. {yields} The thermal and spectroscopic behavior has been studied. {yields} The compound shows AFM interactions which has been fitted to a magnetic model of lineal chains. -- Abstract: The three-dimensional hybrid compound Ni{sub 3}(C{sub 4}H{sub 4}N{sub 2}){sub 3}(V{sub 8}O{sub 23}) has been synthesized by mild hydrothermal methods under autogenous pressure at 170 {sup o}C. The structure of the phase is stable until 380 {sup o}C. The removal of the pyrazine molecules from the structure induces its collapse. The IR spectrum shows the vibration modes of the pyrazine molecule and those of the [VO{sub 4}]{sup 3-} groups. A UV-visible spectrum shows the characteristic bands of the Ni(II) d{sup 8}-high-spin cation in a slightly distorted octahedral coordination. Magnetic measurements indicate the existence of antiferromagnetic interactions that can be fitted with a chain model to give g = 2.31, J/k = -5.3, and zJ'/k = -5.5.

  10. 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.

  11. Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction

    DEFF Research Database (Denmark)

    Stanyer, Lee; Jørgensen, Wenche; Hori, Osamu;

    2008-01-01

    more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex....... Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress....

  12. Photosynthesis of C3, C3-C4, and C4 grasses at glacial CO2.

    Science.gov (United States)

    Pinto, Harshini; Sharwood, Robert E; Tissue, David T; Ghannoum, Oula

    2014-07-01

    Most physiology comparisons of C3 and C4 plants are made under current or elevated concentrations of atmospheric CO2 which do not reflect the low CO2 environment under which C4 photosynthesis has evolved. Accordingly, photosynthetic nitrogen (PNUE) and water (PWUE) use efficiency, and the activity of the photosynthetic carboxylases [Rubisco and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] were compared in eight C4 grasses with NAD-ME, PCK, and NADP-ME subtypes, one C3 grass, and one C3-C4 grass grown under ambient (400 μl l(-1)) and glacial (180 μl l(-1)) CO2. Glacial CO2 caused a smaller reduction of photosynthesis and a greater increase of stomatal conductance in C4 relative to C3 and C3-C4 species. Panicum bisulcatum (C3) acclimated to glacial [CO2] by doubling Rubisco activity, while Rubisco was unchanged in Panicum milioides (C3-C4), possibly due to its high leaf N and Rubisco contents. Glacial CO2 up-regulated Rubisco and PEPC activities in concert for several C4 grasses, while NADP-ME and PEP-CK activities were unchanged, reflecting the high control exerted by the carboxylases relative to the decarboxylases on the efficiency of C4 metabolism. Despite having larger stomatal conductance at glacial CO2, C4 species maintained greater PWUE and PNUE relative to C3-C4 and C3 species due to higher photosynthetic rates. Relative to other C4 subtypes, NAD-ME and PEP-CK grasses had the highest PWUE and PNUE, respectively; relative to C3, the C3-C4 grass had higher PWUE and similar PNUE at glacial CO2. Biomass accumulation was reduced by glacial CO2 in the C3 grass relative to the C3-C4 grass, while biomass was less reduced in NAD-ME grasses compared with NADP-ME and PCK grasses. Under glacial CO2, high resource use efficiency offers a key evolutionary advantage for the transition from C3 to C4 photosynthesis in water- and nutrient-limited environments.

  13. The Resolved Outflow from 3C 48

    Science.gov (United States)

    Shih, Hsin-Yi; Stockton, Alan

    2014-10-01

    We investigate the properties of the high-velocity outflow driven by the young radio jet of 3C 48, a compact-steep-spectrum source. We use the Space Telescope Imaging Spectrograph on board the Hubble Space Telecope to obtain (1) low-resolution UV and optical spectra and (2) multi-slit medium-resolution spectra of the ionized outflow. With supporting data from ground-based spectrographs, we are able to accurately measure the ratios of diagnostic emission lines such as [O III] λ5007, [O III] λ3727, [N II] λ6548, Hα, Hβ, [Ne V] λ3425, and [Ne III] λ3869. We fit the observed emission-line ratios using a range of ionization models, powered by active galactic nucleus (AGN) radiation and shocks, produced by the MAPPINGS code. We have determined that AGN radiation is likely the dominant ionization source. The outflow's density is estimated to be in the range n = 103-104 cm-3, the mass is ~6 × 106 M ⊙, and the metallicity is likely equal to or higher than solar. Compared with the typical outflows associated with more evolved radio jets, this young outflow is denser, less massive, and more metal rich. Multi-slit observations allow us to construct a two-dimensional velocity map of the outflow that shows a wide range of velocities with distinct velocity components, suggesting a wide-angle clumpy outflow. Based in part on observations made with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS 5-26555. These observations are associated with program GO-11574. Some of the data presented herein were obtained at the W. M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California and the National Aeronautics and Space Administration. The Observatory was made possible by the generous financial support of the W. M. Keck Foundation. Some of the

  14. A class of luminescent cyclometalated alkynylgold(III) complexes: synthesis, characterization, and electrochemical, photophysical, and computational studies of [Au(C=N=C)(C triple bond C-R)] (C=N=C = kappa(3)C,N,C bis-cyclometalated 2,6-diphenylpyridyl).

    Science.gov (United States)

    Wong, Keith Man-Chung; Hung, Ling-Ling; Lam, Wai Han; Zhu, Nianyong; Yam, Vivian Wing-Wah

    2007-04-11

    A new class of luminescent cyclometalated alkynylgold(III) complexes, [Au(RC=N(R')=CR)(CCR' ')], i.e., [Au(C=N=C)(C triple bond CR'')] (HC=N=CH = 2,6-diphenylpyridine) R' ' = C6H5 1, C6H4-Cl-p 2, C6H4-NO2-p 3, C6H4-OCH3-p 4, C6H4-NH2-p 5, C6H4-C6H13-p 6, C6H13 7, [Au(tBuC=N=CtBu)(C triple bond CC6H5)] 8 (HtBuC=N=CtBuH = 2,6-bis(4-tert-butylphenyl)pyridine), and [Au(C=NTol=C)(CCC6H4-C6H13-p)] 9 (HC=NTol=CH = 2,6-diphenyl-4-p-tolylpyridine), have been synthesized and characterized. The X-ray crystal structures of most of the complexes have also been determined. Electrochemical studies show that, in general, the first oxidation wave is an alkynyl ligand-centered oxidation, while the first reduction couple is ascribed to a ligand-centered reduction of the cyclometalated ligand with the exception of 3 in which the first reduction couple is assigned as an alkynyl ligand-centered reduction. Their electronic absorption and luminescence behaviors have also been investigated. In dichloromethane solution at room temperature, the low-energy absorption bands are assigned as the pi-pi* intraligand (IL) transition of the cyclometalated RC=N(R')=CR ligand with some mixing of a [pi(C triple bond CR'') --> pi*(RC=N(R')=CR)] ligand-to-ligand charge transfer (LLCT) character. The low-energy emission bands of all the complexes, with the exception of 5, are ascribed to origins mainly derived from the pi-pi* IL transition of the cyclometalated RC=N(R')=CR ligand. In the case of 5 that contains an electron-rich amino substituent on the alkynyl ligand, the low-energy emission band was found to show an obvious shift to the red. A change in the origin of emission is evident, and the emission of 5 is tentatively ascribed to a [pi(CCC6H4NH2) --> pi*(C=N=C)] LLCT excited-state origin. DFT and TDDFT computational studies have been performed to verify and elucidate the results of the electrochemical and photophysical studies.

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

    OpenAIRE

    Cupp-Enyard, Carrie

    2009-01-01

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

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

    Science.gov (United States)

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

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

  17. Multifrequency radio observations of 3C 28, 76. 1, 186 and 319

    Energy Technology Data Exchange (ETDEWEB)

    Macklin, J.T. (Mullard Radio Astronomy Observatory, Cambridge (UK). Cavendish Lab.)

    1983-04-01

    Multifrequency observations of 3C 28, 76.1, 186 and 319, made with the One-Mile and 5-km telescopes, enable the spectral-index distributions of each source and the polarization properties of 3C 76.1 to be studied. 3C 28 is shown to have the structure of a Fanaroff-Riley class I source, but it is 10 times more luminous than expected for this class. Although 3C 76.1 is also in Fanaroff-Riley class I, it contains a feature which has the properties of a 'hotspot'. The spectral-index distribution in 3C 76.1 is complex; the magnetic field is perpendicular to the axes of the jets, and the polarization properties are consistent with a tangled-field model. The morphology of 3C 186 remains unclear, and 3C 319 is a double source with only one 'hotspot', rather than being a tail source. It is suggested that the different properties of 3C 28 and 76.1 are due to their different environments; this explanation would be consistent with a general scheme which can account for all the observed dependences of source properties on cluster membership.

  18. Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang; Wang, Yong; Brunzelle, Joseph; Kovari, Iulia A.; Kovari, Ladislau C. (WSU-MED); (NWU)

    2012-03-27

    Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

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

    Science.gov (United States)

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

    2015-01-01

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

  20. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna S 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...... 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...... kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1...

  3. Analysis list: Nr3c1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Nr3c1 Adipocyte,Blood,Breast,Embryo,Embryonic fibroblast,Liver,Neural + mm9 http://...dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Nr3c1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Nr3c1....5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Nr3c1.10.tsv http://dbarchive.bioscie...ncedbc.jp/kyushu-u/mm9/colo/Nr3c1.Adipocyte.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Nr3c1....Blood.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Nr3c1.Breast.tsv,http

  4. Theoretical studies of the proton transfer behaviors in molecular complexes analogous to catalytic triad of serine protease:Toward understanding the existence and significance of the low-barrier hydrogen-bond in enzymatic catalysis

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A representative acetate-(5-methylimidazole)-methanol system has been employed as a model of catalytic triad in serine protease to validate the formation processes of lowbarrier H-bonds(LBHB) at the B3LYP/6-311++G level of theory,and variable H-bonding characters from conventional ones to LBHBs have been represented along with the proceedings of proton transfer.Solvent effect is an important factor in modulation of the existence of an LBHB,where an LBHB(or a conventional H-bond) in the gas phase can be changed into a non-LBHB(an LBHB) upon solvation.The origin of the additional stabili-zation energy arising from the LBHB may be attributed to the H-bonding energy difference before and after proton transfer because the shared proton can freely move between the proton donor and proton acceptor.Most importantly,the order of magnitude of the stabilization energy depends on the studied systems.Furthermore,the nonexistence of LBHBs in the catalytic triad of serine proteases has been verified in a more sophisticated model treated using the ONIOM method.As a result,only the single proton transfer mechanism in the catalytic triad has been confirmed and the origin of the powerful catalytic efficiency of serine proteases should be attributed to other factors rather than the LBHB.

  5. Inflammation, proteases and cancer.

    NARCIS (Netherlands)

    Kempen, L.C. van; Visser, K.E. de; Coussens, L.M.

    2006-01-01

    Tumours are complex tissues composed of ever-evolving neoplastic cells, matrix proteins that provide structural support and sequester biologically active molecules, and a cellular stromal component. Reciprocal interactions between neoplastic cells, activated host cells and the dynamic micro-environm

  6. New redshift determinations for three 3C radio sources.

    Science.gov (United States)

    Reynaldi, V.

    2017-01-01

    I report the new redshift determinations of three radio sources 3C 196.1, 3C 268.2 and 3C 303.1 by using GMOS/Gemini North long-slit optical spectroscopy. The details of the observations are summarized in the following table (the B600 grating was used for the three observations): Object | RA(J2000) | DEC(J2000) | Date of obs. | width-slit(arcsec) | PA(deg) | Exp.Time(sec) 3C 196.1 | 8:15:27.8 | -03:08:27 | Mar 2012 | 0.5 | 50 | 2560 3C 268.2| |12:00:59.1 | 31:33:28 | Feb 2011 | 0.5 | 165 | 2576 3C 303.1 | 14:43:14.5 | 77:07:28 | Feb 2012 | 1 | 145 | 2560 The three of the sources have extended regions of ionized gas that do not obey a spherical distribution.

  7. 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.

  8. Structural determinants of MALT1 protease activity.

    Science.gov (United States)

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

    2012-05-25

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

  9. Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells.

    Science.gov (United States)

    Okano, K; Aoki, Y; Sakurai, T; Kajitani, M; Kanai, S; Shimazu, T; Shimizu, H; Naruto, M

    1987-07-01

    Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.

  10. Structural basis for substrate specificity of alphavirus nsP2 proteases.

    Science.gov (United States)

    Russo, Andrew T; Malmstrom, Robert D; White, Mark A; Watowich, Stanley J

    2010-08-24

    The alphavirus nsP2 protease is essential for correct processing of the alphavirus nonstructural polyprotein (nsP1234) and replication of the viral genome. We have combined molecular dynamics simulations with our structural studies to reveal features of the nsP2 protease catalytic site and S1'-S4 subsites that regulate the specificity of the protease. The catalytic mechanism of the nsP2 protease appears similar to the papain-like cysteine proteases, with the conserved catalytic dyad forming a thiolate-imidazolium ion pair in the nsP2-activated state. Substrate binding likely stabilizes this ion pair. Analysis of bimolecular complexes of Venezuelan equine encephalitis virus (VEEV) nsP2 protease with each of the nsP1234 cleavage sites identified protease residues His(510), Ser(511), His(546) and Lys(706) as critical for cleavage site recognition. Homology modelling and molecular dynamics simulations of diverse alphaviruses and their cognate cleavage site sequences revealed general features of substrate recognition that operate across alphavirus strains as well as strain specific covariance between binding site and cleavage site residues. For instance, compensatory changes occurred in the P3 and S3 subsite residues to maintain energetically favourable complementary binding surfaces. These results help explain how alphavirus nsP2 proteases recognize different cleavage sites within the nonstructural polyprotein and discriminate between closely related cleavage targets.

  11. VLBA polarimetric observations of the CSS quasar 3C147

    CERN Document Server

    Rossetti, A; Dallacasa, D; Junor, W; Salter, C J; Saikia, D J; 10.1051/0004-6361/200811190

    2009-01-01

    Aims. We report new VLBA polarimetric observations of the compact steep-spectrum (CSS) quasar 3C147 (B0538+498) at 5 and 8.4GHz. Methods. By using multifrequency VLBA observations, we derived milliarcsecond-resolution images of the total intensity, polarisation, and rotation measure distributions, by combining our new observations with archival data. Results. The source shows a one-sided structure, with a compact region, and a component extending about 200 mas to the south-west. The compact region is resolved into two main components with polarised emission, a complex rotation measure distribution, and a magnetic field dominated by components perpendicular to the source axis. Conclusions. By considering all the available data, we examine the possible location of the core component, and discuss two possible interpretations of the observed structure of this source: core-jet and lobe-hot spot. Further observations to unambiguously determine the location of the core would help distinguish between the two possibil...

  12. Complement C3c as a Biomarker in Heart Failure

    Directory of Open Access Journals (Sweden)

    A. Frey

    2013-01-01

    Full Text Available Introduction. Experimental data indicates an important role of the innate immune system in cardiac remodeling and heart failure (HF. Complement is a central effector pathway of the innate immune system. Animals lacking parts of the complement system are protected from adverse remodeling. Based on these data, we hypothesized that peripheral complement levels could be a good marker for adverse remodeling and prognosis in patients with HF. Methods and Results. Since complement activation converges on the complement factor C3, we measured serum C3c, a stable C3-conversion product, in 197 patients with stable systolic HF. Subgroups with normal and elevated C3c levels were compared. C3c levels were elevated in 17% of the cohort. Patients with elevated C3c levels exhibited a trend to better survival, slightly higher LVEF, and lower NTpro-BNP values in comparison to patients with normal C3c values. No differences were found regarding NYHA functional class. Significantly more patients with elevated C3c had preexisting diabetes. The prevalence of CAD, arterial hypertension, and atrial fibrillation was not increased in patients with elevated C3c. Conclusion. Elevated C3c levels are associated with less adverse remodeling and improved survival in patients with stable systolic heart failure.

  13. Mex3c mutation reduces adiposity and increases energy expenditure.

    Science.gov (United States)

    Jiao, Yan; George, Sunil K; Zhao, Qingguo; Hulver, Matthew W; Hutson, Susan M; Bishop, Colin E; Lu, Baisong

    2012-11-01

    The function of MEX3C, the mammalian homolog of Caenorhabditis elegans RNA-binding protein muscle excess 3 (MEX-3), was unknown until our recent report that MEX3C is necessary for normal postnatal growth and enhances the expression of local bone Igf1 expression. Here we report the pivotal role of Mex3c in energy balance regulation. Mex3c mutation caused leanness in both heterozygous and homozygous transgenic mice, as well as a more beneficial blood glucose and lipid profile in homozygous transgenic mice, in both sexes. Although transgenic mice showed normal food intake and fecal lipid excretion, they had increased energy expenditure independent of physical activity. Mutant mice had normal body temperature, Ucp1 expression in brown adipose tissue, and muscle and liver fatty acid oxidation. Mex3c is expressed in neurons and is detectable in the arcuate nucleus, the ventromedial nucleus, and the dorsomedial nucleus of the hypothalamus. Mex3c was not detected in NPY or POMC neurons but was detected in leptin-responsive neurons in the ventromedial nucleus. Mex3c and Leptin double mutant mice were growth retarded and obese and had blood profiles similar to those of ob/ob mice but showed none of the steatosis observed in ob/ob mice. Our data show that Mex3c is involved in energy balance regulation.

  14. Chaotic Feature in the Light Curve of 3C 273

    Institute of Scientific and Technical Information of China (English)

    Lei Liu

    2006-01-01

    Some nonlinear dynamical techniques, including state-space reconstruction and correlation integral, are used to analyze the light curve of 3C 273. The result is compared with a chaotic model. The similarities between them suggest there is a low-dimension chaotic attractor in the light curve of 3C 273.

  15. Biotechnology of cold-active proteases.

    Science.gov (United States)

    Joshi, Swati; Satyanarayana, Tulasi

    2013-05-03

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

  16. 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.

  17. VLBI, MERLIN and HST observations of the giant radio galaxy 3C 236

    NARCIS (Netherlands)

    Schilizzi, RT; Tian, WW; Conway, JE; Nan, R; Miley, GK; Barthel, PD; Normandeau, M; Dallacasa, D; Gurvits, LI

    2001-01-01

    We present VLBI and MERLIN data at 1.66 and 4.99 GHz on the central component coincident with the nucleus of the giant radio galaxy, 3C 236. The nuclear radio structure is composed of two complexes of emission which are resolved on scales from 1 milli-arcsec (mas) to 1 arcsec. Oscillations with an a

  18. Functional characterization of the cleavage specificity of the sapovirus chymotrypsin-like protease.

    Science.gov (United States)

    Robel, Ivonne; Gebhardt, Julia; Mesters, Jeroen R; Gorbalenya, Alexander; Coutard, Bruno; Canard, Bruno; Hilgenfeld, Rolf; Rohayem, Jacques

    2008-08-01

    Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2' positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2' positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.

  19. Reaction mechanism of -acylhydroxamate with cysteine proteases

    Indian Academy of Sciences (India)

    R Shankar; P Kolandaivel

    2007-09-01

    The gas-phase reaction mechanism of -acylhydroxamate with cysteine proteases has been investigated using ab initio and density functional theory. On the irreversible process, after breakdown of tetrahedral intermediate (INT1), small 1-2 anionotropic has been formed and rearranged to give stable by-products sulfenamide (P1) and thiocarbamate (P2) with considerable energy loss. While, on the reversible part of this reaction mechanism, intermediate (INT2) breaks down on oxidation, to form a stable product (P3). Topological and AIM analyses have been performed for hydrogen bonded complex in this reaction profile. Intrinsic reaction coordinates [IRC, minimum-energy path (MEP)] calculation connects the transition state between R-INT1, INT1-P1 and INT1-P2. The products P1, P2 and P3 are energetically more stable than the reactant and hence the reaction enthalpy is found to be exothermic.

  20. Effective Nanoparticle-based Gene Delivery by a Protease Triggered Charge Switch

    DEFF Research Database (Denmark)

    Gjetting, Torben; Jølck, Rasmus Irming; Andresen, Thomas Lars

    2014-01-01

    investigation in vivo including a PEG layer and a net negative charge that should ensure long-circulating properties before being activated by proteases in diseased tissue. Protease activation leads to detachment of PEG and a charge switching where the LNPs become positive due to the presence of glutamates...... in the cleaved peptide moiety. The cationic lipid DOTAP is used mainly to complex DNA and proton titratable DODAP is used to increase endosomal escape and enhance transfection efficiency. The idea of using a mixture of permanently charged and titratable cationic lipids shielded by a protease sensitive negatively...

  1. 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.

  2. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  3. Nelfinavir: fourth protease inhibitor approved.

    Science.gov (United States)

    1997-01-01

    The Food and Drug Administration (FDA) has granted accelerated approval to nelfinavir in both adult and pediatric formulations. Agouron, the manufacturer, used innovative computerized drug design techniques to discover, design, and refine the nelfinavir molecule. Nelfinavir is marketed under the trade name Viracept, and costs $5,000 per year. Early clinical trials find it to be as powerful as the other protease inhibitors, but with a different resistance profile. The drug has relatively few drug indications; however, several compounds have been contraindicated.

  4. Targeting exosites on blood coagulation proteases

    Directory of Open Access Journals (Sweden)

    Monteiro Robson Q.

    2005-01-01

    Full Text Available The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed in this short revision. Bothrojaracin is a snake venom-derived protein that binds to thrombin exosites 1 and 2. Complex formation impairs several exosite-dependent activities of thrombin including fibrinogen cleavage and platelet activation. Bothrojaracin also interacts with proexosite 1 on prothrombin thus decreasing the zymogen activation by the prothrombinase complex (FXa/FVa. Ixolaris is a two Kunitz tick salivary gland inhibitor, that is homologous to tissue factor pathway inhibitor. Recently it was demonstrated that ixolaris binds to heparin-binding exosite of FXa, thus impairing the recognition of prothrombin by the enzyme. In addition, ixolaris interacts with FX possibly through the heparin-binding proexosite. Differently from FX, the ixolaris-FX complex is not recognized as substrate by the intrinsic tenase complex (FIXa/FVIIIa. We conclude that these inhibitors may serve as tools for the study of coagulation exosites as well as prototypes for new anticoagulant drugs.

  5. From galaxy-scale fueling to nuclear-scale feedback: the merger-state of radio galaxies 3C293, 3C305 & 4C12.50

    CERN Document Server

    Emonts, Bjorn; Villar-Martin, Montserrat; Hodgson, Jeff; Brogt, Erik; Tadhunter, Clive; Mahony, Elizabeth; Oosterloo, Tom

    2016-01-01

    Powerful radio galaxies are often associated with gas-rich galaxy mergers. These mergers may provide the fuel to trigger starburst and active galactic nuclear (AGN) activity. In this Research Note, we study the host galaxies of three seemingly young or re-started radio sources that drive fast outflows of cool neutral hydrogen (HI) gas, namely 3C 293, 3C 305 and 4C 12.50 (PKS 1345+12). Our aim is to link the feedback processes in the central kpc-scale region with new information on the distribution of stars and gas at scales of the galaxy. For this, we use deep optical V-band imaging of the host galaxies, complemented with HI emission-line observations to study their gaseous environments. We find prominent optical tidal features in all three radio galaxies, which confirm previous claims that 3C 293, 3C 305 and 4C 12.50 have been involved in a recent galaxy merger or interaction. Our data show the complex morphology of the host galaxies, and identify the companion galaxies that are likely involved in the merger...

  6. Privacy Issues of the W3C Geolocation API

    CERN Document Server

    Doty, Nick; Wilde, Erik

    2010-01-01

    The W3C's Geolocation API may rapidly standardize the transmission of location information on the Web, but, in dealing with such sensitive information, it also raises serious privacy concerns. We analyze the manner and extent to which the current W3C Geolocation API provides mechanisms to support privacy. We propose a privacy framework for the consideration of location information and use it to evaluate the W3C Geolocation API, both the specification and its use in the wild, and recommend some modifications to the API as a result of our analysis.

  7. Molecular Gas in the Powerful Radio Galaxies 3C~31 and 3C~264 Major or Minor Mergers?

    CERN Document Server

    Lim, J; Combes, F

    2000-01-01

    We report the detection of $^{12}$CO~($1 \\to 0$) and $^{12}$CO~($2 \\to 1$) emission from the central regions ($\\lesssim 5$--$10 {\\rm kpc}$) of the two powerful radio galaxies 3C~31 and 3C~264. Their individual CO emission exhibits a double-horned line profile that is characteristic of an inclined rotating disk with a central depression at the rising part of its rotation curve. The inferred disk or ring distributions of the molecular gas is consistent with the observed presence of dust disks or rings detected optically in the cores of both galaxies. For a CO to H$_2$ conversion factor similar to that of our Galaxy, the corresponding total mass in molecular hydrogen gas is $(1.3 \\pm 0.2) \\times 10^9 {\\rm M_{\\odot}}$ in 3C~31 and $(0.31 \\pm 0.06) \\times 10^9 {\\rm M_{\\odot}}$ in 3C~264. Despite their relatively large molecular-gas masses and other peculiarities, both 3C~31 and 3C~264, as well as many other powerful radio galaxies in the (revised) 3C catalog, are known to lie within the fundamental plane of normal...

  8. Optical Periodicity Analysis of 3C 446 using Period04

    Indian Academy of Sciences (India)

    Fei Guo; Hao Jing Zhang

    2014-09-01

    All the data of the blazar 3C446 at 8, 4.8, 14 and 22 GHz, presented in publications from 1977 to 2006, have been compiled to generate light curves. The light curves show violent activity of 3C446. Using Period04 analysis method, we have found that there is a period of 7.2 yr, which is consistent with the results that we found using wavelet analysis method. We get the instability region as = 123.83.

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  10. Orchestration of an uncommon maturation cascade of the house dust mite protease allergen quartet

    Directory of Open Access Journals (Sweden)

    Marie-Eve eDumez

    2014-03-01

    Full Text Available In more than 20% of the world population, sensitization to house dust mite (HDM allergens triggers typical allergic diseases such as allergic rhinitis and asthma. Amongst the 23 mite allergen groups hitherto identified, groups 1 are cysteine proteases belonging to the papain-like family whereas groups 3, 6 and 9 are serine proteases displaying trypsin, chymotrypsin and collagenolytic activities, respectively. While these proteases are more likely to be involved in the mite digestive system, they also play critical roles in the initiation and in the chronicity of the allergic response notably through the activation of innate immune pathways. All these allergenic proteases are expressed in mite as inactive precursor form. Until recently, the exact mechanisms of their maturation into active proteases remained to be fully elucidated. Recent breakthroughs in the understanding of the activation mechanisms of mite allergenic protease precursors have highlighted an uncommon and unique maturation pathway orchestrated by group 1 proteases that tightly regulates the proteolytic activities of groups 1, 3, 6 and 9 through complex intra- or intermolecular mechanisms. This review presents and discusses the currently available knowledge of the activation mechanisms of group 1, 3, 6 and 9 allergens of Dermatophagoides pteronyssinus laying special emphasis on their localization, regulation and interconnection.

  11. Generation of a C3c specific monoclonal antibody and assessment of C3c as a putative inflammatory marker derived from complement factor C3

    DEFF Research Database (Denmark)

    Palarasah, Yaseelan; Skjodt, Karsten; Brandt, Jette;

    2010-01-01

    complex (C5b-C9) and quantification of complement split products by precipitation-in-gel techniques (e.g. C3d). We have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C3c without interference from other products generated from the complement component C3. The C3c specific m......Ab was tested in different ELISA combinations with various types of in vitro activated sera and with plasma or serum samples from factor I deficient patients. The specificity of the mAb was evaluated in immunoprecipitation techniques and by analysis of eluted fragments of C3 after immunoaffinity chromatography...

  12. Carbonization and transition layer effects on 3C-SiC film residual stress

    Science.gov (United States)

    Anzalone, R.; Litrico, G.; Piluso, N.; Reitano, R.; Alberti, A.; Fiorenza, P.; Coffa, S.; La Via, F.

    2017-09-01

    In this work an extended study of the carbonization process of the silicon surface and of a low temperature transition layer in the temperature rump on the 3C-SiC epitaxial growth has been reported. It has been observed that increasing the C/H2 ratio the voids density decreases, the thickness of the carbonization layer and the density increase and the morphology improves. The low temperature transition layer, grown during the ramp between the carbonization step and the real growth process, produce a further reduction of the voids at the 3C-SiC/Si interface and a considerable reduction of the stress of the 3C-SiC film. This stress reduction is related to a large change of the film morphology. No effect of the interface silicon layer on the stress is observed. This study has shown the complex connection between the first steps of the 3C-SiC growth process and the properties of the film in term of stress and superficial morphology. The residual stress has important implications with regard to the processing (wafer bow) and quality of the epitaxy. Residual stress also changes the mechanical response and/or the resonant frequency of the thin-film structure and may degrade the performance in MEMS-based devices. Therefore, a better understanding of the stress relaxation mechanism could improve the performances of 3C-SiC devices and sensor technologies.

  13. 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...

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

    Science.gov (United States)

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

    2012-12-01

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

  15. Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

    Directory of Open Access Journals (Sweden)

    Sungho Ghil

    Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.

  16. Complexity

    CERN Document Server

    Gershenson, Carlos

    2011-01-01

    The term complexity derives etymologically from the Latin plexus, which means interwoven. Intuitively, this implies that something complex is composed by elements that are difficult to separate. This difficulty arises from the relevant interactions that take place between components. This lack of separability is at odds with the classical scientific method - which has been used since the times of Galileo, Newton, Descartes, and Laplace - and has also influenced philosophy and engineering. In recent decades, the scientific study of complexity and complex systems has proposed a paradigm shift in science and philosophy, proposing novel methods that take into account relevant interactions.

  17. 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.

  18. Chronic Pancreatitis, Type 3c Diabetes, and Pancreatic Cancer Risk

    Directory of Open Access Journals (Sweden)

    David C Whitcomb

    2014-09-01

    Full Text Available About half of all patients with chronic pancreatitis (CP develop diabetes mellitus (DM due to the loss of islet cell mass, not just beta cells as in Type 1 DM (T1DM, or due to insulin resistance, as in Type 2 DM (T2DM. Patients with DM from loss of islets due to pancreatic disease or resection are diagnosed with pancreatogenic or Type 3c DM (T3cDM. Patients with T3cDM also lose counter-regulatory hormones, such as glucagon and pancreatic polypeptide, and experience maldigestion associated with pancreatic exocrine insufficiency. Patients with T3cDM are therefore more susceptible to hypoglycemia and a mismatch (asynchrony between food ingestion and nutrient absorption. At the same time, the use of incretin therapy is likely useless, since maldigestion leads to the release of higher levels of hind gut hormones, including GLP1. Thus, T3cDM caused by CP or destruction of the islets involves a special class of potential risks and comorbidity that may be overlooked if the CP has not been diagnosed.

  19. Phonons in A3C60 Lattice and Structural Dynamics

    Directory of Open Access Journals (Sweden)

    Sven Larsson

    2010-01-01

    Full Text Available The critical temperature ( of superconductivity in A3C60 compounds is generally lower smaller with alkali atoms (A. Furthermore decreases with applied pressure. In the BCS model, these trends are explained by the lower density of states at the Fermi level for a decreased lattice constant (R. There is more than one counterexample, however, suggesting that BCS does not give the whole truth. The most important one is that the compound with the largest lattice constant, Cs3C60, is not superconducting at all at ambient pressure. In this paper we derive a novel model where a negative lattice contribution to Hubbard U, proportional to 1/R, is taken into account. It is possible to explain why A3C60 compounds with A = Li, and Na have a low or are not superconducting at all, and why Cs3C60 is superconducting only at applied pressure and then with the highest of all C60 alkali fullerides. It is concluded that the density of states mechanism derived in the BCS model is in doubt. Nevertheless superconductivity in A3C60 depends on electron-phonon coupling. The dominating phonon is the bond stretching Ag phonon, a breathing phonon for the whole fullerene molecular ion.

  20. A Proposal for a Structural Model of the Feline Calicivirus Protease Bound to the Substrate Peptide under Physiological Conditions

    Directory of Open Access Journals (Sweden)

    Masaru Yokoyama

    2017-07-01

    Full Text Available Feline calicivirus (FCV protease functions to cleave viral precursor proteins during productive infection. Previous studies have mapped a protease-coding region and six cleavage sites in viral precursor proteins. However, how the FCV protease interacts with its substrates remains unknown. To gain insights into the interactions, we constructed a molecular model of the FCV protease bound with the octapeptide containing a cleavage site of the capsid precursor protein by homology modeling and docking simulation. The complex model was used to screen for the substrate mimic from a chemical library by pharmacophore-based in silico screening. With this structure-based approach, we identified a compound that has physicochemical features and arrangement of the P3 and P4 sites of the substrate in the protease, is predicted to bind to FCV proteases in a mode similar to that of the authentic substrate, and has the ability to inhibit viral protease activity in vitro and in the cells, and to suppress viral replication in FCV-infected cells. The complex model was further subjected to molecular dynamics simulation to refine the enzyme-substrate interactions in solution. The simulation along with a variation study predicted that the authentic substrate and anti-FCV compound share a highly conserved binding site. These results suggest the validity of our in silico model for elucidating protease-substrate interactions during FCV replication and for developing antivirals.

  1. A Proposal for a Structural Model of the Feline Calicivirus Protease Bound to the Substrate Peptide under Physiological Conditions.

    Science.gov (United States)

    Yokoyama, Masaru; Oka, Tomoichiro; Takagi, Hirotaka; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Tohya, Yukinobu; Sato, Hironori

    2017-01-01

    Feline calicivirus (FCV) protease functions to cleave viral precursor proteins during productive infection. Previous studies have mapped a protease-coding region and six cleavage sites in viral precursor proteins. However, how the FCV protease interacts with its substrates remains unknown. To gain insights into the interactions, we constructed a molecular model of the FCV protease bound with the octapeptide containing a cleavage site of the capsid precursor protein by homology modeling and docking simulation. The complex model was used to screen for the substrate mimic from a chemical library by pharmacophore-based in silico screening. With this structure-based approach, we identified a compound that has physicochemical features and arrangement of the P3 and P4 sites of the substrate in the protease, is predicted to bind to FCV proteases in a mode similar to that of the authentic substrate, and has the ability to inhibit viral protease activity in vitro and in the cells, and to suppress viral replication in FCV-infected cells. The complex model was further subjected to molecular dynamics simulation to refine the enzyme-substrate interactions in solution. The simulation along with a variation study predicted that the authentic substrate and anti-FCV compound share a highly conserved binding site. These results suggest the validity of our in silico model for elucidating protease-substrate interactions during FCV replication and for developing antivirals.

  2. Structural insights into the unique inhibitory mechanism of the silkworm protease inhibitor serpin18

    Science.gov (United States)

    Guo, Peng-Chao; Dong, Zhaoming; Zhao, Ping; Zhang, Yan; He, Huawei; Tan, Xiang; Zhang, Weiwei; Xia, Qingyou

    2015-01-01

    Serpins generally serve as inhibitors that utilize a mobile reactive center loop (RCL) as bait to trap protease targets. Here, we present the crystal structure of serpin18 from Bombyx mori at 1.65 Å resolution, which has a very short and stable RCL. Activity analysis showed that the inhibitory target of serpin18 is a cysteine protease rather than a serine protease. Notably, this inhibitiory reaction results from the formation of an intermediate complex, which then follows for the digestion of protease and inhibitor into small fragments. This activity differs from previously reported modes of inhibition for serpins. Our findings have thus provided novel structural insights into the unique inhibitory mechanism of serpin18. Furthermore, one physiological target of serpin18, fibroinase, was identified, which enables us to better define the potential role for serpin18 in regulating fibroinase activity during B. mori development. PMID:26148664

  3. Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease from Candida parapsilosis.

    Science.gov (United States)

    Dostál, Jiří; Pecina, Adam; Hrušková-Heidingsfeldová, Olga; Marečková, Lucie; Pichová, Iva; Řezáčová, Pavlina; Lepšík, Martin; Brynda, Jiří

    2015-12-01

    The virulence of the Candida pathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p from Candida parapsilosis was determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundant C. parapsilosis secreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.

  4. C1A cysteine protease-cystatin interactions in leaf senescence.

    Science.gov (United States)

    Díaz-Mendoza, Mercedes; Velasco-Arroyo, Blanca; González-Melendi, Pablo; Martínez, Manuel; Díaz, Isabel

    2014-07-01

    Senescence-associated proteolysis in plants is a crucial process to relocalize nutrients from leaves to growing or storage tissues. The massive net degradation of proteins involves broad metabolic networks, different subcellular compartments, and several types of proteases and regulators. C1A cysteine proteases, grouped as cathepsin L-, B-, H-, and F-like according to their gene structures and phylogenetic relationships, are the most abundant enzymes responsible for the proteolytic activity during leaf senescence. Besides, cystatins as specific modulators of C1A peptidase activities exert a complex regulatory role in this physiological process. This overview article covers the most recent information on C1A proteases in leaf senescence in different plant species. Particularly, it is focussed on barley, as the unique species where the whole gene family members of C1A cysteine proteases and cystatins have been analysed.

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

    Directory of Open Access Journals (Sweden)

    Shuaiyu Wang

    2016-12-01

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

  6. New infrared spectral component of the quasar 3C273

    Energy Technology Data Exchange (ETDEWEB)

    Robson, E.I.; Gear, W.K.; Brown, L.M.J.; Courvoisier, T.J.-L.; Smith, M.G.; Griffin, M.J.; Blecha, A.

    1986-09-11

    Following the dramatic infrared to millimetre-wavelength flare seen in the quasar 3C273 during 1983, the authors have continued to monitor its overall continuum emission. Recent measurements show that the 10-..mu..m to 3-mm emission has decayed to a level well below any seen previously, while the 1-4-..mu..m emission has remained relatively constant. This behaviour has revealed the presence of an apparently non-variable component which dominates the near-infrared emission in 3C273 and includes the small 'bump' at approx. 3.5 ..mu..m in the power-law continuum.

  7. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang;

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...

  8. 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

  9. Protease-degradable electrospun fibrous hydrogels

    Science.gov (United States)

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-03-01

    Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

  10. Complex

    African Journals Online (AJOL)

    CLEMENT O BEWAJI

    Schiff bases and their complex compounds have been studied for their .... establishing coordination of the N–(2 – hydroxybenzyl) - L - α - valine Schiff base ..... (1967); “Spectrophotometric Identification of Organic Compounds”, Willey, New.

  11. Discovery of a faint optical jet in 3C 120

    DEFF Research Database (Denmark)

    Hjorth, J.; Vestergaard, Marianne; Sorensen, A. N.;

    1995-01-01

    the optical condensation A of the galaxy, which includes the bright 4" radio knot, is found to be 12 % polarized with the electric field vectors perpendicular to the jet. These findings indicate that 3C 120 contains the 6th known extragalactic optical synchrotron jet, quite similar in its properties...

  12. A microelectromechanical system digital 3C array seismic cone penetrometer

    NARCIS (Netherlands)

    Ghose, R.

    2012-01-01

    A digital 3C array seismic cone penetrometer has been developed for multidisciplinary geophysical and geotechnical applications. Seven digital triaxial microelectromechanical system accelerometers are installed at 0.25-m intervals to make a 1.5-m-long downhole seismic array. The accelerometers have

  13. Microwave Radiometer – 3 Channel (MWR3C) Handbook

    Energy Technology Data Exchange (ETDEWEB)

    Cadeddu, MP

    2012-05-04

    The microwave radiometer 3-channel (MWR3C) provides time-series measurements of brightness temperatures from three channels centered at 23.834, 30, and 89 GHz. These three channels are sensitive to the presence of liquid water and precipitable water vapor.

  14. Search for exotic events from the L3+C data

    Institute of Scientific and Technical Information of China (English)

    DING Lin-Kai; HE Zuo-Xiu; HUO An-Xiang; JING Cai-Liu; KUANG Hao-Huai; LEI Yu; LI Li; MA Xin-Hua; MA Yu-Qian; QING Cheng-Rui; WANG Rui-Guang; YAO Zhi-Guo; YU Zhong-Qiang; ZHANG Chao; ZHANG Feng; ZHANG Jing; ZHU Qing-Qi

    2009-01-01

    An effort to search for Kolar-like events within the data set of the L3+C experiment is reported. From a total of 0.89×1010 triggered events there are no reliable two-prong Kolar-like events observed. The some reasonable assumptions.

  15. The Double–Double Radio Galaxy 3C293

    Indian Academy of Sciences (India)

    S. A. Joshi; S. Nandi; D. J. Saikia; C. H. Ishwara-Chandra; C. Konar

    2011-12-01

    We present the results of radio continuum observations at frequencies ranging from ∼ 150–5000 MHz of the misaligned double–double radio galaxy (DDRG) 3C293 (J1352+3126) using the GMRT and the VLA, and estimate the time-scale of interruption of jet activity to be less than ∼ 0.1 Myr.

  16. A decade of 3C technologies: insights into nuclear organization

    NARCIS (Netherlands)

    de Wit, E.; de Laat, W.

    2012-01-01

    Over the past 10 years, the development of chromosome conformation capture (3C) technology and the subsequent genomic variants thereof have enabled the analysis of nuclear organization at an unprecedented resolution and throughput. The technology relies on the original and, in hindsight, remarkably

  17. Position Measurements of the Core in 3C 66B

    Indian Academy of Sciences (India)

    G.-Y. Zhao; Y.-J. Chen; Z.-Q. Shen; H. Sudou; S. Iguchi; Y. Murata; Y. Taniguchi

    2011-03-01

    It was argued that 3C 66B, a nearby radio galaxy, harbors a supermassive black hole binary (SMBHB). To investigate this, a 4-epoch VLBA phase referencing imaging observation was performed in 2004–2005. Here we present some preliminary results of this project. We found a large position difference compared to previous results.

  18. Data of evolutionary structure change: 1V37A-2YW3C [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1V37A-2YW3C 1V37 2YW3 A C --------MELWLVRH--------GETLWNREGRLLGWT...41 ALA CA 498 2YW3 C 2YW3C...CA 453 2YW3 C 2YW3C.../pdbChain> 2YW3C LEITL-RTEKG EEE - HH...VID> 3 2YW3 C 2YW3C

  19. Connection Between X-Ray Emission and Relativistic Jets in the Radio Galaxies 3C 111 and 3C 120

    Science.gov (United States)

    Aller, Margo F.

    2005-01-01

    This work represents a part of a longterm study of the X-ray flux variability in radio galaxies and its relation to flux and structural changes in the associated radio jet. The work described here included: 1) continued study of the emission properties of the FR I radio galaxy 3C 120 known to exhibit a jet/disk connection from our past work; and 2) the commencement of monitoring of a second radio galaxy, the FR I1 object 3C 111 which was selected because of similar radio and X-ray properties to 3C 120, including the presence of Fe K a emission. The association between X-ray dips and new superluminal components, suggesting a picture in which the radio jet is fed by accretion events near the black hole, was identified in 3C 120 using combined RXTE and radio flux monitoring data and bi-monthly to monthly imaging data from the VLBA at 43 GHz. Such data were also obtained for both targets during the period described here. Specific goals were to more broadly investigate the X-ray dip/superluminal connection in 3C 120, thereby determining the epochs of X-ray minima and superluminal ejections more accurately (and hence more precisely determining the distance between the accretion disk and the core of the radio jet), and to determine whether a similar pattern is present in the data for a second radio galaxy. In 3C 111 a different time scale (longer time delays between X-ray dips and superluminal ejections) was expected due to the higher black hole mass implied by its higher radio luminosity: no black hole mass is published for this object but one can be determined from a PDS analysis of the RXTE data. The addition of the second source to the study would identify whether a similar connection was present in other sources and, if found, would provide important information on how time scale (and hence size scale) of accretion disk/jet systems depends on black hole mass. The grant included funding for the reduction and analysis of data obtained during the time period of Rossi

  20. Mast cell proteases as pharmacological targets.

    Science.gov (United States)

    Caughey, George H

    2016-05-05

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the

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

    Science.gov (United States)

    2010-01-01

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

  2. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  3. Mannan-binding lectin (MBL)-associated serine protease-1 (MASP-1), a serine protease associated with humoral pattern-recognition molecules

    DEFF Research Database (Denmark)

    Thiel, Steffen; Degn, Søren Egedal; Nielsen, H J;

    2012-01-01

    The pattern-recognition molecules mannan-binding lectin (MBL) and the three ficolins circulate in blood in complexes with MBL-associated serine proteases (MASPs). When MBL or ficolin recognizes a microorganism, activation of the MASPs occurs leading to activation of the complement system, an impo...

  4. Ahp cyclodepsipeptides: the impact of the Ahp residue on the "canonical inhibition" of S1 serine proteases.

    Science.gov (United States)

    Stolze, Sara C; Meltzer, Michael; Ehrmann, Michael; Kaiser, Markus

    2013-07-22

    S1 serine proteases are by far the largest and most diverse family of proteases encoded in the human genome. Although recent decades have seen an enormous increase in our knowledge, the biological functions of most of these proteases remain to be elucidated. Chemical inhibitors have proven to be versatile tools for studying the functions of proteases, but this approach is hampered by the limited availability of inhibitor scaffold structures with the potential to allow rapid discovery of selective, noncovalent small-molecule protease inhibitors. The natural product class of Ahp cyclodepsipeptides is an unusual class of small-molecule canonical inhibitors; the incorporation of protease cleavage sequences into their molecular scaffolds enables the design of specific small-molecule inhibitors that simultaneously target the S and S' subsites of the protease through noncovalent mechanisms. Their synthesis is tedious, however, so in this study we have investigated the relevance of the Ahp moiety for achieving potent inhibition. We found that although the Ahp residue plays an important role in inhibition potency, appropriate replacement with β-hydroxy amino acids results in structurally less complex derivatives that inhibit serine proteases in the low micromolar range.

  5. Synthesis of C3/C1-Substituted Tetrahydroisoquinolines

    Directory of Open Access Journals (Sweden)

    Mohamed Mihoubi

    2015-08-01

    Full Text Available A broad biological screening of the natural alkaloid N-methylisosalsoline (2 extracted from Hammada scoparia leaves against a panel of human and parasitic proteases revealed an interesting activity profile of 2 towards human 20S proteasome. This outcome suggests that the 1,2,3,4-tetrahydroisoquinoline skeleton may be exploited as a template for the development of novel anticancer agents. In this article, we report the synthesis and chemical characterization of a new series of isosalsoline-type alkaloids (10–11 with variations at N2 and C3 positions with respect to the natural Compound 2, obtained by a synthetic strategy that involves the Bischler-Napieralski cyclization. The substrate for the condensation to the tetrahydroisoquinoline system, i.e., a functionalized β-arylethyl amine, was obtained through an original double reduction of nitroalkene. The synthetic strategy can be directed to the construction of highly substituted and functionalized 1,2,3,4-tetrahydroisoquinolines.

  6. 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.

  7. 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.

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

    Science.gov (United States)

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

    2007-11-01

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

  9. (Processing and targeting of the thiol protease aleurain)

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, J.C.

    1990-01-01

    Our goal for work during the past two years under this Grant was to characterize the barley thiol protease, aleurain, to determine if it is secreted or retained intracellularly in aleurone cells, and to begin to elucidate structural features that might control targeting of the protein to its final destination. We have shown that aleurain is synthesized as a proenzyme with two N-linked oligosaccharide chains, one high mannose-type and one complex-type. Aleurain undergoes processing to mature form by removal of an Nterminal prosegment, and is retained intracellularly; it cannot be detected among proteins secreted from aleurone cells. Treatment of aleurone cells with tunicamycin to prevent glycosylation of aleurain does not prevent processing of the unglycosylated form. The N-terminal portion of aleurain's prosegment is homologous to the comparable region in two yeast vacuolar proteases, where that region is known to contain the signal necessary for targeting the proteases to the vacuole. 18 refs., 7 figs.

  10. Structure-function relationship of Chikungunya nsP2 protease: A comparative study with papain.

    Science.gov (United States)

    Ramakrishnan, Chandrasekaran; Kutumbarao, Nidamarthi H V; Suhitha, Sivasubramanian; Velmurugan, Devadasan

    2016-11-07

    Chikungunya virus is a growing human pathogen transmitted by mosquito bite. It causes fever, chills, nausea, vomiting, joint pain, headache, and swelling in the joints. Its replication and propagation depend on the protease activity of the Chikungunya virus-nsP2 protein, which cleaves the nsP1234 polyprotein replication complex into individual functional units. The N-terminal segment of papain is structurally identical with the Chikungunya virus-nsP2 protease. Hence, molecular dynamics simulations were performed to compare molecular mechanism of these proteases. The Chikungunya virus-snP2 protease shows more conformational changes and adopts an alternate conformation. However, N-terminal segment of these two proteases has identical active site scaffold with the conserved catalytic diad. Hence, some of the non-peptide inhibitors of papain were used for induced fit docking at the active site of the nsP2 to assess the binding mode. In addition, the peptides that connect different domains/protein in Chikungunya virus poly-protein were also subjected for docking. The overall results suggest that the active site scaffold is the same in both the proteases and a possibility exists to experimentally assess the efficacy of some of the papain inhibitors to inhibit the Chikungunya virus-nsP2.

  11. Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus

    Science.gov (United States)

    Burchacka, Ewa; Zdzalik, Michal; Niemczyk, Justyna-Stec; Pustelny, Katarzyna; Popowicz, Grzegorz; Wladyka, Benedykt; Dubin, Adam; Potempa, Jan; Sienczyk, Marcin; Dubin, Grzegorz; Oleksyszyn, Jozef

    2014-01-01

    Staphylococcus aureus is responsible for a variety of human infections, including life-threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of α-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-PheP-(OC6H4−4-SO2CH3)2 and Suc-Val-Pro-PheP-(OC6H5)2 are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of α-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases. PMID:24375505

  12. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors.

    Science.gov (United States)

    Serrano-Luna, Jesús; Piña-Vázquez, Carolina; Reyes-López, Magda; Ortiz-Estrada, Guillermo; de la Garza, Mireya

    2013-01-01

    The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms.

  13. Structures of HIV Protease Guide Inhibitor Design to Overcome Drug Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Irene T.; Kovalevsky, Andrey Y.; Harrison, Robert W. (GSU)

    2008-06-03

    The HIV/AIDS infection continues to be a major epidemic worldwide despite the initial promise of antiviral drugs. Current therapy includes a combination of drugs that inhibit two of the virally-encoded enzymes, the reverse transcriptase and the protease. The first generation of HIV protease inhibitors that have been in clinical use for treatment of AIDS since 1995 was developed with the aid of structural analysis of protease-inhibitor complexes. These drugs were successful in improving the life span of HIV-infected people. Subsequently, the rapid emergence of drug resistance has necessitated the design of new inhibitors that target mutant proteases. This second generation of antiviral protease inhibitors has been developed with the aid of data from medicinal chemistry, kinetics, and X-ray crystallographic analysis. Traditional computational methods such as molecular mechanics and dynamics can be supplemented with intelligent data mining approaches. One approach, based on similarities to the protease interactions with substrates, is to incorporate additional interactions with main chain atoms that cannot easily be eliminated by mutations. Our structural and inhibition data for darunavir have helped to understand its antiviral activity and effectiveness on drug resistant HIV and demonstrate the success of this approach.

  14. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors

    Science.gov (United States)

    Serrano-Luna, Jesús; Piña-Vázquez, Carolina; Reyes-López, Magda; Ortiz-Estrada, Guillermo

    2013-01-01

    The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms. PMID:23476670

  15. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors

    Directory of Open Access Journals (Sweden)

    Jesús Serrano-Luna

    2013-01-01

    Full Text Available The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms.

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

    Science.gov (United States)

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

    2009-01-01

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

  17. HST Polarimetry of the 3C 273 Jet

    Science.gov (United States)

    Clautice, Devon; Perlman, Eric S.; Sparks, William B.; Biretta, John A.; O'Dea, Christopher P.; Baum, Stefi Alison; Cheung, Chi C.; Birkinshaw, Mark; Worrall, Diana M.; Martel, Andre; Urry, C. Megan; Stawarz, Lukasz; Coppi, Paolo S.; Uchiyama, Yasunobu; Cara, Mihai; Meisenheimer, Klaus; Begelman, Mitchell C.

    2017-01-01

    We present preliminary results using HST polarimetry of the jet of 3C 273. Polarization is a critical parameter for understanding jet flows, and has proven essential in characterizing the physics of FR I jets; high-quality HST polarimetry has been done for just two other FR II jets previously. Our recent work on two quasar jets, where we measured high optical polarization in the brightest X-ray knots, has favored the synchrotron emission model over the alternative IC/CMB model for their optical to X-ray emission. These new observations of 3C 273 allow for the determination of the magnetic field structure and confirmation of which emission mechanisms are operating to create its optical to X-ray emission, and will allow us to greatly advance modeling efforts for this jet and nail down its kinetic power, a key unknown parameter for understanding quasars and their cosmological effects.

  18. A Complete Set of VSOP Observations of 3C279

    Science.gov (United States)

    Pant, N. D.; Piner, B. G.; Edwards, P. G.; Hirabayashi, H.; Wehrle, A. E.; Unwin, S. C.

    2009-08-01

    We have compiled a complete set of VSOP observations of 3C279, consisting of eight 5 GHz, and six 1.6 GHz, VSOP observations, all of which include the VLBA in the ground array. We are using the data-set to determine brightness temperature limits from model-fits to the visibilities, the transverse structure of the jet over its first 20 milliarcseconds, and parsec-scale spectral index maps.

  19. Hydrodynamical Simulations of Colliding Jets: Modeling 3C 75

    Science.gov (United States)

    Molnar, S. M.; Schive, H.-Y.; Birkinshaw, M.; Chiueh, T.; Musoke, G.; Young, A. J.

    2017-01-01

    Radio observations suggest that 3C 75, located in the dumbbell shaped galaxy NGC 1128 at the center of Abell 400, hosts two colliding jets. Motivated by this source, we perform three-dimensional hydrodynamical simulations using a modified version of the GPU-accelerated Adaptive-MEsh-Refinement hydrodynamical parallel code (GAMER) to study colliding extragalactic jets. We find that colliding jets can be cast into two categories: (1) bouncing jets, in which case the jets bounce off each other keeping their identities, and (2) merging jets, when only one jet emerges from the collision. Under some conditions the interaction causes the jets to break up into oscillating filaments of opposite helicity, with consequences for their downstream stability. When one jet is significantly faster than the other and the impact parameter is small, the jets merge; the faster jet takes over the slower one. In the case of merging jets, the oscillations of the filaments, in projection, may show a feature that resembles a double helix, similar to the radio image of 3C 75. Thus we interpret the morphology of 3C 75 as a consequence of the collision of two jets with distinctly different speeds at a small impact parameter, with the faster jet breaking up into two oscillating filaments.

  20. The Trails of Superluminal Jet Components in 3C 111

    Science.gov (United States)

    Kadler, M.; Ros, E.; Perucho, M.; Kovalev, Y. Y.; Homan, D. C.; Agudo, I.; Kellermann, K. I.; Aller, M. F.; Aller, H. D.; Lister, M. L.; Zensus, J. A.

    2007-01-01

    The parsec-scale radio jet of the broad-line radio galaxy 3C 111 has been monitored since 1995 as part of the 2cm Survey and MOJAVE monitoring observations conducted with the VLBA. Here, we present results from 18 epochs of VLBA observations of 3C 111 and from 18 years of radio flux density monitoring observations conducted at the University of Michigan. A major radio flux-density outburst of 3C 111 occurred in 1996 and was followed by a particularly bright plasma ejection associated with a superluminal jet component. This major event allows us to study a variety of processes associated with outbursts of radio-loud AGN in much greater detail than possible in other cases: the primary perturbation gives rise to the formation of a forward and a backward-shock, which both evolve in characteristically different ways and allow us to draw conclusions about the workflow of jet-production events; the expansion, acceleration and recollimation of the ejected jet plasma in an environment with steep pressure and density gradients are revealed; trailing components are formed in the wake of the primary perturbation as a result of Kelvin- Helmholtz instabilities from the interaction of the jet with the external medium. The jet-medium interaction is further scrutinized by the linear-polarization signature of jet components traveling along the jet and passing a region of steep pressure/density gradients.

  1. The inner kiloparsec of the jet in 3C264

    CERN Document Server

    Lara, L; Cotton, B; Feretti, L; Venturi, T; Lara, Lucas; Giovannini, Gabriele; Cotton, Bill; Feretti, Luigina; Venturi, Tiziana

    2003-01-01

    We present new multi-frequency EVN, MERLIN and VLA observations of the radio source 3C264, sensitive to linear scales ranging from the parsec to several kiloparsecs. The observations confirm the existence of regions with different properties in the first kiloparsec of the jet. The most remarkable feature is the transition between a well collimated narrow jet at distances from the core below 80 pc, to a conical-shaped wide jet, with an opening angle of 20 degrees. Another change of properties, consisting of an apparent deflection of the jet ridge line and a diminution of the surface brightness, occurs at a distance of 300 pc from the core, coincident with the radius of a ring observed at optical wavelengths. Our observations add new pieces of information on the spectrum of the radio-optical jet of 3C264, with results consistent with a synchrotron emission mechanism and a spectrum break frequency in the infrared. Brightness profiles taken perpendicularly to the jet of 3C264 are consistent with a spine brightene...

  2. Infrared spectrophotometry of three Seyfert galaxies and 3C 273

    Science.gov (United States)

    Cutri, R. M.; Puetter, R. C.; Rudy, R. J.; Willner, S. P.; Aitken, D. K.; Jones, B.; Merrill, K. M.; Roche, P. F.; Russell, R. W.; Soifer, B. T.

    1981-01-01

    Spectrophotometry in the range 2.1-4.0 microns is presented for the Seyfert galaxies NGC 1068, NGC 4151 and Mrk 231 and the quasar 3C 273, together with broadband and narrowband observations of the Seyfert galaxies in the range 8-13 microns. The spectra of NGC 1068 and NGC 4151 are found to contain a significant component due to starlight, especially at shorter wavelengths. The nonstellar component in NGC 1068 is observed to fall off rapidly at wavelengths shorter than 4 microns, consistent with the interpretation of the excess beyond 5 microns as thermal reradiation by dust. Observations confirm the variability of NGC 4151, and indicate the presence of two components of the flux other than starlight: a nonthermal variable component predominant at shorter wavelengths and a constant, probably thermal component at wavelengths greater than 3 microns. Mrk 231 and 3C 273 exhibit no discernable stellar component and were not observed to vary by more than 10%. Evidence is obtained for a broad minimum in the 8 to 13 micron spectrum of Mrk 231, as well as possible structure between rest wavelengths of 2.8 and 2.9 microns, and the spectrum is not a power law. The spectrum of 3C 273 is consistent with a power law from 1.2 to 10 microns, with small but significant deviations.

  3. Epstein-Barr virus nuclear antigen 3C stabilizes Gemin3 to block p53-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Qiliang Cai

    2011-12-01

    Full Text Available The Epstein-Barr nuclear antigen 3C (EBNA3C, one of the essential latent antigens for Epstein-Barr virus (EBV-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103 is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs. Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities.

  4. The role of proteases in the differentiation of Acanthamoeba castellanii.

    Science.gov (United States)

    Dudley, Ricky; Alsam, Selwa; Khan, Naveed Ahmed

    2008-09-01

    Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii. Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.

  5. 3C380: a powerful radio source seen end-on

    Energy Technology Data Exchange (ETDEWEB)

    Wilkinson, P.N. (Nuffield Radio Astronomy Labs., Jodrell Bank (UK)); Akujor, C.E. (Nuffield Radio Astronomy Labs., Jodrell Bank (UK) Nigeria Univ., Nsukka (Nigeria). Dept of Physics); Cornwell, T.J. (National Radio Astronomy Observatory, Socorro, N.M. (USA)); Saikia, D.J. (Nuffield Radio Astronomy Labs., Jodrell Bank (UK) Tata Inst. of Fundamental Research, GMRT Project, Pune (India))

    1991-01-01

    We present new VLA, MERLIN and VLBI observations of 3C380, a powerful compact steep-spectrum radio source with complex extended structure, and attempt to clarify whether it is a larger source seen end-on or whether is is intrinsically of galactic dimensions and distorted. The extended structure, which could be a pair of overlapping lobes, exhibits a strong depolarization asymmetry of the kind found in powerful double sources, while the core exhibits superluminal motion. 3C380 therefore has features to be expected of a Fanaroff-Riley class II source seen approximately end-on. However, from a detailed consideration of the new evidence we infer that the source is intrinsically small with an overall extent {le}60 kpc. (author).

  6. Lipase and protease extraction from activated sludge

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  7. 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.

  8. Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

    Directory of Open Access Journals (Sweden)

    Louise S Goupil

    2016-08-01

    Full Text Available Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi. In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites.

  9. Multiwavelength and Polarimetric Analysis of the Flat Spectrum Radio Quasars 3C 273 and 3C 279

    Science.gov (United States)

    Fernandes, Sunil; Patiño-Álvarez, Victor; Chavushyan, Vahram; Schlegel, Eric M.; Lopez-Rodriguez, Enrique; León-Tavares, Jonathan; Carrasco, Luis; Valdés, José; Carramiñana, Alberto

    2017-01-01

    This poster presents results of multiwavelength analyses of 3C 273 and 3C 279. The main goals were to identify the gamma-ray emission region and dominant high-energy emission processes. Our methodology consisted of analyzing light curves from radio to gamma-rays over 6 - 8 years and polarimetric, spectral and line emission behavior.In 3C 279, we found that the emission from millimeter to ultraviolet was simultaneous and therefore co-spatial. We identified two active states where different high-energy emission processes were dominant. We found multiwavelength flaring events consistent with component ejections and shocks. We proposed that the gamma-ray emission region changed over time based on observations of both simultaneous and delayed gamma-rays emission with respect to low-energy emission during different time-frames.In 3C 273, we identified a non-thermal flare related to a component ejection and a thermal flare related to accretion. From reverberation mapping we found that the broad line region dynamical behavior over time possibly affects the derived supermassive black hole mass.In both objects we found that the gamma-ray spectral index was variable, and a trend of harder spectral index with higher gamma-ray luminosity. From the identification of different dominant high-energy emission processes, we concluded that the dominant high-energy emission mechanism changes with time. Overall, we concluded that similar results from both objects points to behavior that is potentially common to flat spectrum radio quasars. Increasing the sample size of objects analyzed with similar methodologies will provide more results to confirm or refine our conclusions.

  10. MALT1 protease: equilibrating immunity versus tolerance

    OpenAIRE

    Bertossi, Arianna; Krappmann, Daniel

    2014-01-01

    MALT1 paracaspase links signaling cascades emanating from adaptive or innate immune receptors to the canonical NF-κB pathway. Now, Jaworski et al (2014) investigate the physiological role of MALT1 protease activity in mice. Besides the expected requirement of MALT1 activity for immune activation, the study unveils a novel function for MALT1 activity for the development of peripheral tolerance. Thus, MALT1 protease can act immunogenic or tolerogenic, and this interplay will be highly relevant ...

  11. Solid-state characterization of the HIV protease inhibitor

    CERN Document Server

    Kim, Y A

    2002-01-01

    The LB71350, (3S, 4R)-Epoxy-(5S)-[[N-(1-methylethoxy) carbonyl]-3-(methylsulfonyl)-L-valinyl]amin= o]-N-[2-methyl-(1R)-[(phenyl)carbonyl]propyl-6-phenylhexanamide, is a novel HIV protease inhibitor. Its equilibrium solubility at room temperature was less than 40 mu g/mL. It was speculated that the low aqueous solubility might be due to the high crystalline lattice energy resulting from intermolecular hydrogen bonds. The present study was carried out to learn the solid-state characteristics of LB71350 using analytical methods such as NMR, FT-IR and XRD. sup 1 sup 3 C Solid-state NMR, solution NMR, and FT-IR spectra of the various solid forms of LB71350 were used to identify the conformation and structure of the solid forms. The chemical shifts of sup 1 sup 3 C solid-state NMR spectra suggest that the crystalline form might have 3 intermolecular hydrogen bondings between monomers.

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

    Science.gov (United States)

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

    1983-01-01

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

  13. 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.

  14. ADAM Proteases and Gastrointestinal Function.

    Science.gov (United States)

    Jones, Jennifer C; Rustagi, Shelly; Dempsey, Peter J

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis.

  15. ADAM Proteases and Gastrointestinal Function

    Science.gov (United States)

    Jones, Jennifer C.; Rustagi, Shelly; Dempsey, Peter J.

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  16. Extreme entropy-enthalpy compensation in a drug-resistant variant of HIV-1 protease.

    Science.gov (United States)

    King, Nancy M; Prabu-Jeyabalan, Moses; Bandaranayake, Rajintha M; Nalam, Madhavi N L; Nalivaika, Ellen A; Özen, Ayşegül; Haliloğlu, Türkan; Yilmaz, Neşe Kurt; Schiffer, Celia A

    2012-09-21

    The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5-15 kcal/mol, while losing only 1-3 kcal/mol in total binding free energy for any of six FDA-approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wild-type protease and another drug-resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug-resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design.

  17. 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.

  18. Experience with Server Self Service Center (S3C)

    CERN Document Server

    Sucik, J; CERN. Geneva. IT Department

    2010-01-01

    CERN has a successful experience with running Server Self Service Center (S3C) for virtual server provisioning which is based on Microsoft® Virtual Server 2005. With the introduction of Windows Server 2008 and its built-in hypervisor based virtualization (Hyper-V) there are new possibilities for the expansion of the current service. This paper describes the architecture of the redesigned virtual Server Self Service based on Hyper-V which provides dynamically scalable virtualized resources on demand as needed and outlines the possible implications on the future use of virtual machines at CERN.

  19. Delta-function Approximation SSC Model in 3C 273

    Indian Academy of Sciences (India)

    S. J. Kang; Y. G. Zheng; Q. Wu

    2014-09-01

    We obtain an approximate analytical solution using approximate calculation on the traditional one-zone synchrotron self-Compton (SSC) model. In this model, we describe the electron energy distribution by a broken power-law function with a sharp cut-off, and non-thermal photons are produced by both synchrotron and inverse Compton scattering of synchrotron photons. We calculate the radiation energy spectrum of electrons by the function. We apply this model to the multi-wavelength Spectral Energy Distributions (SED) of the 3C 273 in different states, and obtain excellent fits to the observed spectra of this source.

  20. Experience with Server Self Service Center (S3C)

    CERN Multimedia

    Sucik, J

    2009-01-01

    CERN has a successful experience with running Server Self Service Center (S3C) for virtual server provisioning which is based on Microsoft® Virtual Server 2005. With the introduction of Windows Server 2008 and its built-in hypervisor based virtualization (Hyper-V) there are new possibilities for the expansion of the current service. This paper describes the architecture of the redesigned virtual Server Self Service based on Hyper-V which provides dynamically scalable virtualized resources on demand as needed and outlines the possible implications on the future use of virtual machines at CERN.

  1. Structure and Properties of the Nonface-Spiral Fullerenes T-C380, D3-C384, D3-C440, and D3-C672 and Their Halma and Leapfrog Transforms

    DEFF Research Database (Denmark)

    Wirz, Lukas; Tonner, Ralf; Avery, James Emil;

    2013-01-01

    The structure and properties of the three smallest nonface-spiral (NS) fullerenes NS-T-C380, NS-D3-C384, NS-D3-C440, and the first isolated pentagon NS-fullerene, NS-D3-C672, are investigated in detail. They are constructed by either a generalized face-spiral algorithm or by vertex insertions fol...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  3. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    Science.gov (United States)

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  4. Mixed ligand copper(II) complexes of N,N-bis(benzimidazol-2-ylmethyl)amine (BBA) with diimine co-ligands: efficient chemical nuclease and protease activities and cytotoxicity.

    Science.gov (United States)

    Loganathan, Rangasamy; Ramakrishnan, Sethu; Suresh, Eringathodi; Riyasdeen, Anvarbatcha; Akbarsha, Mohamad Abdulkadhar; Palaniandavar, Mallayan

    2012-05-21

    A series of mononuclear mixed ligand copper(II) complexes [Cu(bba)(diimine)](ClO(4))(2)1-4, where bba is N,N-bis(benzimidazol-2-ylmethyl)amine and diimine is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp) (3), or dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) (4), have been isolated and characterized by analytical and spectral methods. The coordination geometry around copper(II) in 2 is described as square pyramidal with the two benzimidazole nitrogen atoms of the primary ligand bba and the two nitrogen atoms of phen (2) co-ligand constituting the equatorial plane and the amine nitrogen atom of bba occupying the apical position. In contrast, the two benzimidazole nitrogen atoms and the amine nitrogen atom of bba ligand and one of the two nitrogen atoms of 5,6-dmp constitute the equatorial plane of the trigonal bipyramidal distorted square based pyramidal (TBDSBP) coordination geometry of 3 with the other nitrogen atom of 5,6-dmp occupying the apical position. The structures of 1-4 have been optimized by using the density functional theory (DFT) method at the B3LYP/6-31G(d,p) level. Absorption spectral titrations with Calf Thymus (CT) DNA reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand, dpq (4) > 5,6-dmp (3) > phen (2) > bpy (1). The DNA binding affinity of 4 is higher than 2 revealing that the π-stacking interaction of the dpq ring in between the DNA base pairs with the two bzim moieties of the bba ligand stacked along the DNA surface is more intimate than that of phen. The complex 3 is bound to DNA more strongly than 1 and 2 through strong hydrophobic interaction of the methyl groups on 5,6-positions of the phen ring in the DNA grooves. The extent of the decrease in relative emission intensities of DNA-bound ethidium bromide (EB) upon adding the complexes parallels the trend in DNA binding affinities. The large enhancement in relative viscosity of DNA upon binding

  5. Clusters of galaxies associated with quasars. I. 3C 206

    Energy Technology Data Exchange (ETDEWEB)

    Ellingson, E.; Yee, H.K.C.; Green, R.F.; Kinman, T.D. (Steward Observatory, Tucson, AZ (USA); Montreal Universite (Canada); Kitt Peak National Observatory, Tucson, AZ (USA))

    1989-06-01

    Multislit spectroscopy and three-color CCD photometry of the galaxies in the cluster associated with the quasar 3C 206 (PKS 0837-12) at z = 0.198 are presented. This cluster is the richest environment of any low-redshift quasar observed in an Abell richness class 1 cluster. The cluster has a very flattened structure and a very concentrated core about the quasar. Most of the galaxies in this field have colors and luminosities consistent with normal galaxies at this redshift. The background-corrected blue fraction of galaxies is consistent with values for other rich clusters. The existence of several blue galaxies in the concentrated cluster core is an anomaly for a region of such high galaxy density, however, suggesting the absence of a substantial intracluster medium. This claim is supported by the Fanaroff-Riley (1974) class II morphology of the radio source. The velocity dispersion calculated from 11 spectroscopically confirmed cluster members is 500 + or - 110 km/s, which is slightly lower than the average for Abell class 1 clusters. A high frequency of interaction between the quasar host galaxy and cluster core members at low relative velocities, and a low intracluster gas pressure, may comprise a favorable environment for quasar activity. The properties of the cluster of galaxies associated with 3C 206 are consistent with this model. 59 refs.

  6. Photoluminescence Properties of Nanocrystalline 3C-SiC Films

    Institute of Scientific and Technical Information of China (English)

    YU Wei; LU Xue-qin; LU Wan-bing; HAN Li; FU Guang-sheng

    2006-01-01

    Nanocrystalline (nc) 3C-SiC films on the Si substrate were prepared by the helicon wave plasma enhanced chemical vapor deposition (HW-PECVD) technique. With the SiH4-CH4 gas flow ratio changing, the films exhibit different photoluminescence (PL) characteristics. Under the stoichiometric condition, the PL peak redshift from 470 nm to 515 nm is detected with the increase of excitation wavelength, which can be attributed to the quantum confinement effect radiation of 3C-SiC nanocrystals of different sizes. However, the appearance of an additional PL band at 436 nm in Si-rich film might be sourced back to the excess of Si defect centers in it. This is also the case for C-rich film for its PL band lying at 570 nm. The results above quoted indicate an important influence of gas flow ratio on the PL properties of the SiC films providing an effective guidance for analyzing the luminescence mechanism and exploring the high-efficiency light emission of the SiC films.

  7. Modelling the $\\gamma$-ray variability of 3C 273

    CERN Document Server

    Zheng, Y G; Huang, B R; Kang, S J

    2016-01-01

    We investigate MeV-GeV $\\gamma$-ray outbursts in 3C 273 in the frame of a time-dependent one-zone synchrotron self-Compton (SSC) model. In this model, electrons are accelerated to extra-relativistic energy through the stochastic particle acceleration and evolve with the time, nonthermal photons are produced by both synchrotron and inverse Compton scattering of synchrotron photons. Moreover, nonthermal photons during a quiescent are produced by the relativistic electrons in the steady state and those during a outburst are produced by the electrons whose injection rate is changed at some time interval. We apply the model to two exceptionally luminous $\\gamma$-ray outbursts observed by the Fermi-LAT from 3C 273 in September, 2009 and obtain the multi-wavelength spectra during the quiescent and during the outburst states, respectively. Our results show that the time-dependent properties of outbursts can be reproduced by adopting the appropriate injection rate function of the electron population.

  8. The Trails of Superluminal Jet Components in 3C111

    CERN Document Server

    Kadler, M; Perucho, M; Kovalev, Y Y; Homan, D C; Agudo, I; Kellermann, K I; Aller, M F; Aller, H D; Lister, M L; Zensus, J A

    2008-01-01

    In 1996, a major radio flux-density outburst occured in the broad-line radio galaxy 3C111. It was followed by a particularly bright plasma ejection associated with a superluminal jet component, which has shaped the parsec-scale structure of 3C111 for almost a decade. Here, we present results from 18 epochs of Very Long Baseline Array (VLBA) observations conducted since 1995 as part of the VLBA 2 cm Survey and MOJAVE monitoring programs. This major event allows us to study a variety of processes associated with outbursts of radio-loud AGN in much greater detail than has been possible in other cases: the primary perturbation gives rise to the formation of a leading and a following component, which are interpreted as a forward and a backward-shock. Both components evolve in characteristically different ways and allow us to draw conclusions about the work flow of jet-production events; the expansion, acceleration and recollimation of the ejected jet plasma in an environment with steep pressure and density gradien...

  9. The high energy spectrum of 3C 273

    CERN Document Server

    Esposito, V; Jean, P; Tramacere, A; Türler, M; Lähteenmäki, A; Tornikoski, M

    2015-01-01

    Aims. The high energy spectrum of 3C 273 is usually understood in terms of inverse-Compton emission in a relativistic leptonic jet. This model predicts variability patterns and delays that could be tested with simultaneous observations from the radio to the GeV range. Methods. The instruments IBIS, SPI, JEM-X on board INTEGRAL, PCA on board RXTE, and LAT on board Fermi have enough sensitivity to follow the spectral variability of 3C 273 from the keV to the GeV. We looked for correlations between the different energy bands, including radio data at 37 GHz collected at the Mets\\"ahovi Radio Observatory and built quasi-simultaneous multiwavelength spectra in the high energy domain when the source is flaring either in the X-rays or in the {\\gamma} rays. Results. Both temporal and spectral analysis suggest a two-component model to explain the complete high energy spectrum. X-ray emission is likely dominated by a Seyfert-like component while the {\\gamma}-ray emission is dominated by a blazar-like component produced ...

  10. Far-infrared polarisation of the quasar 3C 279

    CERN Document Server

    Klaas, U; Clavel, J; Klaas, Ulrich; Laureijs, Rene J.; Clavel, Jean

    1999-01-01

    We present the first FIR polarisation results of the OVV quasar 3C 279 obtained with ISOPHOT for two epochs in 1996 and 1997. We describe its integral polarisation properties at a wavelength of 170 micron where the source shows a maximum in its energy distribution. After a gamma-ray flare in January 1996, a polarisation of 23 % closely aligned with the radio jet axis was measured in July 1996. In June 1997, the polarisation degree had decreased to 6.5 % with a less good alignment. On the other hand, the total 170 micron flux is the same for both epochs. Our measurements provide additional constraints for the multi-wavelength properties of synchrotron emission in radio jets and the temporal evolution of these properties: they show that the FIR radiation of 3C 279 is optically thin and that its origin is very close to the core. The variability of the FIR polarisation without any change of the total FIR flux can be explained by a disordering of the magnetic field in between the core and the first stationary VLBI...

  11. XMM-Newton observations of 3C 273

    CERN Document Server

    Page, K L; Done, C; O'Brien, P T; Reeves, J N; Sembay, S; Stuhlinger, M

    2004-01-01

    A series of nine XMM-Newton observations of the radio-loud quasar 3C 273 are presented, concentrating mainly on the soft excess. Although most of the individual observations do not show evidence for iron emission, co-adding them reveals a weak, broad line (EW ~ 56 eV). The soft excess component is found to vary, confirming previous work, and can be well fitted with multiple blackbody components, with temperatures ranging between ~40 and ~330 eV, together with a power-law. Alternatively, a Comptonisation model also provides a good fit, with a mean electron temperature of ~350 eV, although this value is higher when the soft excess is more luminous over the 0.5-10 keV energy band. In the RGS spectrum of 3C 273, a strong detection of the OVII He-alpha absorption line at zero redshift is made; this may originate in warm gas in the local intergalactic medium, consistent with the findings of both Fang et al. (2003) and Rasmussen et al. (2003).

  12. Design, Synthesis, and Evaluation of Novel Prodrugs of Transition State Inhibitors of Norovirus 3CL Protease.

    Science.gov (United States)

    Galasiti Kankanamalage, Anushka C; Kim, Yunjeong; Rathnayake, Athri D; Alliston, Kevin R; Butler, Michelle M; Cardinale, Steven C; Bowlin, Terry L; Groutas, William C; Chang, Kyeong-Ok

    2017-07-27

    Ester and carbamate prodrugs of aldehyde bisulfite adduct inhibitors were synthesized in order to improve their pharmacokinetic and pharmacodynamic properties. The inhibitory activity of the compounds against norovirus 3C-like protease in enzyme and cell-based assays was determined. The ester and carbamate prodrugs displayed equivalent potency to those of the precursor aldehyde bisulfite adducts and precursor aldehydes. Furthermore, the rate of ester cleavage was found to be dependent on alkyl chain length. The generated prodrugs exhibited low cytotoxicity and satisfactory liver microsomes stability and plasma protein binding. The methodology described herein has wide applicability and can be extended to the bisulfite adducts of common warheads employed in the design of transition state inhibitors of serine and cysteine proteases of medical relevance.

  13. The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

    Science.gov (United States)

    Hook, V Y; Sei, C; Yasothornsrikul, S; Toneff, T; Kang, Y H; Efthimiopoulos, S; Robakis, N K; Van Nostrand, W

    1999-01-29

    Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

  14. 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...

  15. The Infrared Detection of the Pulsar Wind Nebula in the Galactic Supernova Remnant 3C 58

    Science.gov (United States)

    Slane, P.; Helfand, D. J.; Reynolds, S. P.; Gaensler, B. M.; Lemiere, A.; Wang, Z.

    2008-03-01

    We present infrared observations of 3C 58 with the Spitzer Space Telescope and the Canada-France-Hawaii Telescope. Using the IRAC camera, we have imaged the entire source, which results in clear detections of the nebula at 3.6 and 4.5 μm. The derived flux values are consistent with extrapolation of the X-ray spectrum to the infrared band, demonstrating that any cooling break in the synchrotron spectrum must occur near the soft X-ray band. We also detect the torus surrounding PSR J0205+6449, the 65 ms pulsar that powers 3C 58. The torus spectrum requires a break between the infrared and X-ray bands, and perhaps multiple breaks. This complex spectrum, which is an imprint of the particles injected into the nebula, has considerable consequences for the evolution of the broadband spectrum of 3C 58. We illustrate these effects and discuss the impact of these observations on the modeling of broadband spectra of pulsar wind nebulae.

  16. The Infrared Detection of the Pulsar Wind Nebula in the Galactic Supernova Remnant 3C 58

    CERN Document Server

    Slane, P; Reynolds, S P; Gaensler, B M; Lemiere, A; Wang, Z

    2008-01-01

    We present infrared observations of 3C 58 with the Spitzer Space Telescope and the Canada-France-Hawaii Telescope. Using the IRAC camera, we have imaged the entire source resulting in clear detections of the nebula at 3.6 and 4.5 microns. The derived flux values are consistent with extrapolation of the X-ray spectrum to the infrared band, demonstrating that any cooling break in the synchrotron spectrum must occur near the soft X-ray band. We also detect the torus surrounding PSR J0205+6449, the 65 ms pulsar that powers 3C 58. The torus spectrum requires a break between the infrared and X-ray bands, and perhaps multiple breaks. This complex spectrum, which is an imprint of the particles injected into the nebula, has considerable consequences for the evolution of the broadband spectrum of 3C 58. We illustrate these effects and discuss the impact of these observations on the modeling of broadband spectra of pulsar wind nebulae.

  17. From galaxy-scale fueling to nuclear-scale feedback. The merger-state of radio galaxies 3C 293, 3C 305, and 4C 12.50

    Science.gov (United States)

    Emonts, B. H. C.; Morganti, R.; Villar-Martín, M.; Hodgson, J.; Brogt, E.; Tadhunter, C. N.; Mahony, E.; Oosterloo, T. A.

    2016-11-01

    Powerful radio galaxies are often associated with gas-rich galaxy mergers. These mergers may provide the fuel to trigger starburst and active galactic nuclear (AGN) activity. In this Research Note, we study the host galaxies of three seemingly young or re-started radio sources that drive fast outflows of cool neutral hydrogen (H i) gas, namely 3C 293, 3C 305 and 4C 12.50 (PKS 1345+12). Our aim is to link the feedback processes in the central kpc-scale region with new information on the distribution of stars and gas at scales of the galaxy. For this, we use deep optical V-band imaging of the host galaxies, complemented with H i emission-line observations to study their gaseous environments. We find prominent optical tidal features in all three radio galaxies, which confirm previous claims that 3C 293, 3C 305, and 4C 12.50 have been involved in a recent galaxy merger or interaction. Our data show the complex morphology of the host galaxies and identify the companion galaxies that are likely involved in the merger or interaction. The radio sources appear to be (re-)triggered at a different stage of the merger; 4C 12.50 is a pre-coalescent and possibly multiple merger, 3C 293 is a post-coalescent merger that is undergoing a minor interaction with a close satellite galaxy, while 3C 305 appears to be shaped by an interaction with a gas-rich companion. For 3C 293 and 3C 305, we do not detect H i beyond the inner 30-45 kpc region, which shows that the bulk of the cold gas is concentrated within the host galaxy, rather than along the widespread tidal features.

  18. Assembly and maturation of the bacteriophage lambda procapsid: gpC is the viral protease.

    Science.gov (United States)

    Medina, Elizabeth; Wieczorek, Doug; Medina, Eva Margarita; Yang, Qin; Feiss, Michael; Catalano, Carlos Enrique

    2010-09-03

    Viral capsids are robust structures designed to protect the genome from environmental insults and deliver it to the host cell. The developmental pathway for complex double-stranded DNA viruses is generally conserved in the prokaryotic and eukaryotic groups and includes a genome packaging step where viral DNA is inserted into a pre-formed procapsid shell. The procapsids self-assemble from monomeric precursors to afford a mature icosahedron that contains a single "portal" structure at a unique vertex; the portal serves as the hole through which DNA enters the procapsid during particle assembly and exits during infection. Bacteriophage lambda has served as an ideal model system to study the development of the large double-stranded DNA viruses. Within this context, the lambda procapsid assembly pathway has been reported to be uniquely complex involving protein cross-linking and proteolytic maturation events. In this work, we identify and characterize the protease responsible for lambda procapsid maturation and present a structural model for a procapsid-bound protease dimer. The procapsid protease possesses autoproteolytic activity, it is required for degradation of the internal "scaffold" protein required for procapsid self-assembly, and it is responsible for proteolysis of the portal complex. Our data demonstrate that these proteolytic maturation events are not required for procapsid assembly or for DNA packaging into the structure, but that proteolysis is essential to late steps in particle assembly and/or in subsequent infection of a host cell. The data suggest that the lambda-like proteases and the herpesvirus-like proteases define two distinct viral protease folds that exhibit little sequence or structural homology but that provide identical functions in virus development. The data further indicate that procapsid assembly and maturation are strongly conserved in the prokaryotic and eukaryotic virus groups.

  19. Multifrequency observations of the superluminal quasar 3C 345

    Science.gov (United States)

    Bregman, J. N.; Glassgold, A. E.; Huggins, P. J.; Neugebauer, G.; Soifer, B. T.; Matthews, K.; Roellig, T. P. L.; Bregman, J. D.; Witteborn, F. C.; Lester, D. F.

    1986-01-01

    Attention is given to the continuum properties of the superluminal quasar 3C 345, on the basis of radio, optical, IR, and X-ray frequency monitorings, as well as by means of simultaneous multifrequency spectra extending from the radio through the X-ray bands. Radio outbursts, which appear to follow IR-optical outbursts by about one year, first occur at the highest frequencies, as expected from optical depth effects; the peak flux is nevertheless often reached at several frequencies at once. The beginning of outbursts, as defined by mm-measurements, corresponds to the appearance of the three known 'superluminal' components. An increase in the X-ray flux during 1979-1980 corresponds to increased radio flux, while the IR flux changes in the opposite sense.

  20. The Multifrequency Campaign on 3C 279 in January 2006

    CERN Document Server

    Collmar, W; Krichbaum, T P; Agudo, I; Bottacini, E; Bremer, M; Burwitz, V; Cuccchiara, A; Grupe, D; Gurwell, M

    2010-01-01

    We present the results of a multifrequency campaign from radio to hard X-ray energies on the blazar 3C 279 during an optical high-state in January 2006. We give the observational results (multifrequency light curves and spectra) and compile an SED. This complements an SED from an optical low-state in June 2003. Surprisingly the two SEDs differ only in their high-energy synchrotron emission (near-IR - UV), while the low-energy inverse-Compton emission (X- to Gamma-rays) remained unchanged. By interpreting with a steady-state leptonic emission model, the variability among the SED can be reproduced by a change solely of the low-energy cutoff of the relativistic electron distribution. In an internal shock model for blazar emission, such a change could e.g. achieved through a varying relative Lorentz factor of colliding shells producing internal shocks in the jet.

  1. An optical inverse-Compton hotspot in 3C196?

    CERN Document Server

    Hardcastle, M J

    2001-01-01

    Several hotspots of FRII radio sources have previously been detected in the X-ray at a flux level consistent with the X-rays being due to inverse-Compton scattering of radio synchrotron photons (`synchrotron self-Compton'), if the magnetic fields in the hotspots are close to their equipartition values. However, the number of hotspots compact and bright enough to exhibit detectable X-ray emission is small, so it is worth searching for synchrotron self-Compton emission in the optical, in spite of the obvious observational difficulties of such an approach. In this note I report on a possible detection of an optical inverse-Compton hotspot in a deep Hubble Space Telescope observation of the distant quasar 3C196, at a level which implies a hotspot magnetic field strength close to equipartition if the electrons have a low-energy cutoff around gamma ~ 500.

  2. An optical inverse-Compton hotspot in 3C 196?

    Science.gov (United States)

    Hardcastle, M. J.

    2001-07-01

    Several hotspots of FRII radio sources have previously been detected in the X-ray at a flux level consistent with the X-rays being due to inverse-Compton scattering of radio ``synchrotron photons synchrotron self-Compton'', if the magnetic fields in the hotspots are close to their equipartition values. However, the number of hotspots compact and bright enough to exhibit detectable X-ray emission is small, so it is worth searching for synchrotron self-Compton emission in the optical, in spite of the obvious observational difficulties of such an approach. In this note I report on a possible detection of an optical inverse-Compton hotspot in a deep Hubble Space Telescope observation of the distant quasar 3C 196, at a level which implies a hotspot magnetic field strength close to equipartition if the electrons have a low-energy cutoff around gamma ~ 500.

  3. 3C 84 Expanding Radio Lobe Revealed by VSOP Observations

    Science.gov (United States)

    Asada, K.; Kameno, S.; Shen, Z.-Q.; Shinji, H.; Gabuzda, D. C.; Inoue, M.

    2009-08-01

    We report the detection of the expansion and inner proper motions of a young radio lobe associated with the bright radio source 3C 84 in the Seyfert galaxy NGC 1275 using multi-epoch VSOP observation. The observed inner proper motions are consistent with the evolution scenario of classical double radio sources. The apparent expansion velocity is 0.50 ± 0.09 c, and the age of radio lobe is estimated to be 45.7 ± 8.9 years in 2001. The total flux density at 5 GHz increased at the end of the 1950's, with several peaks in the middle of the 1980's, and is in a decay phase now. The decay of total flux density can be naturally explained by an adiabatic cooling due to the expansion of the radio lobe, and previously measured spectral indices suggest that the emission comes from the surface of the radio lobe.

  4. Anisotropies in the HI gas distribution toward 3C 196

    Science.gov (United States)

    Kalberla, P. M. W.; Kerp, J.

    2016-10-01

    Context. The local Galactic Hi gas was found to contain cold neutral medium (CNM) filaments that are aligned with polarized dust emission. These filaments appear to be dominated by the magnetic field and in this case turbulence is expected to show distinct anisotropies. Aims: We use the Galactic Effelsberg-Bonn Hi Survey (EBHIS) to derive 2D turbulence spectra for the Hi distribution in direction to 3C 196 and two more comparison fields. Methods: Prior to Fourier transform we apply a rotational symmetric 50% Tukey window to apodize the data. We derive average as well as position angle dependent power spectra. Anisotropies in the power distribution are defined as the ratio of the spectral power in orthogonal directions. Results: We find strong anisotropies. For a narrow range in position angle, in direction perpendicular to the filaments and the magnetic field, the spectral power is on average more than an order of magnitude larger than parallel. In the most extreme case the anisotropy reaches locally a factor of 130. Anisotropies increase on average with spatial frequency as predicted by Goldreich & Sridhar (1995, ApJ, 438, 763), at the same time the Kolmogorov spectral index remains almost unchanged. The strongest anisotropies are observable for a narrow range in velocity and decay with a power law index close to -8/3, almost identical to the average isotropic spectral index of -2.9 <γ< -2.6. Conclusions: Hi filaments, associated with linear polarization structures in LOFAR observations in direction to 3C 196, show turbulence spectra with marked anisotropies. Decaying anisotropies appear to indicate that we witness an ongoing shock passing the Hi and affecting the observed Faraday depth.

  5. The Jet and Circumnuclear Environment of 3C 293

    Science.gov (United States)

    Floyd, David J. E.; Perlman, Eric; Leahy, J. Patrick; Beswick, R. J.; Jackson, Neal J.; Sparks, William B.; Axon, David J.; O'Dea, Christopher P.

    2006-03-01

    We present new HST NIR polarimetry, broadband and narrowband imaging, and MERLIN 4.5 GHz multifrequency synthesis radio imaging of 3C 293, a unique radio galaxy whose host is an obvious merger remnant, in an exceptionally underdense region of space. We have discovered NIR, optical, and UV synchrotron emission from the jet. In the optical, the jet is mostly obscured by a dust lane, but three knots are clear in our HST NICMOS images at 1.6 and 2.0 μm, clearly aligning with features in the radio. The outer jet knot is highly polarized (~15%) at 2 μm, confirming the synchrotron emission mechanism. The radio-IR spectral index steepens significantly with distance from the nucleus, as in 3C 273 and in contrast to M87. The inner knot is visible (with hindsight) on the WFPC2 and STIS images obtained for the earlier 3CR HST snapshot surveys. There is no [Fe II] emission seen associated with the jet, constraining the role of shock-induced ionization by the jet. Overall there is a strong implication that the NIR jet emission is indeed synchrotron. From our NIR images, the core of the galaxy is clearly identifiable with the main feature in the western extension of the radio ``jet'' image, although no unresolved AGN component is identifiable even at K band, consistent with an FR II-like nucleus obscured by an optically thick torus. The galaxy appears to have a single nucleus, with any multiple nuclei falling within the central PPARC in the UK.

  6. Protease Inhibitors from Plants with Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Yoonkyung Park

    2009-06-01

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

  7. ABT-378, a Highly Potent Inhibitor of the Human Immunodeficiency Virus Protease

    Science.gov (United States)

    Sham, Hing L.; Kempf, Dale J.; Molla, Akhteruzammen; Marsh, Kennan C.; Kumar, Gondi N.; Chen, Chih-Ming; Kati, Warren; Stewart, Kent; Lal, Ritu; Hsu, Ann; Betebenner, David; Korneyeva, Marina; Vasavanonda, Sudthida; McDonald, Edith; Saldivar, Ayda; Wideburg, Norm; Chen, Xiaoqi; Niu, Ping; Park, Chang; Jayanti, Venkata; Grabowski, Brian; Granneman, G. Richard; Sun, Eugene; Japour, Anthony J.; Leonard, John M.; Plattner, Jacob J.; Norbeck, Daniel W.

    1998-01-01

    The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 μM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, ≤0.06 μM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC50 in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS. PMID:9835517

  8. SCREENING OF PROTEASE ENZYME BY CONSTRUCTION OF METAGENOMIC LIBRARY FROM MARINE SOIL SEDIMENTS

    Directory of Open Access Journals (Sweden)

    R.PRABAVATHI

    2012-07-01

    Full Text Available Nonetheless, the cultivable microorganisms constituting these resources correspond to only a small fraction of the microbial diversity less than 1% of the microorganisms in various environments are readily cultivable (Amann et al., 1995. This limits the range of a search for new biocatalysts for the bioprocess industry, so the use of complex communities and the effort to overcome the problem of noncultivability attract not only scientific attention but also biotechnological innovation. Methods have been developed and used toovercome the non-cultivability of environmental microorganisms for biotechnology, namely cloning and the expression of metagenomes in suitable expression hosts. Proteases are present in all living forms as they are involved in various metabolic processes. They are mainly involved in hydrolysis of the peptide bonds (Gupta et al., 2002. Proteases find a wide range of applications in food, pharmaceutical, leather and textile, detergent, diagnostics industries and also in waste management. In order to discover new proteases from metagenomiclibraries, we screened for proteolytic activity from a constructed metagenomic library by direct cloning of environmental DNA of large DNA inserts. A novel gene encoding proteolytic enzyme was picked up,sequenced, expressed in E. coli and characterized. Several microbial proteases from the culturable organisms have been characterized. However, very few proteases have been identified through culture independent metagenomic approach.

  9. NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain

    Directory of Open Access Journals (Sweden)

    Adrian Velazquez-Campoy

    2013-06-01

    Full Text Available The nonstructural protein 3 (NS3 from the hepatitis C virus (HCV is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors, suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data.

  10. Cleavage entropy as quantitative measure of protease specificity.

    Science.gov (United States)

    Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Margreiter, Michael A; Spitzer, Gudrun M; Wallnoefer, Hannes G; Liedl, Klaus R

    2013-04-01

    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.

  11. 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.

  12. Insect response to plant defensive protease inhibitors.

    Science.gov (United States)

    Zhu-Salzman, Keyan; Zeng, Rensen

    2015-01-07

    Plant protease inhibitors (PIs) are natural plant defense proteins that inhibit proteases of invading insect herbivores. However, their anti-insect efficacy is determined not only by their potency toward a vulnerable insect system but also by the response of the insect to such a challenge. Through the long history of coevolution with their host plants, insects have developed sophisticated mechanisms to circumvent antinutritional effects of dietary challenges. Their response takes the form of changes in gene expression and the protein repertoire in cells lining the alimentary tract, the first line of defense. Research in insect digestive proteases has revealed the crucial roles they play in insect adaptation to plant PIs and has brought about a new appreciation of how phytophagous insects employ this group of molecules in both protein digestion and counterdefense. This review provides researchers in related fields an up-to-date summary of recent advances.

  13. 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.

  14. Lipase and protease extraction from activated sludge

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  15. Identification of chikungunya virus nsP2 protease inhibitors using structure-base approaches.

    Science.gov (United States)

    Nguyen, Phuong T V; Yu, Haibo; Keller, Paul A

    2015-04-01

    The nsP2 protease of chikungunya virus (CHIKV) is one of the essential components of viral replication and it plays a crucial role in the cleavage of polyprotein precursors for the viral replication process. Therefore, it is gaining attention as a potential drug design target against CHIKV. Based on the recently determined crystal structure of the nsP2 protease of CHIKV, this study identified potential inhibitors of the virus using structure-based approaches with a combination of molecular docking, virtual screening and molecular dynamics (MD) simulations. The top hit compounds from database searching, using the NCI Diversity Set II, with targeting at five potential binding sites of the nsP2 protease, were identified by blind dockings and focused dockings. These complexes were then subjected to MD simulations to investigate the stability and flexibility of the complexes and to gain a more detailed insight into the interactions between the compounds and the enzyme. The hydrogen bonds and hydrophobic contacts were characterized for the complexes. Through structural alignment, the catalytic residues Cys1013 and His1083 were identified in the N-terminal region of the nsP2 protease. The absolute binding free energies were estimated by the linear interaction energy approach and compared with the binding affinities predicted with docking. The results provide valuable information for the development of inhibitors for CHIKV.

  16. Activation of ADAM 12 protease by copper

    DEFF Research Database (Denmark)

    Loechel, F; Wewer, Ulla M.

    2001-01-01

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

  17. Hydrolysis of Fish Protein by Analkaline Protease

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Cod muscle protein was hydrolyzed by an alkaline protease in our study. The influences of hydrolysis temperature,fish protein concentration,and ratio of protease addition to protein amount on its degree of hy drolysis (DH) of protein were studied in details by applying dual quadratic rotary combinational design. The final results showed that more than 84% cod muscle protein could be hydrolyzed and recovered. Cod protein hydrolysate thus obtained had a balanced amino acid composition and mainly consisted of small peptides with molecule weight less than 6900 dalton.

  18. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang;

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending...... of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden....

  19. Serine Proteases an Ab Initio Molecular Dynamics Study

    CERN Document Server

    De Santis, L

    1999-01-01

    In serine proteases (SP's), the H-bond between His-57 and Asp-102, and that between Gly-193 and the transition state intermediate play a crucial role for enzymatic function. To shed light on the nature of these interactions, we have carried out ab initio molecular dynamics simulations on complexes representing adducts between the reaction intermediate and elastase (one protein belonging to the SP family). Our calculations indicate the presence of a low--barrier H-bond between His-57 and Asp-102, in complete agreement with NMR experiments on enzyme--transition state analog complexes. Comparison with an ab initio molecular dynamics simulation on a model of the substrate--enzyme adduct indicates that the Gly-193--induced strong stabilization of the intermediate is accomplished by charge/dipole interactions and not by H-bonding as previously suggested. Inclusion of the protein electric field in the calculations does not affect significantly the charge distribution.

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

    Science.gov (United States)

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

    2013-01-29

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

  1. 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....

  2. 3C 273 with NuSTAR: Unveiling the AGN

    CERN Document Server

    Madsen, Kristin K; Walton, Dominic J; Harrison, Fiona A; Ballantyne, David R; Boggs, Steve E; Brenneman, Laura W; Christensen, Finn E; Craig, William W; Fabian, Andrew C; Forster, Karl; Grefenstette, Brian W; Guainazzi, Matteo; Hailey, Charles J; Madejski, Greg M; Matt, Giorgio; Stern, Daniel; Zhang, William W

    2015-01-01

    We present results from a 244 ks $NuSTAR$ observation of 3C 273, obtained during a cross-calibration campaign with the $Chandra$, $INTEGRAL$, $Suzaku$, $Swift$, and $XMM-Newton$ observatories. We show that the spectrum, when fit with a power-law model using data from all observatories except $INTEGRAL$ over the 1-78 keV band, leaves significant residuals in the $NuSTAR$ data between 30-78 keV. The $NuSTAR$ 3-78 keV spectrum is well-described by an exponentially cutoff power-law ($\\Gamma = 1.646 \\pm 0.006$, E$_\\mathrm{cutoff} = 202_{-34}^{+51}$ keV) with a weak reflection component from cold, dense material. There is also evidence for a weak ($EW = 23 \\pm 11$ eV) neutral iron line. We interpret these features as arising from coronal emission plus reflection off an accretion disk or distant material. Beyond 80 keV $INTEGRAL$ data show clear excess flux relative to an extrapolation of the AGN model fit to $NuSTAR$. This high-energy power-law is consistent with the presence of a beamed jet, which begins to domina...

  3. Spectroscopic monitoring of the Blazar 3C 454.3

    CERN Document Server

    Benítez, E; Raiteri, C M; Villata, M; Dultzin, D; Martínez, O; Perez-Camargo, B; Torrealba, J

    2009-01-01

    We performed an optical spectroscopic monitoring of the blazar 3C 454.3 from September 2003 to July 2008. Sixteen optical spectra were obtained during different runs, which constitute the first spectroscopic monitoring done in the rest-frame UV region (z=0.859). An overall flux variation of the MgII (2800 A) by a factor ~ 3 was observed, while the corresponding UV continuum (F_cont at 3000 A) changed by a factor ~ 14. The MgII emission lines respond proportionally to the continuum variations when the source is in a low-activity state. In contrast, near the optical outbursts detected in 2005 and 2007, the MgII emission lines showed little response to the continuum flux variations. During the monitored period the UV FeII flux changed by a factor ~ 6 and correlated with F_cont (r = 0.92). A negative correlation between EW(Mg II) and F_cont was found, i.e. the so-called "Intrinsic Baldwin Effect".

  4. Integral field spectroscopy of the radio galaxy 3C 171

    CERN Document Server

    Márquez, I; Durret, F; Petitjean, P

    2000-01-01

    We have performed integral field spectroscopy of the radio galaxy 3C 171 (redshift z=0.238) with the TIGER instrument at the Canada France Hawaii telescope in the Hbeta-[OIII]4959-5007 wavelength region. We present the reconstructed Hbeta and [OIII] images and compare them to the HST and radio maps. We discuss the variations of the [OIII]/Hbeta line ratio throughout the nebulosity. We also analyze the velocity field in detail, in particular the presence of several components. We find that the kinematics derived with emission lines in the central region (inside 1 arcsec) are compatible with a disk-like rotation of low amplitude (50 km/s). The continuum surface brightness profile follows an r^{1/4} law, suggesting that the underlying galaxy is an elliptical with an effective radius of 15 kpc. We have fit two components in the region centered 2.7 arcsec to the West and of extension 3 arcsec^2. We find that the blueshifted component is an extension of the central part, whereas the second one is redshifted by 600 ...

  5. Suzaku observation of the giant radio galaxy 3C 326

    CERN Document Server

    Isobe, Naoki; Gandhi, Poshak; Hayato, Asami; Nagai, Hiroshi; Hada, Kazuhiro; Seta, Hiromi; Matsuta, Keiko

    2009-01-01

    A Suzaku observation of a giant radio galaxy, 3C 326, which has a physical size of about 2 Mpc, was conducted on 2008 January 19 -- 21. In addition to several X-ray sources, diffuse emission was significantly detected associated with its west lobe, but the east lobe was contaminated by an unidentified X-ray source WARP J1552.4+2007. After careful evaluation of the X-ray and Non X-ray background, the 0.4 -- 7 keV X-ray spectrum of the west lobe is described by a power-law model. The photon index and 1 keV flux density was derived as $1.82_{-0.24}^{+0.26}\\pm0.04$ and $19.4_{-3.2}^{+3.3}\\pm 3.0$ nJy, respectively, where the first and second errors represent the statistical and systematic ones. The diffuse X-rays were attributed to be inverse Compton radiation by the synchrotron radio electrons scattering off the cosmic microwave background photons. This radio galaxy is the largest among those with lobes detected through inverse Compton X-ray emission. A comparison of the radio to X-ray fluxes yields the energy d...

  6. 3C 66A: Variability in 2007-2015

    Science.gov (United States)

    Hagen-Thorn, V. A.; Morozova, D. A.; Arkharov, A. A.; Hagen-Thorn, E. I.; Troitsky, I. S.; Troitskaya, Yu. V.; Milanova, Yu. V.; Volkov, E. V.; Takalo, L. O.; Sillanpää, A.

    2017-06-01

    The results of photometric ( BV RIJHK) and polarimetric ( R)monitoring of the blazar 3C 66A performed at the St. Petersburg State University and the Central AstronomicalObservatory of the Russian Academy of Sciences in 2007-2015, radio observations performed by the Boston University team with the Very Long Baseline Array at 43 GHz, and a gamma-ray light curve based on observations with the Fermi SpaceObservatory are presented. Color variations of the object are studied. Changes in the optical spectral energy distribution are observed at some times, indicating the appearance and disappearance of individual variable sources. A variable source with a degree of polarization of 36% is identified, which is responsible for the polarization variations observed during one episode. The correlations between the variations in the different spectral ranges indicate that the optical and gamma-ray radiation originates near the radio core detected at 43 GHz. The presence of five superluminal components emerging from the core is detected.

  7. Anisotropies in the HI gas distribution toward 3C196

    CERN Document Server

    Kalberla, P M W

    2016-01-01

    The local Galactic HI gas was found to contain cold neutral medium (CNM) filaments that are aligned with polarized dust emission. These filaments appear to be dominated by the magnetic field and in this case turbulence is expected to show distinct anisotropies. We use the Galactic Effelsberg--Bonn HI Survey (EBHIS) to derive 2D turbulence spectra for the HI distribution in direction to 3C196 and two more comparison fields. Prior to Fourier transform we apply a rotational symmetric 50% Tukey window to apodize the data. We derive average as well as position angle dependent power spectra. Anisotropies in the power distribution are defined as the ratio of the spectral power in orthogonal directions. We find strong anisotropies. For a narrow range in position angle, in direction perpendicular to the filaments and the magnetic field, the spectral power is on average more than an order of magnitude larger than parallel. In the most extreme case the anisotropy reaches locally a factor of 130. Anisotropies increase on...

  8. Structure-Guided Design and Optimization of Dipeptidyl Inhibitors of Norovirus 3CL Protease. Structure-Activity Relationships and Biochemical, X-ray Crystallographic, Cell-Based, and In Vivo Studies

    Science.gov (United States)

    Kankanamalage, Anushka C. Galasiti; Kim, Yunjeong; Weerawarna, Pathum M.; Uy, Roxanne Adeline Z.; Damalanka, Vishnu C.; Mandadapu, Sivakoteswara Rao; Alliston, Kevin R.; Mehzabeen, Nurjahan; Battaile, Kevin P.; Lovell, Scott; Chang, Kyeong-Ok; Groutas, William C.

    2015-01-01

    Norovirus infection constitutes the primary cause of acute viral gastroenteritis. There are currently no vaccines or norovirus-specific antiviral therapeutics available for the management of norovirus infection. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of anti-norovirus therapeutics. We describe herein the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrate herein in vivo efficacy of an inhibitor using the murine model of norovirus infection. PMID:25761614

  9. A protease storm cleaves a cell-cell adhesion molecule in cancer: multiple proteases converge to regulate PTPmu in glioma cells.

    Science.gov (United States)

    Phillips-Mason, Polly J; Craig, Sonya E L; Brady-Kalnay, Susann M

    2014-09-01

    Cleavage of the cell-cell adhesion molecule, PTPµ, occurs in human glioblastoma multiforme brain tumor tissue and glioma cell lines. PTPµ cleavage is linked to increased cell motility and growth factor independent survival of glioma cells in vitro. Previously, PTPµ was shown to be cleaved by furin in the endoplasmic reticulum to generate membrane associated E- (extracellular) and P- (phosphatase) subunits, and by ADAMs and the gamma secretase complex at the plasma membrane. We also identified the presence of additional extracellular and intracellular PTPµ fragments in brain tumors. We set out to biochemically analyze PTPµ cleavage in cancer cells. We determined that, in addition to the furin-processed form of PTPµ, a pool of 200 kDa full-length PTPµ exists at the plasma membrane that is cleaved directly by ADAM to generate a larger shed form of the PTPµ extracellular segment. Notably, in glioma cells, full-length PTPµ is also subject to calpain cleavage, which generates novel PTPµ fragments not found in other immortalized cells. We also observed glycosylation and phosphorylation differences in the cancer cells. Our data suggest that an additional serine protease also contributes to PTPµ shedding in glioma cells. We hypothesize that a "protease storm" occurs in cancer cells whereby multiple proteases converge to reduce the presence of cell-cell adhesion molecules at the plasma membrane and to generate protein fragments with unique biological functions. As a consequence, the "protease storm" could promote the migration and invasion of tumor cells.

  10. Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes.

    Science.gov (United States)

    Govind, Chhabi K; Qiu, Hongfang; Ginsburg, Daniel S; Ruan, Chun; Hofmeyer, Kimberly; Hu, Cuihua; Swaminathan, Venkatesh; Workman, Jerry L; Li, Bing; Hinnebusch, Alan G

    2010-07-30

    Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are cotranscriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their cotranscriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing cotranscriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is important for nucleosome eviction. Copyright 2010 Elsevier Inc. All rights reserved.

  11. 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...... for the full length protease...

  12. The effect of high glucose levels on the hypermethylation of protein phosphatase 1 regulatory subunit 3C (PPP1R3C) gene in colorectal cancer

    Indian Academy of Sciences (India)

    Soo Kyung Lee; Ji Wook Moon; Yong Woo Lee; Jung Ok Lee; Su Jin Kim; Nami Kim; Jin Kim; Hyeon Soo Kim; Sun-Hwa Park

    2015-03-01

    DNA methylation is an epigenetic event that occurs frequently in colorectal cancer (CRC). Increased glucose level is a strong risk factor for CRC. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) modulates glycogen metabolism, particularly glycogen synthesis. The aim of this study was to investigate the effect of high glucose levels on DNA methylation of PPP1R3C in CRC. PPP1R3C was significantly hypermethylated in CRC tissues (76/105, 72.38%, < 0.05) and colon cancer cell lines ( < 0.05). CRC tissues obtained from patients with high glucose levels showed that the methylation of PPP1R3C was lower than in patients who had normal levels of glucose. When DLD-1 cells were cultured under conditions of high glucose, the methylation of PPP1R3C was repressed. The expression of PPP1R3C was inversely related to methylation status. In addition, a promoter luciferase assay showed that the transcriptional activity of PPP1R3C was increased in high glucose culture conditions. The number of cells decreased when PPP1R3C was silenced in DLD-1 cells. These results suggest that PPP1R3C, a novel hypermethylated gene in CRC, may play a critical role in cancer cell growth in association with glucose levels.

  13. (Benzyldiphenylphosphine-3κP[μ-bis(diphenylarsinomethane-1:2κ2As:As′]nonacarbonyl-1κ3C,2κ3C,3κ3C-triangulo-triruthenium(0

    Directory of Open Access Journals (Sweden)

    Omar bin Shawkataly

    2010-01-01

    Full Text Available The asymmetric unit of the title triangulo-triruthenium compound, [Ru3(C25H22As2(C19H17P(CO9], consists of two crystallographically independent molecules of the triangulo-triruthenium complex, A and B. The bis(diphenylarsinomethane ligand bridges an Ru—Ru bond and the monodentate phosphine ligand bonds to the third Ru atom. Both the phosphine and arsine ligands are equatorial with respect to the Ru3 triangle. In addition, each Ru atom carries one equatorial and two axial terminal carbonyl ligands. With regard to the three phosphine-substituted rings, the benzyl ring makes dihedral angles of 41.0 (3 and 43.9 (3° with the other two benzene rings in molecule A; these angles are 49.8 (3 and 56.8 (3° in molecule B. The dihedral angles between the two benzene rings are 76.1 (3 and 88.2 (3° for the two diphenylarsino groups in molecule A and 71.3 (3 and 78.1 (3° in molecule B. In the crystal packing, molecules are linked into chains via intermolecular C—H...O hydrogen bonds. Weak intermolecular C—H...π interactions further stabilize the crystal structure.

  14. [μ-Bis(diphenylarsinomethane-1:2κ2As:As′]nonacarbonyl-1κ3C,2κ3C,3κ3C-[(pentafluorophenyldiphenylphosphine-3κP]-triangulo-triruthenium(0 chloroform monosolvate

    Directory of Open Access Journals (Sweden)

    Omar bin Shawkataly

    2010-02-01

    Full Text Available The asymmetric unit of the title triangulo-triruthenium compound, [Ru3(C25H22As2(C18H10F5P(CO9]·CHCl3, contains one molecule of the triangulo-triruthenium complex and one molecule of the disordered chloroform solvent. The bis(diphenylarsinomethane ligand bridges an Ru—Ru bond and the monodentate phosphine ligand bonds to the third Ru atom. Both the arsine and phosphine ligands are equatorial with respect to the Ru3 triangle. In addition, each Ru atom carries one equatorial and two axial terminal carbonyl ligands. The phosphine-substituted benzene rings make dihedral angles of 68.43 (15, 65.14 (14 and 89.75 (14° with each other. The dihedral angles between the two benzene rings are 80.70 (15 and 84.53 (16° for the two diphenylarsino groups. In the crystal packing, the molecules are linked into a plane parallel to bc by intermolecular C—H...O and C—H...F hydrogen bonds. Weak intermolecular C—H...π interactions further stabilize the crystal structure.

  15. 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 th

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

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... 2School of Biological and Chemical Engineering, Zhejiang University ... factorial central composite design (CCD) was chosen to explain the combined ... obtained by the statistical analysis showed that corn starch at 19.8 g/l, ... the optimization process. .... would inhibited the thermostable protease synthesis.

  17. Proteases, neutrophils, and periodontitis: the NET effect.

    Science.gov (United States)

    Nauseef, William M

    2014-10-01

    Neutrophils exert potent antimicrobial activities in their role as first-line cellular defenders against infection. The synergistic and collective actions of oxidants and granule proteins, including serine proteases, support the microbial killing in phagosomes, where most neutrophil-mediated antimicrobial action occurs. In addition to phagocytosis, specific stimuli prompt neutrophils to extrude a matrix of DNA, histones, and granule proteins to produce neutrophil extracellular traps (NETs), which can trap microbes. Mice lacking the serine proteases necessary for NET production are more susceptible to infection, an observation suggesting that functional NETs are required for host protection. In this issue of the JCI, Sørensen and colleagues characterize neutrophils from a patient with Papillon-Lefèvre syndrome. The patient has an inactivating mutation in the gene encoding dipeptidyl peptidase I, resulting in neutrophils lacking elastase, a serine protease required for NET production. Despite the inability to form NETS, neutrophils from this patient killed pathogens in vitro, and the patient did not exhibit evidence of an increased propensity toward bacterial infections. Together, these results suggest that proteases in human neutrophils are dispensable for protection against bacterial infection and that the ability to generate NETs in vitro does not compromise host defense.

  18. Bacterial proteases: targets for diagnostics and therapy

    NARCIS (Netherlands)

    Kaman, W.E.; Hays, J.P.; Endtz, H.P.; Bikker, F.J.

    2014-01-01

    Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and po

  19. Multiwavelength Observations of 3C66A in 2003

    Science.gov (United States)

    Boettcher, M.; Joshi, M.; Fossati, G.; Smith, I. A.; Mukherjee, R.; Bramel, D.; Cui, W.; WEBT Collaboration

    2004-08-01

    The radio-selected BL Lac object 3C66A was the target of an intensive multiwavelength observing campaign in the last quarter of 2003 and early 2004. It was monitored by the Whole Earth Blazar Telescope (WEBT) collaboration of optical observers, in tandem with 20 X-ray monitoring observations by the Rossi X-Ray Timing Explorer (RXTE), VHE gamma-ray observations by STACEE and VERITAS, and long-term monitoring at radio frequencies. In addition, 9 high-spatial-resolution observations using the VLA are being carried out during the campaign and throughout the year 2004 to follow possible structural changes of the source. A gradual brightening of the source over the course of the campaign was observed at all optical frequencies, culminating in a very bright flare at the end of January 2004. Optical light curves indicate intraday microvariability on time scales down to about 1.3 hours. No significant color-magnitude correlation for the entire data set was evident, but there is a slight indication of a hardness - intensity anti-correlation on intraday time scales. The X-ray spectrum is consistent with a power-law with a photon spectral index of ˜ 2.1, indicating that the RXTE energy band might be located right at the intersection of the synchrotron and the high-energy emission components of the broadband spectral energy distribution. No significant flux or spectral variability at X-ray energies was detected. We extracted snapshot spectral energy distributions at various times throughout the campaign, and present first spectral fits to those SEDs. This work was partially supported by NASA RXTE GO grant no. NNG 04GB13G.

  20. Observations of Titan 3C-4 Transtage Fragmentation Debris

    Science.gov (United States)

    Cowardin, Heather; Seitzer, P.; Abercromby, K.; Barker, E.; Cardona, T.; Krisko, P.; Lederer, S.

    2013-01-01

    The fragmentation of a Titan 3C-4 Transtage (1968-081) on 21 February 1992 is one of only two known break-ups in or near geosynchronous orbit. The original rocket body and 24 pieces of debris are currently being tracked by the US Space Surveillance Network (SSN). The rocket body (SSN# 3432) and several of the original fragments (SSN# 25000, 25001, 30000, and 33511) were observed in survey mode during 2004-2010 using the 0.6-m Michigan Orbital DEbris Survey Telescope (MODEST) in Chile using a broad R filter. This paper will present a size distribution for all calibrated magnitude data acquired on MODEST. Size distribution plots will also be shown using historical models for small fragmentation debris (down to 10 cm) believed to be associated with the Titan break-up. In November 2010, visible broadband photometry (Johnson/Kron-Cousins BVRI) was acquired with the 0.9-m Small and Moderate Aperture Research Telescope System (SMARTS) at the Cerro Tololo Inter-American Observatory (CTIO) in Chile on several Titan fragments (SSN# 25001, 33509, 33510) and the parent rocket body. Color index data will be used to determine the fragment brightness distribution and how the data compares to spacecraft materials measured in the laboratory using similar photometric measurement techniques. In 2012, the SSN added 16 additional fragments to the catalogue. MODEST acquired magnitude data on ten Titan fragments in late 2012 and early 2013. The magnitude distribution of all the observed fragments are analyzed as a function of time. In order to better characterize the breakup fragments spectral measurements were acquired on the original rocket body and five Titan fragments using the 6.5-m Magellan telescopes at Las Campanas Observatory in Chile. The telescopic spectra are compared with laboratory acquired spectra of materials (e.g., Aluminum and various paints) and categorized based on known absorption features for spacecraft materials.

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

    Science.gov (United States)

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

    2014-10-24

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

  2. Arabidopsis AtSerpin1, Crystal Structure and in Vivo Interaction with Its Target Protease RESPONSIVE TO DESICCATION-21 (RD21)

    Energy Technology Data Exchange (ETDEWEB)

    Lampl, Nardy; Budai-Hadrian, Ofra; Davydov, Olga; Joss, Tom V.; Harrop, Stephen J.; Curmi, Paul M.G.; Roberts, Thomas H.; Fluhr, Robert (WIS-I); (Macquarie); (New South)

    2010-05-25

    In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 {angstrom}. The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a full-length and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor l-trans-epoxysuccinyl-l-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the l-trans-epoxysuccinyl-l-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.

  3. Damage buildup in Ar-ion-irradiated 3C-SiC at elevated temperatures

    Science.gov (United States)

    Wallace, J. B.; Bayu Aji, L. B.; Li, T. T.; Shao, L.; Kucheyev, S. O.

    2015-09-01

    Above room temperature, the accumulation of radiation damage in 3C-SiC is strongly influenced by dynamic defect interaction processes and remains poorly understood. Here, we use a combination of ion channeling and transmission electron microscopy to study lattice disorder in 3C-SiC irradiated with 500 keV Ar ions in the temperature range of 25-250 °C. Results reveal sigmoidal damage buildup for all the temperatures studied. For 150 °C and below, the damage level monotonically increases with ion dose up to amorphization. Starting at 200 °C, the shape of damage-depth profiles becomes anomalous, with the damage peak narrowing and moving to larger depths and an additional shoulder forming close to the ion end of range. As a result, damage buildup curves for 200 and 250 °C exhibit an anomalous two-step shape, with a damage saturation stage followed by rapid amorphization above a critical ion dose, suggesting a nucleation-limited amorphization behavior. Despite their complexity, all damage buildup curves are well described by a phenomenological model based on an assumption of a linear dependence of the effective amorphization cross section on ion dose. In contrast to the results of previous studies, 3C-SiC can be amorphized by bombardment with 500 keV Ar ions even at 250 °C with a relatively large dose rate of ˜2 ×1013 cm-2 s-1, revealing a dominant role of defect interaction dynamics at elevated temperatures.

  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. Investigating the X-ray and Gamma-ray Properties of the Galactic Supernova Remnants Kes 69, 3C 396, 3C 400.2

    CERN Document Server

    Ergin, Tülün; Yamazaki, Ryo

    2016-01-01

    Kes 69, 3C 396, and 3C 400.2 are mixed-morphology (MM) Galactic supernova remnants (SNRs), where Kes 69 and 3C 396 are interacting with molecular clouds (MCs). Previous X-ray studies showed that the emission from these SNRs is thermal. It has been suggested that MM SNRs interacting with MCs are potential candidates for recombining plasma (RP) in X-rays and hadronic gamma-ray emission. Recently, Chandra observations revealed signs of RP in 3C 400.2. Our preliminary analyses show that the X-ray emission of NW and SE region of 3C 400.2 arises from recombining plasma. We detected GeV gamma-ray emission from Kes 69 and 3C 396 above 5$\\sigma$.

  6. Structure and Properties of the Nonface-Spiral Fullerenes T-C380, D3-C384, D3-C440, and D3-C672 and Their Halma and Leapfrog Transforms

    DEFF Research Database (Denmark)

    Wirz, Lukas; Tonner, Ralf; Avery, James Emil

    2013-01-01

    followed by a force-field optimization using the recently introduced program Fullerene. The obtained structures were then further optimized at the density functional level of theory and their stability analyzed with reference to Ih-C60. The large number of hexagons results in a higher stability of the NS......The structure and properties of the three smallest nonface-spiral (NS) fullerenes NS-T-C380, NS-D3-C384, NS-D3-C440, and the first isolated pentagon NS-fullerene, NS-D3-C672, are investigated in detail. They are constructed by either a generalized face-spiral algorithm or by vertex insertions...... of NS-fullerenes. A similar result was found to not hold for the related leapfrog transformation. We also show that the first known NS-fullerene with isolated pentagons, NS-D3-C672, is a halma transform of D3-C168....

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

    Directory of Open Access Journals (Sweden)

    Nathalie Dautin

    2010-05-01

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

  8. Mastering the canonical loop of serine protease inhibitors: enhancing potency by optimising the internal hydrogen bond network.

    Directory of Open Access Journals (Sweden)

    Joakim E Swedberg

    Full Text Available BACKGROUND: Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s. The smallest of these is sunflower trypsin inhibitor (SFTI-1, a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4, a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn(14 which displayed both a 125-fold increased capacity to inhibit KLK4 (K(i = 0.0386±0.0060 nM and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn(14 was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion. CONCLUSION/SIGNIFICANCE: These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.

  9. Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

    Science.gov (United States)

    Cusick, John K; Hager, Elizabeth; Gill, Ronald E

    2015-01-01

    The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program.

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

    Directory of Open Access Journals (Sweden)

    Shinong Long

    2010-01-01

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

  11. ADAM-9 is an insulin-like growth factor binding protein-5 protease produced and secreted by human osteoblasts.

    Science.gov (United States)

    Mohan, Subburaman; Thompson, Garrett R; Amaar, Yousef G; Hathaway, Gary; Tschesche, Harald; Baylink, David J

    2002-12-24

    IGF binding protein-5 (BP-5) is an important bone formation regulator. Therefore, elucidation of the identity of IGF binding protein-5 (BP-5) protease produced by osteoblasts is important for our understanding of the molecular pathways that control the action of BP-5. In this regard, BP-5 protease purified by various chromatographic steps from a conditioned medium of U2 human osteosarcoma cells migrated as a single major band, which comigrated with the protease activity in native PAGE and yielded multiple bands in SDS-PAGE under reducing conditions. N-Terminal sequencing of these bands revealed that three of the bands yielded amino acid sequences that were identical to that of alpha2 macroglobulin (alpha2M). Although alpha2M was produced by human osteoblasts (OBs), it was not found to be a BP-5 protease. Because alpha2M had been shown to complex with ADAM proteases and because ADAM-12 was found to cleave BP-3 and BP-5, we evaluated if one of the members of ADAM family was the BP-5 protease. On the basis of the findings that (1) purified preparations of BP-5 protease from U2 cell CM contained ADAM-9, (2) ADAM-9 is produced and secreted in high abundance by various human OB cell types, (3) purified ADAM-9 cleaved BP-5 effectively while it did not cleave other IGFBPs or did so with less potency, and (4) purified ADAM-9 bound to alpha2M, we conclude that ADAM-9 is a BP-5 protease produced by human OBs.

  12. Comparative Study on the Protease Inhibitors from Fish Eggs

    Institute of Scientific and Technical Information of China (English)

    Ustadi; K.Y.Kim; S.M.Kim

    2005-01-01

    The protease inhibitor was purified from five different fish eggs. The molecular weights of Pacific herring, chum salmon, pond smelt, glassfish, and Alaska pollock egg protease inhibitors were 120, 89, 84.5, 17, and 16.8kDa, respectively. The specific inhibitory activity of glassfish egg protease inhibitor was the highest followed by those of Pacific herring and Alaska pollock in order. The specific inhibitory activity and purity of glassfish egg protease inhibitor were 19.70 U mg-1 protein and 164.70 folds of purification, respectively. Glassfish egg protease inhibitor was reasonably stable at 50 - 65 ℃ and pH 8,which was more stable at high temperature and pH than protease inhibitors from the other fish species. Glassfish egg protease inhibitor was noncompetitive with inhibitor constant (Ki) of 4.44 nmol L-1.

  13. Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD.

    Directory of Open Access Journals (Sweden)

    Lin Qu

    2011-09-01

    Full Text Available Toll-like receptor 3 (TLR3 and cytosolic RIG-I-like helicases (RIG-I and MDA5 sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF, and mitochondrial antiviral signaling protein (MAVS, respectively. Previously, we demonstrated that hepatitis A virus (HAV, a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro, that is derived by auto-processing of the P3 (3ABCD segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C-stimulated dimerization of IFN regulatory factor 3 (IRF-3, IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro and downstream 3D(pol sequence, but not 3D(pol polymerase activity. Cleavage occurs at two non-canonical 3C(pro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol sequence modulates the substrate specificity of the upstream 3C(pro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

  14. Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD.

    Directory of Open Access Journals (Sweden)

    Lin Qu

    2011-09-01

    Full Text Available Toll-like receptor 3 (TLR3 and cytosolic RIG-I-like helicases (RIG-I and MDA5 sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF, and mitochondrial antiviral signaling protein (MAVS, respectively. Previously, we demonstrated that hepatitis A virus (HAV, a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro, that is derived by auto-processing of the P3 (3ABCD segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C-stimulated dimerization of IFN regulatory factor 3 (IRF-3, IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro and downstream 3D(pol sequence, but not 3D(pol polymerase activity. Cleavage occurs at two non-canonical 3C(pro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol sequence modulates the substrate specificity of the upstream 3C(pro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

  15. mm-VLBI Observations of the Active Galaxy 3C 111 in Outburst

    CERN Document Server

    Schulz, Robert; Ros, Eduardo; Krichbaum, Thomas P; Großberger, Christoph; Müller, Cornelia; Mannheim, Karl; Agudo, Iván; Aller, Hugh D; Aller, Margo F

    2013-01-01

    The broad-line radio galaxy 3C 111 exhibited a major flux density outburst in 2007. Here, we present imaging and preliminary kinematic results of the jet, based on three millimetre-VLBI observations at 86 GHz using the Global Millimeter VLBI Array (GMVA) covering one year just after the radio flare. The GMVA data allow us to study this outburst with unprecedented image fidelity at highest (sub-parsec) resolution. On these scales, the outburst is resolved into a complex series of plasma components forming an intriguing bent structure. Within 1 mas from the jet base, ejections vary in position angle and components move with an apparent velocity of ~3.7 c, significantly slower than the maximum velocity observed with cm-VLBI on scales beyond 1 mas.

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

    Science.gov (United States)

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

    2015-07-01

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

  17. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    Energy Technology Data Exchange (ETDEWEB)

    Yedidi, Ravikiran S. [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Muhuhi, Joseck M. [Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); Liu, Zhigang [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Bencze, Krisztina Z. [Department of Chemistry, Fort Hays State University, Hays, KS 67601 (United States); Koupparis, Kyriacos [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); O’Connor, Carrie E.; Kovari, Iulia A. [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Spaller, Mark R. [Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); Kovari, Ladislau C., E-mail: kovari@med.wayne.edu [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States)

    2013-09-06

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

  18. Synthesis and biological evaluation of nucleobase-modified analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd)

    DEFF Research Database (Denmark)

    Hrdlicka, Patrick J; Jepsen, Jan S; Nielsen, Claus

    2005-01-01

    A series of nucleobase-modified analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd) were designed to overcome the strict substrate specificity of the activating uridine-cytidine kinase. EUrd, ECyd and target nucleosides were obtained using a short conver...

  19. Multi-Frequency VLBA Studies of the Parsec-Scale Jets in 3C 66A and 3C 66B

    Indian Academy of Sciences (India)

    G.-Y. Zhao; Y.-J. Chen; Z.-Q. Shen; H. Sudou; S. Iguchi; F. Gao; Y. Murata; Y. Taniguchi

    2014-09-01

    We report multi-frequency VLBA phase-referencing observation results of 3C 66A and 3C 66B, including high resolution maps and relative position measurements. The resulting images show similar morphology with that presented in previous works. We find core shift variations in both sources, indicating some physical condition changes in the jets.

  20. A Two-Dimensional Zirconium Carbide by Selective Etching of Al3C3 from Nanolaminated Zr3Al3C5.

    Science.gov (United States)

    Zhou, Jie; Zha, Xianhu; Chen, Fan Y; Ye, Qun; Eklund, Per; Du, Shiyu; Huang, Qing

    2016-04-11

    The room-temperature synthesis of a new two-dimensional (2D) zirconium-containing carbide, Zr3C2T(z) MXene is presented. In contrast to traditional preparation of MXene, the layered ternary Zr3Al3C5 material instead of MAX phases is used as source under hydrofluoric acid treatment. The structural, mechanical, and electronic properties of the synthesized 2D carbide are investigated, combined with first-principles density functional calculations. A comparative study on the structrual stability of our obtained 2D Zr3C2T(z) and Ti3C2T(z) MXenes at elevated temperatures is performed. The obtained 2D Zr3C2T(z) exhibits relatively better ability to maintain 2D nature and strucural integrity compared to Ti-based Mxene. The difference in structural stability under high temperature condition is explained by a theoretical investigation on binding energy.

  1. Cellular Proteases as Cancer Biomarkers: A Review

    Directory of Open Access Journals (Sweden)

    Sarah R. Röthlisberger

    2010-12-01

    Full Text Available Over the past few decades a variety of biomolecules have been proposed as diagnostic biomarkers and predictors of severity for transmissible and nontransmissible diseases. Studies in a range of cancers have revealed many biomarkers with great potential in cancer diagnosis, in establishing tumor stage, progression, and response to therapies; such as the Kallikrein and Metalloproteinase families. Traditionally blood (serum and tissue have been the main biological sources of biomarker discovery, but in the past decade urine has emerged as a promising source of cancer biomarkers. In this review we will focus on two large families, the Kallikrein family of serine proteases discovered in serum, and the Metalloproteinase family of zinc proteases discovered in urine, as potential cancer biomarkers.

  2. 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......, the disulfide bridge was replaced with amide bonds of various lengths. The novel peptides did not retain their inhibitory activity, but formed the basis for another strategy. Second, bicyclic peptides were obtained by creating head-to-tail cyclized peptides that were made bicyclic by the addition of a covalent...... increased. Finally, the effect of multivalent display of upain-2 was investigated. Several dimers of upain-2 were made and the attachment of upain-2 via the Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) onto an alkyne functionalized carbohydrate scaffold was investigated. Besides the synthesis...

  3. 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.

  4. 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.

  5. 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

  6. Evolution of cyclic peptide protease inhibitors.

    Science.gov (United States)

    Young, Travis S; Young, Douglas D; Ahmad, Insha; Louis, John M; Benkovic, Stephen J; Schultz, Peter G

    2011-07-05

    We report a bacterial system for the evolution of cyclic peptides that makes use of an expanded set of amino acid building blocks. Orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pairs, together with a split intein system were used to biosynthesize a library of ribosomal peptides containing amino acids with unique structures and reactivities. This peptide library was subsequently used to evolve an inhibitor of HIV protease using a selection based on cellular viability. Two of three cyclic peptides isolated after two rounds of selection contained the keto amino acid p-benzoylphenylalanine (pBzF). The most potent peptide (G12: GIXVSL; X=pBzF) inhibited HIV protease through the formation of a covalent Schiff base adduct of the pBzF residue with the ε-amino group of Lys 14 on the protease. This result suggests that an expanded genetic code can confer an evolutionary advantage in response to selective pressure. Moreover, the combination of natural evolutionary processes with chemically biased building blocks provides another strategy for the generation of biologically active peptides using microbial systems.

  7. 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.

  8. 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.

  9. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    M El Bakkouri; A Pow; A Mulichak; K Cheung; J Artz; M Amani; S Fell; T de Koning-Ward; C Goodman; et al.

    2011-12-31

    The Clpchaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clpchaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  10. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  11. 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.

  12. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals

    Directory of Open Access Journals (Sweden)

    Folasade M. Olajuyigbe

    2016-10-01

    Full Text Available Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

  13. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

    DEFF Research Database (Denmark)

    Porta, Claudine; Xu, Xiaodong; Loureiro, Silvia;

    2013-01-01

    Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release...

  14. Xylanase and Protease Increase Solubilization of Non-Starch Polysaccharides and Nutrient Release of Corn- and Wheat Distillers Dried Grains with Solubles

    DEFF Research Database (Denmark)

    Pedersen, Mads Brøgger; Dalsgaard, Søren; Arent, Susan

    2015-01-01

    and protease yielded the highest degree of enzymatic degradation, indicating close association of arabinoxylan and protein within the cell wall matrix. Collectively, the GH10 xylanase degraded DDGS more efficiently than the GH11 xylanases, due to the complexity of the substrate and the substrate affinity...... of this xylanase. The current in vitro results indicate a high potential of xylanase in combination with protease to efficiently degrade DDGS and promote nutrient release in diets for non-ruminant animals....

  15. Molecular Recognition of Cobalt(III)-ligated Peptides by Serine Proteases: The Role of Electrostatic Effects

    DEFF Research Database (Denmark)

    Bagger, Sven; Wagner, Kim

    1998-01-01

    A series of peptides with a positively charged cobalt(III)-complex group attached to the carboxylate terminal was synthesized. The behaviour of these metallopeptides as acceptor nucleophiles in acyl transfer reactions catalyzed by the three serine proteases bovine pancreatic à-chymotrypsin, porci...... and charged residues on the enzyme surfaces. The idea of using the metallopeptides in practical enzymatic peptide synthesis is put forward....

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

    OpenAIRE

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

    2015-01-01

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

  17. High Variability of Plasma Drug Concentrations in Dual Protease Inhibitor Regimens

    OpenAIRE

    Guiard-Schmid, Jean-Baptiste; Poirier, Jean-Marie; Meynard, Jean-Luc; Bonnard, Philippe; Gbadoe, Ayi Hola; Amiel, Corinne; Calligaris, Frédérique; Abraham, Bruno; Pialoux, Gilles; Girard, Pierre-Marie; Jaillon, Patrice; Rozenbaum, Willy

    2003-01-01

    Ritonavir (RTV) strongly increases the concentrations of protease inhibitors (PIs) in plasma in patients given a combination of RTV and another PI. This pharmacological interaction is complex and poorly characterized and shows marked inter- and intraindividual variations. In addition, RTV interacts differently with saquinavir (SQV), indinavir (IDV), amprenavir (APV), and lopinavir (LPV). In this retrospective study on 542 human immunodeficiency virus-infected patients, we compared inter- and ...

  18. High temperature wear performance of HVOF-sprayed Cr3C2-WC-NiCoCrMo and Cr3C2-NiCr hardmetal coatings

    Science.gov (United States)

    Zhou, Wuxi; Zhou, Kesong; Li, Yuxi; Deng, Chunming; Zeng, Keli

    2017-09-01

    A novel Cr3C2-WC-NiCoCrMo and commercial Cr3C2-NiCr thermal spray-grade powders with particle size of -45 + 15 μm were prepared by an agglomeration and sintering process. Cr3C2-WC-NiCoCrMo and Cr3C2-NiCr coatings were deposited by high velocity oxygen fuel (HVOF) spraying. The fundamental properties of both coatings were evaluated and friction wear test against Al2O3 counterbodies of both coatings at high temperatures (450 °C, 550 °C, 650 °C) were carried out ball-on-disk high temperature tribometer. All specimens were characterized by optical microscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS) and 3D non-contact surface mapping profiler. The results have shown that the Cr3C2-WC-NiCoCrMo coating exhibited lower porosity, higher micro-hardness compared to the Cr3C2-NiCr coating. The Cr3C2-WC-NiCoCrMo coating also exhibited better wear resistance and higher friction coefficient compared to the Cr3C2-NiCr coating when sliding against the Al2O3 counterpart. Wear rates of both coatings increased with raising temperature. Both coatings experienced abrasive wear; hard phase particles (WC and Cr3C2) with different sizes, distributed in the matrix phase, will effectively improve the resistance against wear at high temperatures.

  19. Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus.

    Science.gov (United States)

    Vass, Robert H; Chien, Peter

    2013-11-01

    Chromosome replication relies on sliding clamps that are loaded by energy-dependent complexes. In Escherichia coli, the ATP-binding clamp loader subunit DnaX exists as both long (τ) and short (γ) forms generated through programmed translational frameshifting, but the need for both forms is unclear. Here, we show that in Caulobacter crescentus, DnaX isoforms are unexpectedly generated through partial proteolysis by the AAA+ protease casein lytic proteinase (Clp) XP. We find that the normally processive ClpXP protease partially degrades DnaX to produce stable fragments upon encountering a glycine-rich region adjacent to a structured domain. Increasing the sequence complexity of this region prevents partial proteolysis and generates a τ-only form of DnaX in vivo that is unable to support viability on its own. Growth is restored when γ is provided in trans, but these strains are more sensitive to DNA damage compared with strains that can generate γ through proteolysis. Our work reveals an unexpected mode of partial processing by the ClpXP protease to generate DnaX isoforms, demonstrates that both τ and γ forms of DnaX are required for Caulobacter viability, and identifies a role for clamp loader diversity in responding to DNA damage. The conservation of distinct DnaX isoforms throughout bacteria despite fundamentally different mechanisms for producing them suggests there may be a conserved need for alternate clamp loader complexes during DNA damaging conditions.

  20. Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin.

    Science.gov (United States)

    Murakami, Yoji; Wada, Yoshihiro; Kobayashi, Hidetomo; Irie, Atsushi; Hasegawa, Makoto; Yamanaka, Hiroyasu; Okamoto, Keinosuke; Eto, Masatoshi; Imamura, Takahisa

    2012-10-01

    ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.

  1. Heighten the Study on Factor Seven Activating Protease

    Institute of Scientific and Technical Information of China (English)

    贺石林; 陈方平; 张广森; 文志斌

    2008-01-01

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

  2. Economic Methods of Ginger Protease'sextraction and Purification

    Science.gov (United States)

    Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

    This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

  3. Serum mannan-binding lectin-associated serine protease 2 levels in colorectal cancer: relation to recurrence and mortality

    DEFF Research Database (Denmark)

    Ytting, Henriette; Christensen, Ib Jarle; Thiel, Steffen;

    2005-01-01

    PURPOSE: Mannan-binding lectin-associated serine protease 2 (MASP-2) is a plasma protein involved in inflammatory processes. MASP-2 circulates in complex with the protein mannan-binding lectin (MBL) or ficolins, and is activated to recruit the complement system when MBL binds to its targets...

  4. A HARD GAMMA-RAY FLARE FROM 3C 279 IN 2013 DECEMBER

    Energy Technology Data Exchange (ETDEWEB)

    Paliya, Vaidehi S.; Stalin, C. S. [Indian Institute of Astrophysics, Block II, Koramangala, Bangalore-560034 (India); Diltz, Chris; Böttcher, Markus [Astrophysical Institute, Department of Physics and Astronomy, Ohio University, Athens, OH 45701 (United States); Buckley, David, E-mail: vaidehi@iiap.res.in [South African Astronomical Observatory, P.O. Box 9, Observatory 7935, Cape Town (South Africa)

    2016-01-20

    The blazar 3C 279 exhibited twin γ-ray flares of similar intensity in 2013 December and 2014 April. In this work, we present a detailed multi-wavelength analysis of the 2013 December flaring event. Multi-frequency observations reveal the uncorrelated variability patterns with X-ray and optical–UV fluxes peaking after the γ-ray maximum. The broadband spectral energy distribution (SED) at the peak of the γ-ray activity shows a rising γ-ray spectrum but a declining optical–UV flux. This observation along with the detection of uncorrelated variability behavior rules out the one-zone leptonic emission scenario. We, therefore, adopt two independent methodologies to explain the SED: a time-dependent lepto-hadronic modeling and a two-zone leptonic radiative modeling approach. In the lepto-hadronic modeling, a distribution of electrons and protons subjected to a randomly orientated magnetic field produces synchrotron radiation. Electron synchrotron is used to explain the IR to UV emission while proton synchrotron emission is used to explain the high-energy γ-ray emission. A combination of both electron synchrotron self-Compton emission and proton synchrotron emission is used to explain the X-ray spectral break seen during the later stage of the flare. In the two-zone modeling, we assume a large emission region emitting primarily in IR to X-rays and γ-rays to come primarily from a fast-moving compact emission region. We conclude by noting that within a span of four months, 3C 279 has shown the dominance of a variety of radiative processes over each other and this reflects the complexity involved in understanding the physical properties of blazar jets in general.

  5. The High Energy view of the Broad Line Radio Galaxy 3C 111

    CERN Document Server

    Ballo, L; Reeves, J N; Sambruna, R M; Tombesi, F

    2011-01-01

    We present the analysis of Suzaku and XMM-Newton observations of the broad-line radio galaxy (BLRG) 3C 111. Its high energy emission shows variability, a harder continuum with respect to the radio quiet AGN population, and weak reflection features. Suzaku found the source in a minimum flux level; a comparison with the XMM-Newton data implies an increase of a factor of 2.5 in the 0.5-10 keV flux, in the 6 months separating the two observations. The iron K complex is detected in both datasets, with rather low equivalent width(s). The intensity of the iron K complex does not respond to the change in continuum flux. An ultra-fast, high-ionization outflowing gas is clearly detected in the XIS data; the absorber is most likely unstable. Indeed, during the XMM-Newton observation, which was 6 months after, the absorber was not detected. No clear roll-over in the hard X-ray emission is detected, probably due to the emergence of the jet as a dominant component in the hard X-ray band, as suggested by the detection above...

  6. Timing of Events in the Central Engine and Jets of the Radio Galaxies 3c 111 and 3c 120 (core Program)

    Science.gov (United States)

    The investigators request continuation of their long-term monitoring of the X-ray flux of the radio galaxies 3C 111 (FR 2) and 3C 120 (FR 1) 2 and 4 times per week, respectively, throughout Cycle 12, as well as a 90 days of daily monitoring of 3C 111. In both objects, dips in X-ray flux precede the appearance of bright superluminal knots in the radio jet. The long-term multiwaveband light curves and sequences of 7 mm VLBA images will record the changing pattern of multiwaveband emission in these two AGN. 3C 111 is a probable EGRET source; if the ID is correct, GLAST will measure its flux daily, allowing relative timing of gamma-ray variations with X-ray and optical events from the central engine plus radio events in the jet.

  7. Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Marcotte,L.; Wass, A.; Gohara, D.; Pathak, H.; Arnold, J.; Filman, D.; Cameron, C.; Hogle, J.

    2007-01-01

    Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3Cpro and the viral polymerase 3Dpol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4- Angstroms resolution and the G64S fidelity mutant of 3Dpol at a 3.0- Angstroms resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3Dpol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3Dpol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.

  8. GADD45A inhibits autophagy by regulating the interaction between BECN1 and PIK3C3.

    Science.gov (United States)

    Zhang, Dongdong; Zhang, Weimin; Li, Dan; Fu, Ming; Chen, Runsheng; Zhan, Qimin

    2015-01-01

    GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the function of GADD45A in autophagy remains unknown. In this report, we demonstrate that GADD45A plays an important role in regulating the process of autophagy. GADD45A is able to decrease LC3-II expression and numbers of autophagosomes in mouse tissues and different cancer cell lines. Using bafilomycin A1 treatment, we have observed that GADD45A regulates autophagosome initiation. Likely, GADD45A inhibition of autophagy is through its influence on the interaction between BECN1 and PIK3C3. Immunoprecipitation and GST affinity isolation assays exhibit that GADD45A directly interacts with BECN1, and in turn dissociates the BECN1-PIK3C3 complex. Furthermore, we have mapped the 71 to 81 amino acids of the GADD45A protein that are necessary for the GADD45A interaction with BECN1. Knockdown of BECN1 can abolish autophagy alterations induced by GADD45A. Taken together, these findings provide the novel evidence that GADD45A inhibits autophagy via impairing the BECN1-PIK3C3 complex formation.

  9. Bacterial retropepsin-like proteases : the evidence from Legionella pneumophila

    OpenAIRE

    Teixeira, Paulo Alexandre Gonçalves

    2013-01-01

    Dissertação de mestrado em Bioquímica apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra. A2 family of aspartic proteases harbors mostly proteases found in retroviruses – the retropepsins. The evolution theories regarding these proteases usually state that these proteins are related to pepsin-like proteases from family A1 by two different hypotheses. By the first (and usually most accepted) theory, upon infection of a e...

  10. Importance of lysosomal cysteine proteases in lung disease

    Directory of Open Access Journals (Sweden)

    Chapman Harold A

    2000-11-01

    Full Text Available Abstract The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically their roles in matrix remodeling and in regulating the immune response, and their relationship to lung diseases.

  11. 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.

  12. Primary structural analysis of sulfhydryl protease inhibitors from pineapple stem.

    Science.gov (United States)

    Reddy, M N; Keim, P S; Heinrikson, R L; Kezdy, F J

    1975-03-10

    Pineapple stem acetone powder provides a rich source of the sulfhydryl protease bromelain and of a family of compositionally similar but chromatographically distinct polypeptide inihibtors of this enzyme. The isoinhibitors have molecular weights of 5600, and they contain five disulfide bonds and about 50 amino acids each (Perlstein, S. H., AND Kezdy, F.J. (1973) J. Supramol. Struct. 1, 249-254). Primary structural analysis of one of the seven inhibitor fractions (VII) revealed extensive microheterogeneity. Each of the inhibitor molecules in Fraction VII was shown to be composed of two peptide chains joined by disulfide bonds. These chains, designated A and B on the basis of size, comprise 41 and 10-11 residues, respectively, and the amino acid sequence of one of each are given below: (see article for formular). On the basis of ionization properties and yields of the A and B chains, it would appear that one of the major inhibitor species in Fraction VII is the covalently linked complex of the two chains shown, namely [A-1, B-2]. The second major inhibitor component of Fraction VII is identical in structure with [A-1, B-2i1 except that residues 1 and 8 in the A chain are pyroglutamate and threonine, respectively, and in the B chain glutamine 11 is replaced by arginine. The third inhibitor in Fraction VII is a minor constituent identical with the second, except that residue 1 in the A chain is glutamate rather than pyroglutamate. This microheterogeneity in the inhibitors of Fraction VII is further increased by the fact that B chains may lack threonine 1, in which case they are decapeptides beginning with alanine. On the basis of the striking homology of the cysteine residues with those of other protease inhibitors, it is proposed that the bromelain inhibitors are generated enzymatically from single chain precursors by excision of a "bridge" paptide which links the NH-2 termal A chain to the COOH-terminal B chain.

  13. Synthesis and biological evaluation of branched and conformationally restricted analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd)

    DEFF Research Database (Denmark)

    Hrdlicka, Patrick J; Andersen, Nicolai K; Jepsen, Jan S;

    2005-01-01

    The synthesis of branched and conformationally restricted analogs of the anticancer nucleosides 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd) is presented. Molecular modeling and (1)H NMR coupling constant analysis revealed that the furanose rings of all analogs except the LNA analog...... are conformationally biased towards South conformation, and are thus mimicking the structure of ECyd. All target nucleosides were devoid of anti-HIV or anticancer activity....

  14. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells.

    Science.gov (United States)

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-03-17

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3C(pro)s) of picornaviruses share similar spatial structures and it is becoming apparent that 3C(pro) plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3C(pro) are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3C(pro) can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3C(pro) and these essential factors, 3C(pro) is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3C(pro) are ongoing and a better understanding of the roles played by 3C(pro) may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3C(pro) is summarized.

  15. Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy.

    Science.gov (United States)

    Khera, Tanvi; Todt, Daniel; Vercauteren, Koen; McClure, C Patrick; Verhoye, Lieven; Farhoudi, Ali; Bhuju, Sabin; Geffers, Robert; Baumert, Thomas F; Steinmann, Eike; Meuleman, Philip; Pietschmann, Thomas; Brown, Richard J P

    2017-03-01

    Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations ( 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-17

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

  17. Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy

    Science.gov (United States)

    Khera, Tanvi; Todt, Daniel; Vercauteren, Koen; McClure, C. Patrick; Verhoye, Lieven; Farhoudi, Ali; Bhuju, Sabin; Geffers, Robert; Baumert, Thomas F.; Steinmann, Eike; Meuleman, Philip; Pietschmann, Thomas; Brown, Richard J.P.

    2017-01-01

    Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations ( 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic

  18. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    Science.gov (United States)

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies.

  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. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.

    Science.gov (United States)

    Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D

    2015-03-01

    Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of

  1. HvPap-1 C1A Protease Participates Differentially in the Barley Response to a Pathogen and an Herbivore

    Directory of Open Access Journals (Sweden)

    Mercedes Diaz-Mendoza

    2017-09-01

    Full Text Available Co-evolutionary processes in plant–pathogen/herbivore systems indicate that protease inhibitors have a particular value in biotic interactions. However, little is known about the defensive role of their targets, the plant proteases. C1A cysteine proteases are the most abundant enzymes responsible for the proteolytic activity during different processes like germination, development and senescence in plants. To identify and characterize C1A cysteine proteases of barley with a potential role in defense, mRNA and protein expression patterns were analyzed in response to biotics stresses. A barley cysteine protease, HvPap-1, previously related to abiotic stresses and grain germination, was particularly induced by flagellin or chitosan elicitation, and biotic stresses such as the phytopathogenic fungus Magnaporthe oryzae or the phytophagous mite Tetranychus urticae. To elucidate the in vivo participation of this enzyme in defense, transformed barley plants overexpressing or silencing HvPap-1 encoding gene were subjected to M. oryzae infection or T. urticae infestation. Whereas overexpressing plants were less susceptible to the fungus than silencing plants, the opposite behavior occurred to the mite. This unexpected result highlights the complexity of the regulatory events leading to the response to a particular biotic stress.

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

    Directory of Open Access Journals (Sweden)

    Laurent Chatel-Chaix

    2010-08-01

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

  3. Powders with superparamagnetic Fe{sub 3}C particles studied with Moessbauer spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    David, B; Schneeweiss, O [Institute of Physics of Materials, Academy of Sciences of the Czech Republic, v.v.i., Ziz' kova 22, CZ-61662 Brno (Czech Republic); Dumitrache, F; Fleaca, C; Alexandrescu, R; Morjan, I [National Institute for Lasers, Plasma and Radiation Physics, PO Box MG-36, 077125 Bucharest-Magurele (Romania)

    2010-03-01

    Two nanopowders with superparamagnetic Fe{sub 3}C particles were synthesised by the method of laser-induced pyrolysis of gaseous precursors. Both were characterised by X-ray diffraction, Moessbauer spectrometry and standard magnetic measurements. The mean crystallite size of Fe{sub 3}C was 3 nm for the first sample and 10 nm for the second sample (Scherrer formula), i.e. it was lower than in our previously studied ferromagnetic Fe{sub 3}C-based sample. Fe{sub 3}C phase in both present samples exhibited by {approx}20 K reduced Curie temperature which is interpreted as a nanosize effect. After annealing of the samples at 1073 K for 30 minutes the Curie temperature of the Fe{sub 3}C phase in both samples matched its standard bulk value. Beside Fe{sub 3}C phase also Fe{sub 3}O{sub 4} and carbon black were present in the synthesised samples.

  4. Powders with superparamagnetic Fe3C particles studied with Mössbauer spectrometry

    Science.gov (United States)

    David, B.; Schneeweiss, O.; Dumitrache, F.; Fleaca, C.; Alexandrescu, R.; Morjan, I.

    2010-03-01

    Two nanopowders with superparamagnetic Fe3C particles were synthesised by the method of laser-induced pyrolysis of gaseous precursors. Both were characterised by X-ray diffraction, Mössbauer spectrometry and standard magnetic measurements. The mean crystallite size of Fe3C was 3 nm for the first sample and 10 nm for the second sample (Scherrer formula), i.e. it was lower than in our previously studied ferromagnetic Fe3C-based sample. Fe3C phase in both present samples exhibited by ~20 K reduced Curie temperature which is interpreted as a nanosize effect. After annealing of the samples at 1073 K for 30 minutes the Curie temperature of the Fe3C phase in both samples matched its standard bulk value. Beside Fe3C phase also Fe3O4 and carbon black were present in the synthesised samples.

  5. Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini.

    Directory of Open Access Journals (Sweden)

    Porntip Pinlaor

    Full Text Available BACKGROUND: The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa, prosegment (95 aa, and mature protease (213 aa. BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%, Paragonimus westermani (58%, Schistosoma mansoni and S. japonicum (52%, and with vertebrate cathepsin F (51%. Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant

  6. Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.

    Directory of Open Access Journals (Sweden)

    George Kostallas

    Full Text Available Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp. The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y and P1 (glutamine, Q within the substrate peptide were confirmed as being the most important specificity determinants. In position P1', glycine (G, serine (S, cysteine (C, alanine (A and arginine (R were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of

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

    Science.gov (United States)

    Zeng, F; Cohen, A C

    2000-05-01

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

  8. Viral protease cleavage of inhibitor of κBα triggers host cell apoptosis

    Science.gov (United States)

    Zaragoza, Carlos; Saura, Marta; Padalko, Elizaveta Y.; Lopez-Rivera, Ester; Lizarbe, Tania R.; Lamas, Santiago; Lowenstein, Charles J.

    2006-01-01

    Apoptosis is an innate immune response to viral infection that limits viral replication. However, the mechanisms by which cells detect viral infection and activate apoptosis are not completely understood. We now show that during Coxsackievirus infection, the viral protease 3Cpro cleaves inhibitor of κBα (IκBα). A proteolytic fragment of IκBα then forms a stable complex with NF-κB, translocates to the nucleus, and inhibits NF-κB transactivation, increasing apoptosis and decreasing viral replication. In contrast, cells with reduced IκBα expression are more susceptible to viral infection, with less apoptosis and more viral replication. IκBα thus acts as a sensor of viral infection. Cleavage of host proteins by pathogen proteases is a novel mechanism by which the host recognizes and responds to viral infection. PMID:17138672

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-05-19

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

  11. 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...

  12. 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

  13. Control of exocellular proteases in dermatophytes and especially Trichophyton rubrum.

    Science.gov (United States)

    Meevootisom, V; Niederpruem, D J

    1979-06-01

    The production of proteases was investigated during growth of dermatophytic fungi with special emphasis on Trichophyton rubrum. Exogenous glucose suppressed elastase production in all dermatophytes examined. The production of protease active guinea pig hair in keratin-salts broth by Microsporum gypseum. Trichophyton mentagrophytes and T. rubrum was also suppressed by glucose. Various carbohydrates added to keratin-salts broth curtailed protease production by T. rubrum as did individual amino acids but ammonium phosphate did not. Enzyme activities against guinea pig hair were compared in twenty-one diverse clinical isolates of T. rubrum cultured in keratin-salts broth. Activity also occurred towards casein, bovine serum albumin, keratin, collagen and elastin after keratin-growth. Studies concerning the properties of enzyme activities in culture filtrates of T. rubrum after keratin-growth suggested that multiple proteases occurred here. Hydrolysis of guinea pig hair and elastin were optimal at pH7 while keratinase was most active at alkaline pH. Divalent cations stimulated protease(s). Ferric ion and mercuric ion stimulated keratinase but were inhibitory to guinea pig hair hydrolysis and elastase. Chelating agents inhibited elastase and the hydrolysis of guinea pig hair more severely than keratinase and all of those effects were reversed by excess calcium. A serine-protease inhibitor, phenylmethylsulfonylfluoride (PMSF), curtailed keratinase but was less inhibitory to elastase and guinea pig hair hydrolysis. Soybean trypsin inhibitor arrested each protease.

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

    African Journals Online (AJOL)

    Nasir

    2016-10-12

    Oct 12, 2016 ... Fermentation medium (by using sub-merged fermentation technique) was incubated for 48 h at ... of protease, and recovery of final product free of protease ... Nutrient broth (0.8 g) was weighed and then dissolved in 50 ml of.

  15. 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

    2015-01-01

    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

  16. 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...

  17. 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 with and w...

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

    OpenAIRE

    Karlsson, Anna; Arvidson, Staffan

    2002-01-01

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

  19. Proteomic protease specificity profiling of clostridial collagenases reveals their intrinsic nature as dedicated degraders of collagen.

    Science.gov (United States)

    Eckhard, Ulrich; Huesgen, Pitter F; Brandstetter, Hans; Overall, Christopher M

    2014-04-04

    Clostridial collagenases are among the most efficient degraders of collagen. Most clostridia are saprophytes and secrete proteases to utilize proteins in their environment as carbon sources; during anaerobic infections, collagenases play a crucial role in host colonization. Several medical and biotechnological applications have emerged utilizing their high collagenolytic efficiency. However, the contribution of the functionally most important peptidase domain to substrate specificity remains unresolved. We investigated the active site sequence specificity of the peptidase domains of collagenase G and H from Clostridium histolyticum and collagenase T from Clostridium tetani. Both prime and non-prime cleavage site specificity were simultaneously profiled using Proteomic Identification of protease Cleavage Sites (PICS), a mass spectrometry-based method utilizing database searchable proteome-derived peptide libraries. For each enzyme we identified >100 unique-cleaved peptides, resulting in robust cleavage logos revealing collagen-like specificity patterns: a strong preference for glycine in P3 and P1', proline at P2 and P2', and a slightly looser specificity at P1, which in collagen is typically occupied by hydroxyproline. This specificity for the classic collagen motifs Gly-Pro-X and Gly-X-Hyp represents a remarkable adaptation considering the complex requirements for substrate unfolding and presentation that need to be fulfilled before a single collagen strand becomes accessible for cleavage. We demonstrate the striking sequence specificity of a family of clostridial collagenases using proteome derived peptide libraries and PICS, Proteomic Identification of protease Cleavage Sites. In combination with the previously published crystal structures of these proteases, our results represent an important piece of the puzzle in understanding the complex mechanism underlying collagen hydrolysis, and pave the way for the rational design of specific test substrates and

  20. Purification and Characterization of An Alkaline Protease from Acetes chinensis

    Institute of Scientific and Technical Information of China (English)

    XU Jiachao; LIU Xin; LI Zhaojie; XU Jie; XUE Changhu; GAO Xin

    2005-01-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55 ℃ and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+ , EDTA and PMSF could inhibit its activity.

  1. Alkaline Protease Production by a Strain of Marine Yeasts

    Institute of Scientific and Technical Information of China (English)

    WANG Ping; CHI Zhenming; MA Chunling

    2006-01-01

    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 ℃.The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0.The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min -1 and agitation speed 150 r min-1.Under the optimal conditions, 623.1 Umg-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.

  2. Identification of a senescence-related protease in coriander leaves

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Senescence-related protease may play an important role in leaf senescence. By improved SDS-Gela- tin-PAGE assay, a 63 ku senescence-related protease (63 SRP) in coriander leaves was identified. Activity of 63 SRP was increased in parallel to the advance of coriander leaf senescence, and inhibited by treating the leaf with gibberellic acid, and enhanced by ethylene treatment. The 63 SRP was suggested to be a serine protease based on the fact that its activity was inhibited by the protease inhibitor PMSF. The optimal temperature for the activity of the 70 ku protease was 50℃. The maximal activity was observed at pH 6-9, some activity could be observed on the gel slices incubated at pH 5 or 11. The 63 SRP was partly purified by the way of ammonium sulfate precipitation and then gel slicing after gel electrophoresis.

  3. Immunoglobulin A1 protease activity in Gemella haemolysans

    DEFF Research Database (Denmark)

    Lomholt, JA; Kilian, Mogens

    2000-01-01

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

  4. Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

    Science.gov (United States)

    Panigrahi, Rashmi; Lemieux, M Joanne

    2015-01-01

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

  5. 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.

  6. The maize cystatin CC9 interacts with apoplastic cysteine proteases.

    Science.gov (United States)

    van der Linde, Karina; Mueller, André N; Hemetsberger, Christoph; Kashani, Farnusch; van der Hoorn, Renier A L; Doehlemann, Gunther

    2012-11-01

    In a recent study we identified corn cystain9 (CC9) as a novel compatibility factor for the interaction of the biotrophic smut fungus Ustilago maydis with its host plant maize. CC9 is transcriptionally induced during the compatible interaction with U. maydis and localizes in the maize apoplast where it inhibits apoplastic papain-like cysteine proteases. The proteases are activated during incompatible interaction and salicylic acid (SA) treatment and, in turn, are sufficient to induce SA signaling including PR-gene expression. Therefore the inhibition of apoplastic papain-like cysteine proteases by CC9 is essential to suppress host immunity during U. maydis infection. Here were present new experimental data on the cysteine protease-cystatin interaction and provide an in silco analysis of plant cystatins and the identified apoplastic cysteine proteases.

  7. Purification and characterization of an alkaline protease from Acetes chinensis

    Science.gov (United States)

    Xu, Jiachao; Liu, Xin; Li, Zhaojie; Xu, Jie; Xue, Changhu; Gao, Xin

    2005-07-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+, EDTA and PMSF could inhibit its activity.

  8. Spatial distribution of metal emissions in supernova remnant 3C 397 viewed with Chandra and XMM

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    We present X-ray equivalent width imaging of 3C 397 for Mg Heα,Si Heα,S Heα,and Fe Kα complex lines with Chandra and XMM-Newton observations.The images revealed that the heavier the element is,the smaller the extent of the element distribution is.The Mg emission is evidently enhanced in the southeastern blow-out region,well along the radio boundary there,and appears to partially envelope the eastern Fe knot.Two bilateral hat-like Si line-emitting structures are along the northern and southern borders,roughly symmetric with respect to the south-east-northwest elongation axis.An S line-emitting shell is located just inside the northern radio and IR shell,indicating a layer of reversely shocked sulphur in the ejecta.A few enhanced Fe features are basically aligned along the diagonal of the rectangular shape of the SNR,which implicates an early asymmetric SN explosion.

  9. An outburst scenario for the X-ray spectral variability in 3C 111

    CERN Document Server

    Tombesi, F; Reynolds, C S; Garcia, J; Lohfink, A

    2013-01-01

    We present a combined Suzaku and Swift BAT broad-band E=0.6-200keV spectral analysis of three 3C 111 observations obtained in 2010. The data are well described with an absorbed power-law continuum and a weak (R~0.2) cold reflection component from distant material. We constrain the continuum cutoff at E_c~150-200keV, which is in accordance with X-ray Comptonization corona models and supports claims that the jet emission is only dominant at much higher energies. Fe XXVI Ly\\alpha emission and absorption lines are also present in the first and second observations, respectively. The modelling and interpretation of the emission line is complex and we explore three possibilities. If originating from ionized disc reflection, this should be emitted at r_in> 50r_g or, in the lamp-post configuration, the illuminating source should be at a height of h> 30r_g over the black hole. Alternatively, the line could be modeled with a hot collisionally ionized plasma with temperature kT = 22.0^{+6.1}_{-3.2} keV or a photo-ionized...

  10. Probing the Disk-jet Connection of the Radio Galaxy 3C120 Observed with Suzaku

    CERN Document Server

    Kataoka, J; Iwasawa, K; Markowitz, A G; Mushotzky, R F; Arimoto, M; Takahashi, T; Tsubuku, Y; Ushio, M; Watanabe, S; Gallo, L C; Madejski, G M; Terashima, Y; Isobe, N; Tashiro, M S; Kohmura, T; Kataoka, Jun; Reeves, James N.; Iwasawa, Kazushi; Markowitz, Alex G.; Mushotzky, Richard F.; Arimoto, Makoto; Takahashi, Tadayuki; Tsubuku, Yoshihiro; Ushio, Masayoshi; Watanabe, Shin; Gallo, Luigi C.; Madejski, Greg M.; Terashima, Yuichi; Isobe, Naoki; Tashiro, Makoto S.; Kohmura, Takayoshi

    2006-01-01

    Broad line radio galaxies (BLRGs) are a rare type of radio-loud AGN, in which the broad optical permitted emission lines have been detected in addition to the extended jet emission. Here we report on deep (40ksec x4) observations of the bright BLRG 3C~120 using Suzaku. The observations were spaced a week apart, and sample a range of continuum fluxes. An excellent broadband spectrum was obtained over two decades of frequency (0.6 to 50 keV) within each 40 ksec exposure. We clearly resolved the iron K emission line complex, finding that it consists of a narrow K_a core (sigma ~ 110 eV or an EW of 60 eV), a 6.9 keV line, and an underlying broad iron line. Our confirmation of the broad line contrasts with the XMM-Newton observation in 2003, where the broad line was not required. The most natural interpretation of the broad line is iron K line emission from a face-on accretion disk which is truncated at ~10 r_g. Above 10 keV, a relatively weak Compton hump was detected (reflection fraction of R ~ 0.6), superposed ...

  11. The family of Deg/HtrA proteases in plants

    Directory of Open Access Journals (Sweden)

    Schuhmann Holger

    2012-04-01

    Full Text Available Abstract Background The Deg/HtrA family of ATP-independent serine endopeptidases is present in nearly all organisms from bacteria to human and vascular plants. In recent years, multiple deg/htrA protease genes were identified in various plant genomes. During genome annotations most proteases were named according to the order of discovery, hence the same names were sometimes given to different types of Deg/HtrA enzymes in different plant species. This can easily lead to false inference of individual protease functions based solely on a shared name. Therefore, the existing names and classification of these proteolytic enzymes does not meet our current needs and a phylogeny-based standardized nomenclature is required. Results Using phylogenetic and domain arrangement analysis, we improved the nomenclature of the Deg/HtrA protease family, standardized protease names based on their well-established nomenclature in Arabidopsis thaliana, and clarified the evolutionary relationship between orthologous enzymes from various photosynthetic organisms across several divergent systematic groups, including dicots, a monocot, a moss and a green alga. Furthermore, we identified a “core set” of eight proteases shared by all organisms examined here that might provide all the proteolytic potential of Deg/HtrA proteases necessary for a hypothetical plant cell. Conclusions In our proposed nomenclature, the evolutionarily closest orthologs have the same protease name, simplifying scientific communication when comparing different plant species and allowing for more reliable inference of protease functions. Further, we proposed that the high number of Deg/HtrA proteases in plants is mainly due to gene duplications unique to the respective organism.

  12. 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.

  13. Implications for Damage Recognition during Dpo4-Mediated Mutagenic Bypass of m1G and m3C Lesions

    Energy Technology Data Exchange (ETDEWEB)

    Rechkoblit, Olga; Delaney, James C.; Essigmann, John M.; Patel, Dinshaw J. (MIT); (MSKCC)

    2012-05-08

    DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3'-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.

  14. Thermal decomposition of Pr[(C5H8NS2)3(C12H8N2)

    Institute of Scientific and Technical Information of China (English)

    GUO; Pengjiang; JIAO; Baojuan; CHEN; Sanping; HU; Rongzu

    2005-01-01

    The thermal behavior of the complex Pr[(C5H8NS2)3(C12H8N2)] in a dry nitrogen flow was examined by TG-DTG analysis. The TG-DTG investigations indicated that Pr[(C5H8NS2)3-(C12H8N2)] was decomposed into Pr2S3 and deposited carbon in one step where Pr2S3 predominated in the final products. The results of non-isothermal kinetic calculations showed that the decomposition stage was the random nucleation and subsequent growth mechanism (n =nential constant In[A/s] was 7.8697. The empirical kinetics model equation was proposed as f(α) =3/2(1-α)[-ln(1-α)]1/3. The X-ray powder diffraction patterns of the thermal decomposition products at 800℃ under N2 atmosphere show that the product can be indexed to the cubic Pr2S3 phase. The transmission electron microscopy (TEM) of the final product reveals the particle appearance of a diameter within 40 nm. The experimental results show that the praseodymium sulfide nanocrystal can be prepared from thermal decomposition of Pr[(C5H8NS2)3(C12H8N2)].

  15. Synthesis of 1,2,3-tripnictolide anions by reaction of group 15 Zintl ions with acetylene. Isolation of [E3C2H2](-) (E = P, As) and preliminary reactivity studies.

    Science.gov (United States)

    Turbervill, Robert S P; Goicoechea, Jose M

    2012-06-21

    Dimethylformamide solutions of K(3)E(7) (E = P, As) react with acetylene yielding the 1,2,3-tripnictolide anions [E(3)C(2)H(2)](-) (R = P (1), As (2)). Preliminary studies have shown that 1 and 2 displace labile ligands in [Ru(COD){η(3)-CH(3)C(CH(2))(2)}(2)] (COD = 1,5-cyclooctadiene) to yield the novel complexes [Ru(η(5)-E(3)C(2)H(2)){CH(3)C(CH(2))(2)}(2)}](-) (E = P (3), As (4)).

  16. Polypeptide stimulators of the Ms-Lon protease.

    Science.gov (United States)

    Rudyak, S G; Shrader, T E

    2000-09-01

    Both the peptidase activity against small fluorescent peptide substrates and the ATPase activity of Lon (La) proteases are stimulated by unstructured proteins such as alpha-casein. This stimulation reveals the simultaneous interaction of Lon with two proteolytic substrates--alpha-casein and the peptide substrate. To understand the cellular function of this stimulation, it is important to determine the physical properties of Lon stimulators. The abilities of compositionally simple random copolymers of amino acids (rcAAs) to stimulate the peptidase and ATPase activities of the Lon protease from Mycobacterium smegmatis (Ms-Lon) and its N-terminal truncation mutant (N-E226) were determined. We report that cationic but not anionic rcAAs stimulated Ms-Lon's peptidase activity but were themselves poor substrates for the enzyme. Peptidase stimulation by rcAAs correlated approximately with the degree of hydrophobicity of these polypeptides and reached levels >10-fold higher than observed previously for Ms-Lon stimulators such as alpha-casein. In contrast to alpha-casein, which stimulates Ms-Lon's peptidase activity by 40% and ATPase activity by 150%, rcAAs stimulated peptidase activity without concomitant stimulation of ATPase activity. Active site labeling experiments suggested that both rcAAs and ATP increased peptidase activity by increasing accessibility to the peptidase active site. Peptidase activity assays in the presence of both alpha-casein and rcAAs revealed that interactions of rcAAs and alpha-casein with Ms-Lon are extremely complex and not mutually exclusive. Specifically, (1) additions of low concentrations of alpha-casein (rcAA-stimulated peptidase activity; (2) additions of higher concentrations of alpha-casein inhibited Ms-Lon's rcAA-stimulated peptidase activity; (3) additions of all concentrations of alpha-casein inhibited N-E226's rcAA-stimulated peptidase activity. We conclude the Ms-Lon can interact with an rcAA, alpha-casein, and a substrate peptide

  17. A comparison of BCF-12 organic scintillators and Al2O3:C crystals for real-time medical dosimetry

    DEFF Research Database (Denmark)

    Beierholm, Anders Ravnsborg; Andersen, Claus Erik; Lindvold, Lars;

    2008-01-01

    Radioluminescence (RL) from aluminium oxide (Al2O3:C) crystals and organic scintillators such as the blue-emitting BCF-12 can be used for precise real-time dose rate measurements during radiation therapy of cancer patients. Attaching the dosimeters to thin light-guiding fiber cables enables in vivo...... can be circumvented for pulsed beams due to the long life-time of the main luminescence center. In contrast, chromatic removal seems to be the most effective method for organic scintillators, but is found to yield some experimental complexities. In this paper, we report on dose rate measurements using...

  18. The Web Consortium - W3C leading the evolution of the Web Exhibition LEPFest 2000

    CERN Multimedia

    2000-01-01

    Founded in October 1994 to lead the World Wide Web to its full potential by developing common protocols that promote its evolution and ensure its inter- operability.Today,W3C has over 420 Members and nearly 60 full- time staff around the world who contribute to the development of W3C specifications and software.

  19. Stable earthworm serine proteases: application of the protease function and usefulness of the earthworm autolysate.

    Science.gov (United States)

    Nakajima, N; Sugimoto, M; Ishihara, K

    2000-01-01

    The fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N. et al., Biosci. Biotechnol. Biochem., 57, 1726-1730 (1993), 60, 293-300 (1996), and 63, 2031-2033 (1999)] were further characterized to exploit their catalytic functions. These enzymes are stable in solution for long periods at room temperature and strongly resistant to organic solvents, even toluene and n-hexane. The serine proteases can act on various protein substrates such as elastin and hemoglobin as well as fibrin, and also catalyzed the hydrolysis of esters such as ethyl acetate and a bioplastic, poly[(R)-3-hydroxybutyrate] film. The enzymes, in the absence of microbial degradation, contributed to the production of the earthworm autolysate possessing antioxidant ability and protease activity, whose components were similar to those of soy sauce. The extract of the earthworm autolysate could be used as a peptone substitute in media for the cultivation of microorganisms.

  20. Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens.

    Directory of Open Access Journals (Sweden)

    Luis F López-Cortés

    Full Text Available Significant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF while on protease inhibitor (PI -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens.This retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure.A total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis and virological efficacy (on-treatment analysis at week 96 were 79.3% (CI95, 76.8-81.8 and 91.5% (CI95, 89.6-93.4, respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4+ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations.Switching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for the administered drug.

  1. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

    Energy Technology Data Exchange (ETDEWEB)

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A. (UMASS, MED)

    2011-08-16

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

  2. Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

    Science.gov (United States)

    Huesgen, Pitter F.; Miranda, Helder; Lam, XuanTam; Perthold, Manuela; Schuhmann, Holger; Adamska, Iwona; Funk, Christiane

    2011-01-01

    Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803. PMID:21332448

  3. The interaction between radio lobes and hot gas in the nearby radio galaxies 3C285 and 3C442A

    CERN Document Server

    Hardcastle, M J; Worrall, D M; Croston, J H; Evans, D A; Birkinshaw, M; Murray, S S

    2007-01-01

    We present Chandra observations of two nearby radio galaxies in group environments, 3C285 and 3C442A. The host galaxies of both sources are involved in mergers with nearby massive galaxies, and the hot gas in the systems is extended along lines joining the interacting galaxies. Both sources show strong evidence for interactions between the radio lobes and the asymmetrical hot gas. We argue that the structure in the hot gas is independent of the existence of the radio lobes in these systems, and argue that hot gas shaped by an ongoing massive galaxy merger may play an important role in the dynamics of radio lobes in other objects. For 3C442A, our observations show that gas is being driven out of both members of the host interacting galaxy pair, and the implied constraints on galaxy velocities are consistent with mildly supersonic motions with respect to the group-scale hot gas. The previously known filamentary radio structure in the center of 3C442A may be a result of the interaction between hot gas expelled f...

  4. S3C2410A中STN型LCD显示驱动的实现%THE IMPLEMENTATION OF STN- LCD DISPLAY DRIVER IN S3C2410A

    Institute of Scientific and Technical Information of China (English)

    张永安

    2012-01-01

    通过对LCD显示原理的分析和对嵌入式芯片S3C2410A中LCD控制器的研究,设计了STNLCD的硬件驱动电路和软件驱动程序,完成了在S3C2410A中对STN LCD驱动的设计.实验结果显示LCD上图像显示稳定清晰,达到设计要求.该设计可用于大部分嵌入式芯片对STNLCD的驱动,是嵌入式系统中实现LCD驱动的一套较好解决方案.%By analyzing the principles of LCD display and researching embedded chips S3C2410A LCD controller, the STN LCD driver in the S3C2410A is designed ,the driver included a STNLCD hardware driver and software drivers. Experimental results show stable and clear image display on the LCD, meet the design requirements. The design can be used for most of the embedded chip STNLCD drive, it become a better solution way of LCD driver in embedded systems.

  5. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease.

    Science.gov (United States)

    Yedidi, Ravikiran S; Muhuhi, Joseck M; Liu, Zhigang; Bencze, Krisztina Z; Koupparis, Kyriacos; O'Connor, Carrie E; Kovari, Iulia A; Spaller, Mark R; Kovari, Ladislau C

    2013-09-06

    Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: 1TW7), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC50: 4.4nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6a against both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of (15)N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

  6. Simultaneous uncoupled expression and purification of the Dengue virus NS3 protease and NS2B co-factor domain.

    Science.gov (United States)

    Shannon, A E; Chappell, K J; Stoermer, M J; Chow, S Y; Kok, W M; Fairlie, D P; Young, P R

    2016-03-01

    Dengue Virus (DENV) infection is responsible for the world's most significant insect-borne viral disease. Despite an increasing global impact, there are neither prophylactic nor therapeutic options available for the effective treatment of DENV infection. An attractive target for antiviral drugs is the virally encoded trypsin-like serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B-NS3pro complex is responsible for cleaving the viral polyprotein into separate functional viral proteins, and is therefore essential for replication. Recombinant expression of an active NS2B-NS3 protease has primarily been based on constructs linking the C-terminus of the approximately 40 amino acid hydrophilic cofactor domain of NS2B to the N-terminus of NS3pro via a flexible glycine linker. The resulting complex can be expressed in high yield, is soluble and catalytically active and has been used for most in vitro screening, inhibitor, and X-ray crystallographic studies over the last 15 years. Despite extensive analysis, no inhibitor drug candidates have been identified yet. Moreover, the effect of the artificial linker introduced between the protease and its cofactor is unknown. Two alternate methods for bacterial expression of non-covalently linked, catalytically active, NS2B-NS3pro complex are described here along with a comparison of the kinetics of substrate proteolysis and binding affinities of substrate-based aldehyde inhibitors. Both expression methods produced high yields of soluble protein with improved substrate proteolysis kinetics and inhibitor binding compared to their glycine-linked equivalent. The non-covalent association between NS2B and NS3pro is predicted to be more relevant for examining inhibitors that target cofactor-protease interactions rather than the protease active site. Furthermore, these approaches offer alternative strategies for the high yield co-expression of other protein assemblies.

  7. In Vivo Assessment of Protease Dynamics in Cutaneous Wound Healing by Degradomics Analysis of Porcine Wound Exudates*

    Science.gov (United States)

    Sabino, Fabio; Hermes, Olivia; Egli, Fabian E.; Kockmann, Tobias; Schlage, Pascal; Croizat, Pierre; Kizhakkedathu, Jayachandran N.; Smola, Hans; auf dem Keller, Ulrich

    2015-01-01

    Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease

  8. In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.

    Science.gov (United States)

    Sabino, Fabio; Hermes, Olivia; Egli, Fabian E; Kockmann, Tobias; Schlage, Pascal; Croizat, Pierre; Kizhakkedathu, Jayachandran N; Smola, Hans; auf dem Keller, Ulrich

    2015-02-01

    Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease

  9. The HhoA protease from Synechocystis sp. PCC 6803 - Novel insights into structure and activity regulation.

    Science.gov (United States)

    Hall, Michael; Wagner, Raik; Lam, Xuan Tam; Funk, Christiane; Persson, Karina

    2017-06-01

    Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca(2+) or Mg(2+), binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed. Copyright © 2016. Published by Elsevier Inc.

  10. Dual Pressure from Antiretroviral Therapy and Cell-Mediated Immune Response on the Human Immunodeficiency Virus Type 1 Protease Gene

    Science.gov (United States)

    Karlsson, Annika C.; Deeks, Steven G.; Barbour, Jason D.; Heiken, Brandon D.; Younger, Sophie R.; Hoh, Rebecca; Lane, Meghan; Sällberg, Matti; Ortiz, Gabriel M.; Demarest, James F.; Liegler, Teri; Grant, Robert M.; Martin, Jeffrey N.; Nixon, Douglas F.

    2003-01-01

    Human immunodeficiency virus (HIV)-specific CD8+ T-lymphocyte pressure can lead to the development of viral escape mutants, with consequent loss of immune control. Antiretroviral drugs also exert selection pressures on HIV, leading to the emergence of drug resistance mutations and increased levels of viral replication. We have determined a minimal epitope of HIV protease, amino acids 76 to 84, towards which a CD8+ T-lymphocyte response is directed. This epitope, which is HLA-A2 restricted, includes two amino acids that commonly mutate (V82A and I84V) in the face of protease inhibitor therapy. Among 29 HIV-infected patients who were treated with protease inhibitors and who had developed resistance to these drugs, we show that the wild-type PR82V76-84 epitope is commonly recognized by cytotoxic T lymphocytes (CTL) in HLA-A2-positive patients and that the CTL directed to this epitope are of high avidity. In contrast, the mutant PR82A76-84 epitope is generally not recognized by wild-type-specific CTL, or when recognized it is of low to moderate avidity, suggesting that the protease inhibitor-selected V82A mutation acts both as a CTL and protease inhibitor escape mutant. Paradoxically, the absence of a mutation at position 82 was associated with the presence of a high-avidity CD8+ T-cell response to the wild-type virus sequence. Our results indicate that both HIV type 1-specific CD8+ T cells and antiretroviral drugs provide complex pressures on the same amino acid sequence of the HIV protease gene and, thus, can influence viral sequence evolution. PMID:12767994

  11. Exploring a new serine protease from Cucumis sativus L.

    Science.gov (United States)

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process.

  12. Cold-adapted proteases as an emerging class of therapeutics.

    Science.gov (United States)

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications.

  13. Screening and characterization of protease producing actinomycetes from marine saltern.

    Science.gov (United States)

    Suthindhiran, Krish; Jayasri, Mangalam Achuthananda; Dipali, Dipa; Prasar, Apurva

    2014-10-01

    In the course of systematic screening program for bioactive actinomycetes, an alkaline protease producing halophilic strain Actinopolyspora sp. VITSDK2 was isolated from marine saltern, Southern India. The strain was identified as Actinopolyspora based on its phenotypic and phylogenetic characters. The protease was partially purified using ammonium sulfate precipitation and subsequently by DEAE cellulose column chromatography. The enzyme was further purified using HPLC and the molecular weight was found to be 22 kDa as determined by SDS-PAGE analysis. The purified protease exhibited pH stability in a wide range of 4-12 with optimum at 10.0. The enzyme was found to be stable between 25 and 80 °C and displayed a maximum activity at 60 °C. The enzyme activity was increased marginally in presence of Mn(2+) , Mg(2+) , and Ca(2+) and decreased in presence of Cu(2+) . PMSF and DFP completely inhibited the activity suggesting it belongs to serine protease. Further, the proteolytic activity was abolished in presence of N-tosyl-L-lysine chloromethyl ketone suggesting this might be chymotrypsin-like serine protease. The protease was 96% active when kept for 10 days at room temperature. The results indicate that the enzyme belong to chymotrypsin-like serine protease exhibiting both pH and thermostability, which can be used for various applications in industries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Expression and functions of proteases in vascular tissues.

    Science.gov (United States)

    Petzold, H Earl; Zhao, Mingzhe; Beers, Eric P

    2012-05-01

    With the emergence of new models for wood formation and the increasing emphasis on improving the efficiency of cellulosic biofuel production, research on vascular tissue biology has intensified in recent years. Some of the most active areas of research focus on manipulating activity of enzymes in the cellulose, hemicellulose, pectin and lignin pathways. In addition, great strides have been made in the characterization of transcriptional networks controlling genes that affect differentiation, secondary cell wall synthesis and programmed cell death in xylem. Less attention has been devoted to the characterization of proteases that may be important regulators of post-translational events that affect vascular cell differentiation and function and cell wall composition. Several genes for proteases and components of the ubiquitin/26S proteasome pathway are upregulated in xylem and phloem and in cell culture systems for studying the differentiation of xylem tracheary elements (TEs). Although small molecule protease inhibitors have been used to explore the roles of proteases during the differentiation of cultured TEs, only a small number of vascular tissue-associated protease genes have been directly tested to determine whether they play roles in vascular tissue biology. In this report, we review roles for proteases in vascular cell differentiation and function as determined through the use of protease inhibitors and genetic analyses and conclude by identifying opportunities for future research in this area. Copyright © Physiologia Plantarum 2011.

  15. 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.

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

    Science.gov (United States)

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

    2014-02-01

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

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

    Science.gov (United States)

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

    2014-08-01

    The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.

  18. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Ratia, Kiira; Pegan, Scott; Takayama, Jun; Sleeman, Katrina; Coughlin, Melissa; Baliji, Surendranath; Chaudhuri, Rima; Fu, Wentao; Prabhakar, Bellur S.; Johnson, Michael E.; Baker, Susan C.; Ghosh, Arun K.; Mesecar, Andrew D. (Loyola); (Purdue); (UIC)

    2008-10-27

    We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC{sub 50} value of 20 {mu}M, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC{sub 50} of 15 {mu}M and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.

  19. msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus.

    Science.gov (United States)

    Sahukhal, Gyan S; Batte, Justin L; Elasri, Mohamed O

    2015-02-01

    Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm.

  20. 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.

  1. Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation.

    Science.gov (United States)

    Lai, Chih-Hsin; Lai, Ying-Jung J; Chou, Feng-Pai; Chang, Hsiang-Hua D; Tseng, Chun-Che; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2016-01-01

    Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2.

  2. Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation.

    Directory of Open Access Journals (Sweden)

    Chih-Hsin Lai

    Full Text Available Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2.

  3. Doping and stability of 3C-SiC: from thinfilm to bulk growth

    DEFF Research Database (Denmark)

    Jokubavicius, V.; Sun, J.; Linnarsson, M. K.

    Cubic silicon carbide (3C-SiC) could pave the way for development of advanced electronic and optoelectronic devices. It could be an excellent substrate for growth of nitride and epitaxial graphene layers. Boron doped 3C-SiC films could reach up to 60% efficiency and pave the way for a new solar...... cell technology. Nitrogen and boron doped 3C-SiC layers can depict a new infrared LED. Hexagonal SiC is an excellent substrate for heteropeitaxial growth of 3C-SiC due to excellent compatibility in lattice constant and thermal expansion coefficient. However, the growth of 3C-SiC on such substrates...... is still being followed by a number of obstacles like polytype stabilization and high density of double positioning boundaries in the grown material. The polytype stability during epitaxial growth of doped 3C-SiC has not been explored. Consequently, the polytype stability during bulk growth of doped 3C...

  4. Impact of aeration and agitation on metabolic heat and protease secretion of Aspergillus tamarii in a real-time biological reaction calorimeter.

    Science.gov (United States)

    Dhandapani, Balaji; Mahadevan, Surianarayanan; Dhilipkumar, Sathish Sundar; Rajkumar, Suseela; Mandal, Asit Baran

    2012-06-01

    The effects of aeration and agitation on metabolic heat, alkaline protease production and morphology for Aspergillus tamarii MTCC5152 are reported in this manuscript. Measurement of metabolic heat has been attempted by the continuous and dynamic heat balance method in a biological real-time reaction calorimeter. At lower agitation intensities, growth-related processes were dominating. As a result the protease activity and the product heat yields were lower than those for 350 and 450 rpm. Although biomass growth was necessary to obtain maximum protease yield, agitation seemed to play a vital role in the protease production process. Energy dissipation per circulation function of the process is also deduced from power input. At optimal conditions, 350 rpm and 1 vvm, the gassed power required was 0.133 W. Pellet morphology and protease production were studied under different aeration and agitation intensities of A. tamarii. Pellet structure was considerably influenced by DO, a higher DO level resulted in denser pellets (1,018.4 kg/m(3)) leading to higher protease activity. Coupling of hydrodynamics and bio-reaction highlighted the complex relationship between energy dissipation, substrate uptake rate and fungal physiology. This study emphasised the potential of biocalorimetry as a reliable monitoring and robust control tool for aerobic fermentation of A. tamarii, using agricultural by-products.

  5. Towards 3C-3D digital holographic fluid velocity vector field measurement—tomographic digital holographic PIV (Tomo-HPIV)

    Science.gov (United States)

    Soria, J.; Atkinson, C.

    2008-07-01

    Most unsteady and/or turbulent flows of geophysical and engineering interest have a highly three-dimensional (3D) complex topology and their experimental investigation is in pressing need of quantitative velocity measurement methods that are robust and can provide instantaneous 3C-3D velocity field data over a significant volumetric domain of the flow. This paper introduces and demonstrates a new method that uses multiple digital CCD array cameras to record in-line digital holograms of the same volume of seed particles from multiple orientations. This technique uses the same basic equipment as Tomo-PIV minus the camera lenses, it overcomes the depth-of-field problem of digital in-line holography and does not require the complex optical calibration of Tomo-PIV. The digital sensors can be oriented in an optimal manner to overcome the depth-of-field limitation of in-line holograms recorded using digital CCD or CMOS array cameras, resulting in a 3D reconstruction of the seed particles within the volume of interest, which can subsequently be analysed using 3D cross-correlation PIV analysis to yield a 3C-3D velocity field. A demonstration experiment of Tomo-HPIV using uniform translation with nominally 11 µm diameter seed particles shows that the 3D displacement derived from 3D cross-correlation Tomo-HPIV analysis can be measured within 5% of the imposed uniform translation, where the imposed uniform translation has an estimated standard uncertainty of 4.3%. So this paper proposes a multi-camera digital holographic imaging 3C-3D PIV method, which is identified as tomographic digital holographic PIV or Tomo-HPIV.

  6. 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.

  7. 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

  8. 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

  9. OPTIMIZATION OF PROTEASE PRODUCTION FROM FUNGI ISOLATED FROM SOIL

    Directory of Open Access Journals (Sweden)

    Sonia Sethi

    2015-07-01

    Full Text Available Fungal strains isolated from soil by serial dilution method were screened for alkaline protease production. Isolate Penicillium chrysogenum the most potent producer of alkaline protease was identified. The isolate showed highest activity in the optimized medium at pH 9.0, temperature 35ºC, with 1% soycake and peptone incubated for 7 days. Proteases represent one of the largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes.

  10. Characterization of the entire cystatin gene family in barley and their target cathepsin L-like cysteine-proteases, partners in the hordein mobilization during seed germination.

    Science.gov (United States)

    Martinez, Manuel; Cambra, Ines; Carrillo, Laura; Diaz-Mendoza, Mercedes; Diaz, Isabel

    2009-11-01

    Plant cystatins are inhibitors of cysteine-proteases of the papain C1A and legumain C13 families. Cystatin data from multiple plant species have suggested that these inhibitors act as defense proteins against pests and pathogens and as regulators of protein turnover. In this study, we characterize the entire cystatin gene family from barley (Hordeum vulgare), which contain 13 nonredundant genes, and identify and characterize their target enzymes, the barley cathepsin L-like proteases. Cystatins and proteases were expressed and purified from Escherichia coli cultures. Each cystatin was found to have different inhibitory capability against barley cysteine-proteases in in vitro inhibitory assays using specific substrates. Real-time reverse transcription-polymerase chain reaction revealed that inhibitors and enzymes present a wide variation in their messenger RNA expression patterns. Their transcripts were mainly detected in developing and germinating seeds, and some of them were also expressed in leaves and roots. Subcellular localization of cystatins and cathepsin L-like proteases fused to green fluorescent protein demonstrated the presence of both protein families throughout the endoplasmic reticulum and the Golgi complex. Proteases and cystatins not only colocalized but also interacted in vivo in the plant cell, as revealed by bimolecular fluorescence complementation. The functional relationship between cystatins and cathepsin L-like proteases was inferred from their common implication as counterparts of mobilization of storage proteins upon barley seed germination. The opposite pattern of transcription expression in gibberellin-treated aleurones presented by inhibitors and enzymes allowed proteases to specifically degrade B, C, and D hordeins stored in the endosperm of barley seeds.

  11. Antiferromagnetic resonance in the Mott insulator fcc-Cs3C60.

    Science.gov (United States)

    Suzuki, Yuta; Shibasaki, Seiji; Kubozono, Yoshihiro; Kambe, Takashi

    2013-09-11

    The magnetic ground state of the fcc phase of the Mott insulator Cs3C60 was studied using a low-temperature electron spin resonance technique, and antiferromagnetic resonance (AFMR) below 1.57 K was directly observed at ambient pressure. The AFMR modes for the fcc phase of Cs3C60 were investigated using a conventional two-sublattice model with uniaxial anisotropy, and the spin-flop field was determined to be 4.7 kOe at 1.57 K. The static magnetic exchange interactions and anisotropy field for fcc-Cs3C60 were also estimated.

  12. Thermodynamic properties of solid face centered cubic Rb3C60 at high temperature and pressure

    Science.gov (United States)

    Yang, W.; Sun, J. X.; Liu, H.; Yan, G. F.

    2014-03-01

    Analytic equation of state and thermodynamic quantities of solid fcc Rb3C60 are derived by using an analytic mean field potential method. For intermolecular forces, the double-exponential potential is utilized. Four potential parameters are determined by fitting experimental compression data of Rb3C60 up to 14 GPa at 296 K. Various physical quantities including isothermals, thermal expansion, isochoric heat capacity, Helmholtz free energy and internal energy are calculated and analyzed. Calculated results are consistent with available experimental data in literature. Furthermore, spinodal temperature for Rb3C60 is found to be 2,860 K. Results verify that analytic mean field potential method is a useful approach to consider the anharmonic effect at high temperatures. Numerous reasonable predictions and the change trend of the properties for Rb3C60 at high temperature and pressure have been given.

  13. 基于S3C6410的Uboot分析与移植%Uboot Analysis and Transplantation Based on S3C6410

    Institute of Scientific and Technical Information of China (English)

    冯林琳; 耿恒山

    2013-01-01

    This paper analyzes the structure and the starting process of the new edition Uboot, gives a detailed transplant program based on S3C6410 development board, to achieve Uboot start from NAND Flash. Through the transplant of the net card DM9000A, Uboot network communication function is implemented. The paper has certain reference value for similar platform Uboot transplant.%分析Uboot新版源代码结构和其启动流程,给出基于S3C6410芯片开发板上Uboot的详细移植方案,实现从NANDFlash中启动Uboot.通过对DM9000A网卡的移植,完成Uboot的网络通信功能.对类似平台的Uboot移植有一定的参考价值.

  14. Uboot Analysis and Transplantation Based on S3C2440%基于S3C2440的Uboot分析与移植

    Institute of Scientific and Technical Information of China (English)

    申爽

    2012-01-01

    According to the structure and function of the Uboot, and the analysis of starting codes. This paper firstly proposed a transplant program based on s3c2440 high-capacity Nand Flash and Nor Flash. Then, through a multi-step migration, it improved the various functional modules. Eventually, it loaded the kernel and NFS file systems successfully, by the Uboot, given more Details of the Uboot transplantation.%首先根据对Uboot的结构功能和启动分析,提出了一种基于s3c2440大容量Nand Flash和Nor Flash的移植方案,然后通过多步的移植,完善各个功能模块.最终在Uboot下,使用NFS方式成功加载内核和文件系统,详细给出了Uboot移植方法.

  15. Microstructure and mechanical properties of thermal sprayed nanostructured Cr3C2-Ni20Cr coatings

    OpenAIRE

    Cecilio Alvares da Cunha; Nelson Batista de Lima; Jose Roberto Martinelli; Ana Helena de Almeida Bressiani; Armando Guilherme Fernando Padial; Lalgudi Venkataraman Ramanathan

    2008-01-01

    Cr3C2-Ni20Cr coatings have been used for corrosion and wear resistant applications. However, one of the shortcomings of these coatings is its low hardness, and consequent low wear resistance, for long term high temperature applications. Nanostructured coatings of many materials have exhibited higher hardness and strength compared with conventional coatings of the same material. Consequently, nanostructured coatings of other materials, including Cr3C2-Ni20Cr have been attempted to enhance over...

  16. The Extreme Ultraviolet Spectrum of the Kinetically Dominated Quasar 3C 270.1

    CERN Document Server

    Punsly, Brian

    2015-01-01

    Only a handful of quasars have been identified as kinetically dominated, their long term time averaged jet power, $\\overline{Q}$, exceeds the bolometric thermal emission, $L_{bol}$, associated with the accretion flow. This letter presents the first extreme ultraviolet (EUV) spectrum of a kinetically dominated quasar, 3C 270.1. The EUV continuum flux density of 3C 270.1 is very steep, $F_{\

  17. Target cell APOBEC3C can induce limited G-to-A mutation in HIV-1.

    Directory of Open Access Journals (Sweden)

    Khaoula Bourara

    2007-10-01

    Full Text Available The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G and APOBEC3F (A3F are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif, expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition.

  18. Target Cell APOBEC3C Can Induce Limited G-to-A Mutation in HIV-1

    Science.gov (United States)

    Bourara, Khaoula; Liegler, Teri J; Grant, Robert M

    2007-01-01

    The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G) and APOBEC3F (A3F) are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif), expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C)-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition. PMID:17967058

  19. Bowman-Birk Protease Inhibitor from Vigna unguiculata Seeds Enhances the Action of Bradykinin-Related Peptides

    Directory of Open Access Journals (Sweden)

    Alice da Cunha M. Álvares

    2014-10-01

    Full Text Available The hydrolysis of bradykinin (Bk by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M−1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

  20. Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides.

    Science.gov (United States)

    da Cunha Morales Álvares, Alice; Schwartz, Elisabeth Ferroni; Amaral, Nathalia Oda; Trindade, Neidiane Rosa; Pedrino, Gustavo Rodrigues; Silva, Luciano Paulino; de Freitas, Sonia Maria

    2014-10-30

    The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M-1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

  1. Association between variants of 5-hydroxytryptamine receptor 3C (HTR3C) and chemotherapy-induced symptoms in women receiving adjuvant treatment for breast cancer.

    Science.gov (United States)

    Pud, Dorit; Har-Zahav, Gil; Laitman, Yael; Rubinek, Tami; Yeheskel, Adva; Ben-Ami, Sarah; Kaufman, Bella; Friedman, Eitan; Symon, Zvi; Wolf, Ido

    2014-02-01

    Administration of chemotherapy is associated with a wide array of symptoms affecting quality of life. Genetic risk factors for severity of chemotherapy-induced symptoms have not been determined. The present study aimed to explore the associations between polymorphisms in candidate genes and chemotherapy-induced symptoms. Women treated with at least two cycles of adjuvant doxorubicin and cyclophosphamide, with or without paclitaxel for early breast cancer (n = 105) completed the memorial symptom assessment scale and provided blood for genotyping. DNA was extracted from peripheral blood leukocytes and assayed for single nucleotide polymorphisms (SNPs) in GTP cyclohydrolase 1 (GCH1, rs10483639, rs3783641, and rs8007267), catecholamine-o-methyltransferase (COMT, rs4818), and 5-hydroxytryptamine (serotonin) receptor 3C (HTR3C, rs6766410, and rs6807362). Genotyping of HTR3C revealed a significant association between the presence of rs6766410 and rs6807362 SNPs (K163 and G405 variants) and increased severity of symptoms (p = 0.0001 and p = 0.007, respectively). Multiple regressions revealed that rs6766410 and rs6807362, but not age or stage at diagnosis, predicted severity of symptoms (p = 0.001 and p = 0.006, respectively) and explained 12 % of the variance in each regression model. No association was found between the genetic variants of CGH1 or COMT and symptom score. Our study indicates, for the first time, an association between variants of HTR3C and severity of chemotherapy-induced symptoms. Analyzing these genetic variants may identify patients at increased risk for the development of chemotherapy-induced symptoms and targeting the serotonin pathway may serve as a novel treatment strategy for these patients.

  2. The Place of protease inhibitors in antiretroviral treatment

    Directory of Open Access Journals (Sweden)

    S.B. Tenore

    2009-10-01

    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.

  3. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)

    ashok

    2012-08-28

    Aug 28, 2012 ... sodium chloride concentration), production by submerged fermentation and analytical ... subtilis AKRS3 for alkaline protease production indicated that 3% of .... using eight different carbon sources namely, glucose, lactose,.

  4. Isolation and characterization of a protease-producing thermophilic ...

    African Journals Online (AJOL)

    H.O.D Bio

    2012-08-02

    Aug 2, 2012 ... Full Length Research Paper. Isolation and ... emergence of geothermal-heated groundwater from the ... Meanwhile, the microflora from African geothermal .... The protease activity was assayed in duplicate with cell-free culture.

  5. Stability and selectivity of alkaline proteases in hydrophilic solvents

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Ritthitham, Sinthuwat; Pleissner, Daniel

    2008-01-01

    proteases, but at higher concentrations and particularly in anhydrous systems most enzymes including alkaline proteases will denature and consequently loose activity [1]. However, partial denaturing and increased structural flexibility due to the interaction between hydrophilic solvents and alkaline...... proteases has been agued as the primary reasons for increasing activity, influencing regio-selectivity and improving the enantio-selectivity of these enzymes [2]. Alkaline proteases have been shown to be active not only on peptides, but on a wide range of renewable resources for synthesis of biologically...... active molecules and carriers, and in synthesis of carbohydrate derivatives with designed functional properties.  When it comes to regio-selectivity of alkaline proateses on carbohydrates both the properties of the particular enzyme and the influence of the solvent is determining for the position...

  6. 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...

  7. Variable context Markov chains for HIV protease cleavage site prediction.

    Science.gov (United States)

    Oğul, Hasan

    2009-06-01

    Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

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

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

    May 16, 2008 ... activity and stability towards anionic surfactants like SDS and oxidants ... Alkaline protease activity was determined by the method of. Higahara et al. ... rpm, the media was analyzed for cell mass, total protein concentration ...

  9. Optimization of alkaline protease production by Streptomyces sp ...

    African Journals Online (AJOL)

    Hacene

    2016-06-29

    Jun 29, 2016 ... The results showed the presence of an alkaline protease with optimal pH and ... significant effect on the production of the enzyme (fructose and malt extract), then defining theirs ..... Urease test. + ..... terminating inhibitors. Proc.

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

    Science.gov (United States)

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

    1994-06-13

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

  11. Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease.

    Science.gov (United States)

    Louis, John M; Roche, Julien

    2016-07-01

    Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism.

  12. Design of Multimedia System Based on S3C6410%基于S3C6410的多媒体系统设计

    Institute of Scientific and Technical Information of China (English)

    雷丰中; 刘鹏

    2011-01-01

    提出并实现了以S3C6410为核心处理器的嵌入式多媒体系统.详细介绍了多媒体系统整体设计方案、接口资源和S3C6410用于多媒体数据处理的BIT处理器.同时为方便人机交互控制,采用BC7210芯片以一种新的方式设计了红外遥控电路.实验测试表明,该系统为嵌入武多媒体系统开发提供了速度快、稳定性高的系统环境.%In this paper,the embedded multimedia system based on the processor-S3C6410 is presented and implemented. The overall design of multimedia system' s details and interfaces resources and the BIT processor used for multimedia data are given. At the same time,in order to facilitate the interactive control machine by human,a new way of using BC7210 chip to design the infrared remote control circuit is presented. Experimental tests show that the development system provide a fast,high stability system environment for embedded multimedia system development.

  13. Synthesis and electrochemical performance of Ti3C2Tx with hydrothermal process

    Science.gov (United States)

    Wang, Libo; Zhang, Heng; Wang, Bo; Shen, Changjie; Zhang, Chuanxiang; Hu, Qianku; Zhou, Aiguo; Liu, Baozhong

    2016-09-01

    In this study, a simple hydrothermal method has been developed to prepare Ti3C2Tx from Ti3AlC2 as a high-performance electrode material for supercapacitors. This method is environmentally friendly and has a low level of danger. The morphology and structure of the Ti3C2Tx can be controlled by hydrothermal reaction time, temperature and NH4F amounts. The prepared Ti3C2Tx was characterized by X-ray diffraction, field emission scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy and Brunauer-Emmet-Teller. The results show that the prepared Ti3C2Tx is terminated by O, OH, and F groups. The electrochemical properties of the Ti3C2Tx sample exhibit specific capacitance up to 141 Fcm-3 in 3 M KOH aqueous electrolyte, and even after 1000 cycles, no significant degradation of the volumetric capacitance was observed. These results indicate that the Ti3C2Tx material prepared by this hydrothermal method can be used in high performance supercapacitors.

  14. Picornaviral 3C cysteine proteinases have a fold similar to the chymotrypsin-like serine proteinases

    Energy Technology Data Exchange (ETDEWEB)

    Allaire,M.; Chernaia, M.; Malcolm, B.; James, M.

    1994-01-01

    The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 {angstrom} resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an inter-molecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

  15. Mitochondrial cereblon functions as a Lon-type protease

    OpenAIRE

    Kosuke Kataoka; China Nakamura; Toru Asahi; Naoya Sawamura

    2016-01-01

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we ...

  16. Proteomic Substrate Identification for Membrane Proteases in the Brain

    Science.gov (United States)

    Müller, Stephan A.; Scilabra, Simone D.; Lichtenthaler, Stefan F.

    2016-01-01

    Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane-bound precursor proteins and control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the “a disintegrin and metalloprotease” (ADAM), the beta-site amyloid precursor protein cleaving enzymes (BACE), membrane-type matrix metalloproteases (MT-MMP) and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential not only for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer’s disease (AD), schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail, as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even in vivo from minute amounts of mouse cerebrospinal fluid (CSF). This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10 and γ-secretase, as well as ADAM17 and signal peptide peptidase like 3 (SPPL3), we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs.

  17. Novel Procedures for Identification and Characterization of Viral Proteases Inhibitors

    OpenAIRE

    Ehrenberg, Angelica

    2014-01-01

    Viral proteases are often considered to be attractive drug targets because of their crucial function in the viral replication machinery. In order to increase our knowledge of these important targets and to contribute to the discovery and development of new antiviral drugs, the proteases from hepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and their interactions with inhibitors and fragments have been characterized, using enzyme inhibition and SPR biosensor based in...

  18. Proteomic Substrate Identification for Membrane Proteases in the Brain

    Directory of Open Access Journals (Sweden)

    Stephan A Müller

    2016-10-01

    Full Text Available Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane-bound precursor proteins and control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the a disintegrin and metalloprotease (ADAM, the beta-site APP cleaving enzymes (BACE, membrane-type matrix metalloproteases (MT-MMP and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer’s disease, schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even in vivo from minute amounts of mouse cerebrospinal fluid. This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10, and γ-secretase, as well as ADAM17 and SPPL3, we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs.

  19. 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.

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

    Science.gov (United States)

    Schauperl, Michael; Fuchs, Julian E; Waldner, Birgit J; Huber, Roland G; Kramer, Christian; Liedl, Klaus R

    2015-01-01

    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.